NCI dl - Cancer Institute Monographs First Conference on DNA Topoisomerases in Cancer Chemotherapy 1987 Number 4 U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES Public Health Service National Institutes of Health NCI National Cancer Institute, Vincent T. DeVita, Jr., Director International Cancer Information Center, Susan Molloy Hubbard, Director Editorial Board Robert E. Wittes, Editor-in-Chief Associate Editors Joseph Aisner Ross C. Donehower Robert F. Ozols Lewis Aronow Michael Friedman Carl Pinsky Gregory Curt Timothy J. Kinsella David G. Poplack Richard L. Cysyk Richard Simon Advisory Board Charles M. Balch |. David Goldman T. L. Phillips Robert C. Bast, Jr. Heine H. Hansen H. M. Pinedo Gianni Bonadonna Simon Kramer Carol S. Portlock Edwin C. Cadman Susan E. Krown Joseph V. Simone Giulio J. D’Angio John E. Niederhuber Samuel A. Wells, Jr. Publications Branch, Robin A. Atkiss, Chief Editorial Policy Manuscripts from key conferences dealing with cancer and closely related research fields, or a related group of papers on specific subjects of importance to cancer research, are considered for publication, with the understanding that they have not been published previously and are submitted exclusively to NCI Monographs. All material submitted for consideration will be subject to review, when appropriate, by at least two outside reviewers and one member of the JNC/ or Cancer Treatment Reports Editorial Board. Opinions expressed by the authors are not necessarily those of the publisher or the editors. Symposia or related papers in any of the following areas should be submitted to the Editor-in-Chief, Cancer Treatment Reports: surgery, radiotherapy, chemotherapy, biologic response modification, supportive care, pharmacology, mechanisms of drug action, and medicinal chemistry. Symposia or related papers in cancer etiology, molecular biology, prevention, control, or other areas of cancer research should be submitted to the Editor-in-Chief, JNCI. The editorial offices for both journals are located at the R. A. Bloch International Cancer Information Center, Building 82, Room 209, National Cancer Institute, Bethesda, MD 20892. First Conference on DNA Topoisomerases in Cancer Chemotherapy Editors: Milan Potmesil, M.D., Ph.D. Warren E. Ross, M.D. Editorial Board Kurt W. Kohn, M.D., Ph.D. Leroy F. Liu, Ph.D. Alumni Hall New York University Medical Center New York, N.Y. November 19-20, 1986 RCA7/ [5 ALC }2%¢ PUBL Franco M. Muggia, M.D. Robert Silber, M.D. Acknowledgments Sponsors New York University Post-Graduate Medical School Rita and Stanley H. Kaplan Cancer Center Cosponsors National Cancer Institute European Organization for Research and Treatment of Cancer We acknowledge the assistance of the following corporations: Adria Laboratories, Dublin, OH; Anthra Pharmaceuticals, Inc., New York, NY; Bristol-Myers Oncology Division, Evansville, IN; E. |. du Pont de Nemours & Co., Wilmington, DE; Farmitalia Carlo Erba, Milan, Italy; LymphoMed, Inc., Melrose Park, IL; Merck Sharp & Dohme Research Laboratories, West Point, PA; Schering Corp., Bloomfield, NJ; Smith Kline & French Laboratories, Swedeland, PA; Warner-Lambert Co., Ann Arbor, MI; and The Upjohn Co., Kalamazoo, MI. This work was supported in part by Public Health Service grant R13-CA-43843 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. TABLE OF CONTENTS Introduction and Overview Milan Potmesil and Warren E. Ross Biological Aspects of Topoisomerases DNA Topoisomerases: From a Laboratory Curiosity to a Subject in Cancer Chemotherapy James C. Wang Molecular Genetic Analysis of Topoisomerase || Gene From Drosophila melanogaster Tao-shih Hsieh, Maxwell P. Lee, James M. Nolan, and Elizabeth Wyckoff DNA Topoisomerase Activity Is Required as a Swivel for DNA Replication and for Ribosomal RNA Transcription Steven J. Brill, Stephen DiNardo, Karen Voelkel-Meiman, and Rolf Sternglanz Association of Topoisomerase | With Transcriptionally Active Loci in Drosophila David S. Gilmour and Sarah C. R. Elgin Structural Aspects of Mammalian DNA Replication: Topoisomerase II William G. Nelson and Donald S. Coffey Regulation of DNA Topoisomerases During Cellular Differentiation Annette L. Bodley, Hai-Young Wu, and Leroy F. Liu Drug-Topoisomerase Interaction Involvement of Intracellular ATP in Cytotoxicity of Topoisomerase |l-targetting Antitumor Drugs Gary Kupfer, Annette L. Bodley, and Leroy F. Liu In Vivo and In Vitro Stimulation by Antitumor Drugs of the Topoisomerase ll-induced Cleavage Sites in c-myc Proto-oncogene Jean-Francois Riou, Marie-José Vilarem, Christian J. Larsen, Eric Multon, and Guy F. Riou Camptothecin Inhibits Hsp 70 Heat-Shock Transcription and Induces DNA Strand Breaks in hsp 70 Genes in Drosophila Thomas C. Rowe, Elsa Couto, and David J. Kroll Topoisomerase Inhibitors Can Selectively Interfere With Different Stages of Simian Virus 40 DNA Replication Robert M. Snapka Page 11 17 23 31 37 41 49 55 Topoisomerase-mediated DNA Cleavage Topoisomerase Il as a Target of Anticancer Drug Action in Mammalian Cells Kurt W. Kohn, Yves Pommier, Donna Kerrigan, Judith Markovits, and Joseph M. Covey Role of Proliferation in Determining Sensitivity to Topoisomerase |l-active Chemotherapy Agents Daniel M. Sullivan, Kuan-Chih Chow, Bonnie S. Glisson, and Warren E. Ross Topoisomerase Il as a Target of Antileukemic Drugs Leonard A. Zwelling, Elihu Estey, Milorad Bakic, Lynn Silberman, and Diana Chan Topoisomerase-related Mechanisms of Drug Resistance Topoisomerase Alterations Associated With Drug Resistance in a Line of Chinese Hamster Cells Yves Pommier, Donna Kerrigan, and Kurt W. Kohn Mediation of Multi-drug Resistance in a Chinese Hamster Ovary Cell Line by a Mutant Type Il Topoisomerase Bonnie S. Glisson, Daniel M. Sullivan, Radhey Gupta, and Warren E. Ross Elevated Topoisomerase Il Activity and Altered Chromatin in Nitrogen Mustard-resistant Human Cells K. B. Tan, M. R. Mattern, R. A. Boyce, R. P. Hertzberg, and P. S. Schein Heat Shock Proteins: Role in Thermotolerance, Drug Resistance, and Relationship to DNA Topoisomerases Gloria C. Li DNA Topoisomerase Il as a Potential Factor in Drug Resistance of Human Malignancies Milan Potmesil, Yaw-Huei Hsiang, Leroy F. Liu, Hai-Young Wu, Frank Traganos, Bruce Bank, and Robert Silber Application to Cancer Chemotherapy Metabolic Activation of N-Acylanthracyclines Precedes Their Interaction With DNA Topoisomerase II Robert Silber, Leroy F. Liu, Mervyn Israel, Annette L. Bodley, Yaw-Huei Hsiang, Stanley Kirschenbaum, Trevor W. Sweatman, Ramakrishnan Seshadri, and Milan Potmesil Protein-linked DNA Strand Breaks Produced by Etoposide and Teniposide in Mouse L1210 and Human VA-13 and HT-29 Cell Lines: Relationship to Cytotoxicity Donna Kerrigan, Yves Pommier, and Kurt W. Kohn Structure-Activity Relationships of Podophyllin Congeners That Inhibit Topoisomerase || Byron H. Long Treatment Strategies in Relation to Drug Action Franco M. Muggia and Gordon McVie 61 73 79 83 89 95 99 105 113 117 123 129 Introduction and Overview Milan Potmesil'* and Warren E. Ross? The First Conference on DNA Topoisomerases in Cancer Chemotherapy represented the first meeting with the intended goals of assessing present advances in this new field of cancer research, discussing future direc- tions, and accelerating information transfer into developmental studies of new anticancer drugs. The two-day conference was attended by 68 scientists from 44 institutions within the United States and Canada, and by 23 guests from abroad. Twenty slide presentations were complemented by 30 posters discussed in a separate session. The proceedings include most of the formal lectures, and several additional contributions based on poster presentations. It comes as a surprise to realize how much work has been accomplished and how many new leads have been emblazed in the field covered by the meeting. While DNA topoisomerases, a unique class of enzymes partici- pating in vital processes involving DNA, have been studied in eubacteria and eukaryotes over the past fifteen years, it was recognized only a few years ago that some of the most valuable anticancer chemotherapeutics interact with DNA topoisomerase II. Even more recently, topoisomerase I was also identified as a target for the plant alkaloid camptothecin. These observations have enhanced interest in studies of topoisomerases in mammalian cells. The interaction between the drugs and enzymes has some common features: (i) It mostly involves natural products of microbial or plant origin and their analogs. (ii) It leads to the formation of an abortive complex between the enzyme and DNA. (iii) Formation of the complex is very likely only an initial event leading to cell death. Thus the topoisomerase, whether essential for the cell or not, is qualitatively changed by the drug, and as such can interfere with vital processes involving DNA. Our understanding of how cells process drug-stabilized complexes between topoisomerases and DNA could be essential for the elucida- tion of a killing mechanism by topoisomerase-targeted drugs. The bactericidal effect of nalidixic acid may offer an analogy to these studies. The induction of SOS repair in bacteria is crucial for irreversible drug action. A hypothesis has been proposed suggesting that SOS-like repair is also operating in mammalian cells and may play an important role in the cytotoxicity exerted by drugs inhibiting topoisomerase 11. The resistance to chemotherapy is an extremely serious problem in the management of cancer patients. Research of topoisomerases in cancer cells could be making inroads into this area. Quiescent human leukemic cells have very low levels of topoisomerase 11. This could constitute a mechanism of resistance to topoisom- erase [I-targeted drugs, which may also operate in any slow growing cancer with a large population of quiescent cells. Other factors contributing to drug resistance could include changes in enzyme regulation or processing of the complex between the enzyme and DNA, or the presence of a mutant enzyme which renders cancer cells unresponsive to topoisomerase II-directed drugs. As an attractive alternative, the resistance could be bypassed by selecting topoisomerase I as a target for cancer chemotherapy. The mapping analyses of DNA cleavage mediated by topoisomerase II have shown different patterns among various classes of drugs. However, the frequency of DNA cleavage in drug-treated cells generally correlates well with biological effectiveness of congeners. This indicates that the quantitative assessment of DNA cleav- age may be used as a screen for the selection of new potent anticancer drugs. Finally, information gathered at the basic science level should stimulate a review of current and past clinical trials for clues of relevance. Thus, the circle of basic and clinical data will be unbroken and the benefits of observations in the field of topoisomer- ase research could be made available for practical applications. The Second Conference on DNA Topoisomerases in Cancer Chemotherapy will be held on 17-19 October (Monday-Wednesday) 1988, at the New York University Medical Center in New York City. I Department of Radiology, New York University School of Medicine, New York, NY. 2 Department of Pharmacology, College of Medicine, The J. Hillis Miller Health Center, University of Florida, Gainesville. * Reprint requests: Milan Potmesil, M.D., Department of Radiology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Et B oe = iE " Til TCR CS. 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Wang?** ABSTRACT —Recent studies indicate that the type Il DNA topoisomerases of eubacteria and eukaryotes are structurally and evolutionarily related: the amino and carboxyl half of the single polypeptide eukaryotic enzyme are homologous to the B and A subunit of bacterial gyrase, respectively. The active site tyrosine of Escherichia coli DNA gyrase that becomes covalently linked to DNA during catalysis has been identified to be Tyr 122 of the A subunit. From a comparison of nucleotide sequences of the struc- tural genes encoding several other type II topoisomerases, the active site tyrosine in these enzymes can be deduced. For the type I DNA topoisomerases, although the bacterial enzyme and the eukaryotic enzyme are very different in terms of their primary structures, substrate preferences, and mechanisms, it has been shown that the yeast DNA topoisomerase I can substitute for E. coli DNA topoisomerase I in vivo. The unique features of DNA topoisomerases as targets of antibiotics and anti-tumor drugs are discussed. —NCI Monogr 4:3-6, 1987. A decade and a half has elapsed between the discovery of the first DNA topoisomerase and the first conference on these enzymes as targets of cancer chemotherapy. The field of topoisomerase studies has expanded greatly over this period, and the accumulated information has been sum- marized in a number of recent reviews (/-5). The evolution of DNA topoisomerases was probably a consequence of the double-helix structure of DNA. For the thread-like molecule to be the genetic material, all topologi- cal problems arising from the intertwined strands must be solved. A well-known example is the “swivel” problem in the separation of the parental strands during semi-conserva- tive replication, especially when the DNA is in the form of a ring. Other examples can be found in many vital processes, including transcription, recombination and chromosomal organization and decondensation. Supported by a Public Health Service grant from the National Institutes of Health, Department of Health and Human Services, and a grant from the American Cancer Society, as well as support from Harvard University, the Guggenheim Foundation, Academia Sinica, and the National Science Council during sabbatical leave in Taipei, Taiwan, Republic of China. 2 Department of Biochemistry and Molecular Biology, Harvard Univer- sity, Cambridge, MA, and Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China. 31 thank all of my past and present co-workers in studies of DNA topology and DNA topoisomerases and Dr. Leroy Liu in particular for many stimulating discussions on topoisomerases as targets of cancer chemotherapy. * Reprint requests: James C. Wang, Ph.D., Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Ave., Cambridge, MA 02138. Fig. 1 depicts reactions that are known to be catalyzed by DNA topoisomerases. All reactions shown are interconver- sions between topological isomers (topoisomers), and hence the name of the enzymes (I). Although circular DNAs are commonly used as the substrates of the enzymes in biochemical studies in vitro, long linear DNAs organized into many loops share the same topological problems as ring shaped DNA molecules. All reactions shown in Fig. 1 require the transient break- age of a DNA strand or strands. The breakage must be transient because the strands are intact in the products as well as in the reactants. A topoisomerase performs both the breakage of DNA strands and their subsequent rejoining. In between these events, another strand or pair of strands must pass through the transient discontinuity in order to change the topological state of a ring; such a strand passage event may be also mediated by the enzyme. DNA topoisomerases are subdivided into two categories based on their modes of DNA strand breakage: those breaking one strand at a time are termed type I topoisom- erases, and those breaking a pair of strands of a duplex DNA in concert are termed type II topoisomerases (6,7). Reactions A, D, and E depicted in Fig. 1 are often used to distinguish these categories. In Reaction A, catalysis by a type II enzyme changes the linking number of a DNA in steps of 2 (6,7), which is a mathematical prediction if the transiently broken pairs of ends are not allowed to rotate around the helix axis of the DNA and the linking number is altered by the passage of a duplex segment through the DNA gate (11,12); catalysis by a type I enzyme gives prod- ucts with linking numbers that differ from the linking number of the reactant by either odd or even integers (13). In reactions D and E, the type II enzymes can catalyze the reactions whether the duplex DNA ring has intact strands or not; the type I enzyme can catalyze the reactions only if there is a nick or gap in at least one of the strands (14,15). In this report, several recent findings that are relevant to studies of the enzymes as targets of therapeutic agents are described. TYPE Il TOPOISOMERASES OF EUBACTERIA AND EUKARYOTES ARE STRUCTURALLY AND EVOLUTIONARILY RELATED The type II enzyme found in eukaryotes, eukaryotic DNA topoisomerase II, differs from its counterpart in eubacteria, DNA gyrase (bacterial DNA topoisomerase II), in two major aspects. Structurally, the eukaryotic enzyme is a homodimer, whereas bacterial gyrase is an A,B, tetramer. Catalytically, the eukaryotic enzyme can only relax super- coiled DNA even though it requires ATP, whereas the bac- FIGURE 1.—Schematic drawings illustrating reactions that are catalyzed by DNA topoisomerases. Single-stranded DNA is represented by a thin line and double-stranded DNA is represented by a thick line. (A) Relax- ation and supercoiling. (B) Linking of single-stranded rings of comple- mentary sequences into a duplex ring with the strands intertwined. (C) Knotting and unknotting of a single-stranded ring. (D) Knotting and unknotting of a double-stranded ring. (E) Catenation and decatenation of double-stranded rings. The knotted rings and catenane shown are of the simplest kinds for the sake of convenience. More complex structures are usually involved in the actual reactions. Taken from (8). See (1-10) for further details. terial enzyme catalyzes the negative supercoiling of DNA in the presence of ATP. Recent nucleotide sequencing of the TOP2 genes encod- ing the type II topoisomerases of the budding yeast Sac- charomyces cerevisiae (16,17), the fission yeast Schizosac- charomyces pombe (18), and the fruit fly Drosophila melanogaster (T.-S. Hsieh, personal communication) shows clearly, however, that the amino and carboxyl terminal halves of the single-subunit eukaryotic enzyme are homolo- gous to the B and A subunits of bacterial gyrase, respec- tively, at corresponding positions along the polypeptide chains. Sequencing of the type II topoisomerase genes of phage T4 also shows that the phage enzyme shares homolo- gies at corresponding positions with the bacterial and eu- karyotic DNA topoisomerase II (19,20). When the eukaryo- tic TOP2 gene sequences are compared, the homologies are even more apparent. Several short segments of the S. cere- visiae and D. melanogaster sequences, for examples, code identical stretches of amino acids. Both the similarities and dissimilarities between the bac- terial and eukaryotic type II enzyme are likely to be signifi- cant in the search of anti-cancer drugs and antibiotics. The cloning, over-expressing, and sequencing of the genes have also laid the groundwork for the identification of the human TOP2 gene, the mapping of the active sites of the type II topoisomerases and the positions of mutations that lead to resistance to specific drugs, the crystallization of the enzymes for the determination of their three-dimensional structures, and the elucidation of the mechanisms of action of various drugs. THE ACTIVE SITE FOR DNA BREAKAGE AND REJOINING HAS BEEN MAPPED IN E. COLI GYRASE AND INFERRED IN EUKARYOTIC DNA TOPOISOMERASE Ii A hallmark of DNA topoisomerases is that when a topoisomerase cleaves a DNA strand, it becomes covalently linked to a DNA phosphoryl group. The formation of such a protein-DNA covalent intermediate was first postulated on thermodynamic ground (21), and it was later shown that the major type I and type II DNA topoisomerases in eu- karyotes and eubacteria all form such intermediates via phosphotyrosine linkages (4,5). For E. coli DNA gyrase, the active site tyrosine that becomes covalently linked to DNA during catalysis has been recently identified to be tyr 122 of the A-subunit (30). In the most definitive experiment, the gyrase-DNA covalent complex was first formed by adding sodium do- decyl sulfate to DNA-bound gyrase in the presence of oxo- linic acid. The resulting covalent complex was trypsinized and the DNA with a covalently linked tryptic fragment was isolated and used directly in a protein sequenator. Because the entire protein sequence of the E. coli gyrase A-subunit has been determined through sequencing the gene (31), the identification of even a very short stretch of amino acids in the DNA attached fragment is sufficient for the identifica- tion of the fragment and thus the active site tyrosine lo- cated on it. Table 1 shows a segment of amino acid sequence of E. coli gyrase containing Tyr122 and the sequences of a number of other DNA topoisomerases which are similar to the active site gyrase sequence. TABLE 1.—Amino Acid Sequences Around the Active Site Tyrosines of a Number of DNA Topoisomerases* Gene Amino acid sequence E. coli gyrA B. subtilis gyrA Phage T4 gene 52 S. cerevisiae TOP2 D. melanogaster TOP2 -AlaAlaAlaMetArgTyr(122)Thr- -AlaAlaAlaMetArgTyr(123)Thr- -AlaAlaAlaSerArgTyr(116)Ile- -AlaAlaAlaAlaArgTyr(793)Ile- -CysAlaSerAlaArgTyr(785)lle- ¢ Numbering of the tyrosines is from the ATG initiation codon in the sequences of the structural genes encoding the polypeptides. Only Tyr122 of E. coli DNA gyrase A subunit has been shown experimentally to be the active site tyrosine that becomes covalently linked to DNA during catalysis; the others are implicated from sequences. The sequences of B. subtilis gyrA, T4 gene 52 and S. cerevisiae TOP?2 are taken from (29, 19, and 17), respectively; the sequence of D. melanogaster TOP2 was provided by Prof. T.-S. Hsieh (Duke University). NCI MONOGRAPHS, NUMBER 4, 1987 TYPE | TOPOISOMERASES OF EUBACTERIA AND EUKARYOTES ARE VERY DIFFERENT YET YEAST DNA TOPOISOMERASE | CAN COMPLEMENT E. COLI DNA TOPOISOMERASE | DEFICIENCY In contrast to the sequence similarities shown by the prokaryotic and eukaryotic type II topoisomerases, there is little sequence homology between the type I enzymes of these organisms. Search of sequence similarities of the E. coli and S. cerevisiae DNA topoisomerase I reveals only a short lysine-rich segmént from amino acid 44 to amino acid 65 in the E. coli enzyme, which matches partially the yeast sequence starting from amino acid 79 (22). This short stretch of similarity is probably insignificant, particularly in view of the finding that the yeast sequence can be deleted without affecting its catalytic activity (M. A. Bjornsti and J. C. Wang, to be published). Biochemically, the eubacterial enzyme has a high affinity for single-stranded DNA, which underlies its preferential relaxation of negatively supercoiled DNA (21); the eukary- otic enzyme appears to interact preferentially with double- stranded DNA, and relaxes negatively and positively supercoiled DNA with equal efficiency (23,24). Further- more, when the type I enzymes form the covalent inter- mediates with DNA, the bacterial enzyme is linked to a 5’ phosphoryl group, whereas the eukaryotic enzyme is linked to a 3’ phosphoryl group (4,5). Given these dissimilarities, it is surprising that yeast DNA topoisomerase I can complement a conditional lethal top A mutant of E. coli (M. A. Bjornsti and J. C. Wang, unpublished results). The E. coli strain AS17, which carries a topA amber mutation and a plasmid-borne, thermal- sensitive suppressor, does not grow at temperatures above 37°C. Introduction of a plasmid expressing the yeast enzyme, however, rescues the lethal phenotype of the strain at 37°C. This complementarily indicates that the important common denominator of the two enzymes is their breakage and rejoining of DNA strands. Consistent with this notion is the finding, based on the analysis of about 20 E. coli topA mutants, that the catalytic activity of the enzyme correlates with its biological function (25). The genetic complementation also provides strong evi- dence that the eukaryotic enzyme is active in E. coli. This may facilitate studies of drugs that target against eukary- otic topoisomerase I: it might be plausible to carry out genetic analysis of interactions between a eukaryotic type I enzyme and its antagonists in E. coli. For the type II topoisomerases, although it has been shown previously that active yeast enzyme can be isolated from lysates of E. coli cells expressing the enzyme, it remains uncertain whether the eukaryotic enzyme is active inside the bacterial cells (26). ESSENTIALITY OF DNA TOPOISOMERASES AND DRUG DESIGN Genetic evidence shows clearly that both in eubacteria and in eukaryotes, a type II DNA topoisomerase is essen- tial (4,5). In yeast, it appears that inactivation of DNA topoisomerase II interferes with the separation of pairs of newly replicated chromosomes, leading to chromosomal loss and cell death (27). In contrast, the type I topoisomer- DNA TOPOISOMERASES IN CANCER THERAPY ases in bacteria and eukaryotes are dispensable. Although E. coli DNA topoisomerase I appears essential because its inactivation interferes with cell growth, it has been shown that the deficiency in the enzyme can be complemented by mutations elsewhere, some of which map in gyrA or gyrB, the structural genes encoding DNA gyrase (2,4,5). In yeast, deletion of the gene TOP! encoding DNA topoisomerase I has no effect on cell viability (27). The essentiality of the type II enzyme and the non- essentiality of the type I enzyme are probably responsible for the misconception that it would be futile to develop antibiotics and anti-cancer drugs that target against the type I topoisomerases. As exemplified by the extensive stud- ies of antibiotics of the nalidixic acid class, the cytotoxic- ity of a drug may have little to do with the inactivation of the enzyme, but is the result of forming an abortive com- plex (10). The recent finding that the drug camptothecin acts by blocking the DNA rejoining action of eukaryotic DNA topoisomerase I (28) demonstrates nicely this unique aspect of DNA topoisomerases as targets of therapeutic agents: any DNA topoisomerase, whether essential or not, can be converted in the presence of a drug to a poison that interferes with a vital process involving DNA. In addition to the archetype topoisomerases discussed above, several viruses and parasites are known to possess topoisomerases that differ from their host enzymes. Exam- ples are the topoisomerases of phage T4, vaccinia virus, and trypanosomatids. Such viral and parasitic enzymes are potential targets for drug design, whether they are essential or not. A number of enzymes involved in site-specific recombination and the initiation of viral DNA replication also possess topoisomerase activity. Examples are the phage A integrase, the transposons Tn3 and -yd-resolvase, the yeast 2-micron plasmid fIp gene product, and the gene A protein of phage ¢X174. Strictly speaking, enzymes in this category normally function as DNA strand-trans- ferases. Whereas a DNA topoisomerase reforms the same DNA backbone bond it breaks, a strand-transferase breaks a DNA phosphodiester bond, and transfers the DNA strand to a recipient DNA hydroxyl group different from the original one generated by the strand-breakage step. Because of the similarities in the basic steps of DNA strand breakage and rejoining, under certain conditions a DNA strand-transferase can act as a topoisomerase and vice versa. The strand-transferases can therefore be considered as special topoisomerases, and they might provide addi- tional targets for therapeutic agents. CONCLUDING REMARKS In the past decade and a half, the DNA topoisomerases have emerged as a unique class of enzymes that participate in a number of vital processes involving DNA. Interest in the topoisomerases as targets of cancer chemotherapy is a recent happening and here the topoisomerases are unique in that the pharmacological effects are not necessarily dependent on their inactivation, but can be manifested through the conversion of a fraction of the topoisomerase molecules into detonators. The sequence similarity between bacterial and eukaryotic type II topoisomerases suggests that there is common ground for studies of antibiotics tar- geting at bacterial gyrases and studies of anti-cancer agents targeting against eukaryotic DNA topoisomerase II. The 5 dissimilarity between the type I topoisomerases of bacteria and eukaryotes offers a potential advantage in the design of therapeutic agents, especially antibiotics. It is interesting to see that what started as purely a laboratory curiosity is now the topic of discussion in pharmacological and clinical stud- ies; for those of us working on the more basic aspects of the enzyme, we could perhaps feel a bit less guilty, and perhaps even feel a tinge of pride, when we write the justification sections of our research proposals. REFERENCES (1) WANG JC, Liu LF: DNA topoisomerases: Enzymes which catalyze the concerted breaking and rejoining of DNA backbone bonds. In Molecular Genetics (Taylor JH, ed), Part III. New York: Academic Press, 1979, pp 65-88. (2) GELLERT M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981. (3) Liv LF: DNA topoisomerases—Enzymes that catalyze the breaking and rejoining of DNA. Crit Rev Biochem 15:1-24, 1983. (4) VOSBERG H-P: DNA topoisomerases: Enzymes that control DNA conformation. Curr Top Microbiol Immunol 114:19-102, 1985. (5) WANG JC: DNA topoisomerases. Annu Rev Biochem 54:665-697, 1985. (6) Liu LF, Liu CC, ALBERTS BM: Type II DNA topoisom- erases: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-stranded break. Cell 19:697-707, 1980. (7) BROWN PO, CozzARELLI NR: A sign inversion mechanism for enzymatic supercoiling of DNA. Science 206: 1081-1083, 1979. (8) WANG JC: DNA topoisomerases. /n Nucleases (Linn SM, Roberts RJ, eds). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982, pp 41-57. (9) Liu LF: DNA strand passing and the function of type II DNA topoisomerases. In Mechanistic Studies of DNA Replication and Genetic Recombination (Alberts BM, Fox CF, eds), vol 19. New York: Academic Press, 1980, pp 817-831. (10) CozzARELLI NR: DNA topoisomerases. Cell 22:327-328, 1980. (11) Crick FHC: Linking numbers and nucleosomes. Proc Natl Acad Sci USA 73:2639-2643, 1976. (12) FULLER FB: Decomposition of the linking number of a closed ribbon: A problem from molecular biology. Proc Natl Acad Sci USA 75:3557-3561, 1978. (13) SHURE M, PULLEYBLANK DE, VINOGRAD J: The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity. Nucleic Acids Res 4:1183-1205, 1977. (14) TSE YC, WANG JC: E. coli and M. luteus DNA topoisom- erase I can catalyze catenation or decatenation of double- stranded DNA rings. Cell 22:269-276, 1980. (15) BROWN PO, CozzARELLI NR: Catenation and knotting of duplex DNA by type I topoisomerases: A mechanistic parallel with type 2 topoisomerases. Proc Natl Acad Sci USA 78:843-847, 1981. (16) LYNN R, GIAEVER G, SWANBERG SL, et al: Tandem regions of yeast DNA topoisomerase II share homology with different subunits of bacterial gyrase. Science 233: 647-649, 1986. (17) GIAVER G, LYNN R, GOTO T, et al: The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase II. J Biol Chem 261:12448-12454, 1986. (18) UEMURA T, MORIKAWA K, YANAGIDA M: Nucleotide sequence determination of fission yeast DNA topoisom- erase II gene: Structural and functional relations to other DNA topoisomerases. Eur J Biochem. In press. (19) HUANG W-M: The 52-protein subunit of T4 DNA topoisom- erase is homologous to the gyrA-protein of gyrase. Nucleic Acids Res 14:7379-7390, 1986. (20) HUANG W-M: Nucleotide sequence of a type II DNA topoisomerase II gene. Bacteriophage T4 gene 39. Nucleic Acids Res 14:7751-7766, 1986. (21) WANG JC: Interaction between DNA and an Escherichia coli protein w. J Mol Biol 55:523-533, 1971. (22) Tse-DINH Y-C, WANG JC: Complete nucleotide sequence of the topA gene encoding Escherichia coli DNA topo- isomerase I. J Mol Biol 191:321-331, 1986. (23) CHAMPOUX JJ, DULBECCO R: An activity from mammalian cells that untwists superhelical DNA—A possible swivel for DNA replication. Proc Natl Acad Sci USA 69:143-146, 1972. (24) BAASE WA, WANG JC: An w protein from Drosophila melanogaster. Biochemistry 13:4299-4303, 1974. (25) ZUuMSTEIN L, WANG JC: Probing the structural domains and function in vivo of Escherichia coli DNA topoisomerase I by mutagenesis. J Mol Biol 191:333-340, 1986. (26) Goto T, WANG JC: Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell 36:1073-1080, 1984. (27) YANAGIDA M, WANG JC: Yeast DNA topoisomerases and their structural genes. /n Nucleic Acids and Molecular Biology (Eckstein F, Lilley DMJ, eds). Berlin: Springer- Verlag. In press. (28) HSIANG Y-H, HERTZBERG R, HECHT S, et al: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873-14878, 1985. (29) MORIGA S, OGASAWARA N, YOSHIKAWA H: Structure and function of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin. Nucleic Acids Res 13:2251-2265, 1985. (30) Horowitz DS, WANG JC: Mapping the active site tyrosine of Escherichia coli DNA gyrase. J Biol Chem 262:5339- 5344, 1987. (31) SWANBERG SL, WANG JC: The cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase. J Mol Biol. In press. Molecular Genetic Analysis of Topoisomerase Il Gene From Drosophila melanogaster? Tao-shih Hsieh,* Maxwell P. Lee, James M. Nolan, and Elizabeth Wyckoff? ABSTRACT —The gene encoding the Drosophila type Il DNA topoisomerase has been isolated and characterized. The enzyme is coded for by a messenger RNA of about 5000 nucleotides, the expected size for the enzyme with MWr 170,000, and the gene is interrupted by four introns. The cytogenetic location of this gene has been mapped to the left arm of chromosome 2 at 37D2-6 by both in situ hybridization to polytene chromosomes and genomic blot-hybridization. The complete nucleotide sequence of the coding region and the flanking sequence has been determined. The deduced amino acid sequence shows interesting homology with topoisomerases II from bacteria, bacteriophage T4, and yeasts. The sequence homology between the Drosophila enzyme and those from Saccharomyces and Schizosaccharomyces is very extensive. This, in combination with the similar biochemical properties among all the eucaryotic DNA topoisomerases II, indicates that these enzymes are conserved during the course of evolution.— NCI Monogr 4:7-10, 1987. The structural transitions in DNA and chromatin can be mediated by a class of enzymes called DNA topoisomerases. These structural changes can be condensation and decon- densation of chromosomes during cell cycles as well as chromosomal segregation after its replication. They can also be due to any changes in the DNA secondary struc- tures in either a circular, covalently closed chromosome or in a linear chromosome with ends being restricted to form a loop-like structure. DNA topoisomerases facilitate these structural alterations by being able to transiently break and rejoin DNA strands [for reviews, see (/-4)]. They are classified into two types according to their action of mechanism: type I enzymes can nick and rejoin one DNA strand at a time; type II enzymes work by passing a DNA segment through a reversible double-strand break. From a combination of biochemical and genetic ap- proaches, the physiological functions of these enzymes are best analyzed in bacteria and yeasts. The top 4 gene codes for one of the topoisomerases I in bacteria, w protein, a supercoil-relaxation activity first discovered in E. coli extract in 1969 (5). DNA gyrase was discovered in 1976 for I'Supported by a Public Health Service grant from the National Institute of General Medical Sciences, National Institutes of Health, Department of Health and Human Services. T.-S. Hsieh also received support from Duke University, Academia Sinica, and the National Science Council during sabbatical leave in Taipei, Taiwan, Republic of China. 2Department of Biochemistry, Duke University Medical Center, Durham, NC. * Reprint requests: Tao-shih Hsieh, Ph.D., Department of Biochemistry, Duke University Medical Center, Durham, NC 27710. its activity to supercoil closed circular DNA molecules utilizing the energy from the hydrolysis of ATP (6). This bacterial topoisomerase II is encoded by two genetic loci, gyrA and gyrB. Both DNA gyrase and topoisomerase I control the supercoiling of bacterial chromosomes through a dynamic balance between their activities. Both enzymes can affect esentially all aspects in DNA metabolism, including replication, transcription, recombination, and re- pair (1-4). The genes encoding type I and type II DNA topoisom- erases have been identified in both the budding yeast (7-10) and the fission yeast (/1). It appears that rop1 (coding for topoisomerase I) is not an essential gene and top2 (coding for topoisomerasell) is essential for yeast growth. In most parts of the cell cycles, either topl or top2 is required, and one can functionally substitute the other in providing the essential topoisomerase activity. However, top2 is specifi- cally required at the time of cell division, most likely in the step of topologically separating the intertwined daughter chromosomes (7,12,13). Other functions have also been proposed for topoisomer- ase II. Recently, DNA topoisomerase II was identified as a major component in the structural framework of the inter- phase nucleus, the nuclear matrix, and of the metaphase chromosome scaffold (14-16). The possibility that topo- isomerase II itself provides the attachment point in the nucleus to form the looped domain structure of chromo- somes is encouraged by the finding that matrix attachment regions in DNA are enriched in putative topoisomerase II binding sites (17,18). This observation also serves to stimu- late the speculation that topoisomerases can generate a local torsional stress in actively transcribed regions in the chromatin (19-21). This is an attractive model for gene activation in eucaryotes. However, there is no direct evi- dence available to demonstrate the presence of torsional stress in active genes. The available data actually suggest that there is no torsional stress in the bulk of chromatin and essentially all the superhelical turns in the nucleus are retained by the nucleosome structure (22). This does not rule out the possibility that a small fraction of the chroma- tin is under topological tension. We are interested in establishing a genetic system to ana- lyze the functions of DNA topoisomerases during the growth and differentiation of eucaryotic cells. By using immunochemical methods in screening a Drosophila cDNA library constructed with a phage expression vector, Agt 11, we have isolated and characterized the gene coding for the Drosophila type 11 DNA topoisomerase (23). The following is a summary of our current results in this area. MOLECULAR CLONING OF TOPOISOMERASE II GENE (TOP2) IN DROSOPHILA We have purified DNA topoisomerase II from Dro- sophila embryos and prepared monospecific antibodies against it (24). After screening a Drosophila cDNA library constructed with an expression vector system of Agt 11 (25), we isolated the cDNA sequences encoding part of or the whole enzyme molecule. Initially the identity of the cloned gene being the one coding for topoisomerase II was confirmed by the following observations. (1) The positive recombinant phages produced fusion proteins which are antigenic toward topoisomerase II antibodies and the sizes of these fusion proteins correlated with the sizes of inserted Drosophila sequences. We have purified the fraction of antibody which can bind specifically with these fusion proteins, and these affinity-purified antibcdies can recog- nize and bind topoisomerase II in the Drosophila embryo extract. (2) The top2 RNA was prepared from a full-length cDNA clone using the in vitro transcription system with the phage-specific promoters. The in vitro translation of top2 DNA in rabbit reticulocyte lysates generated a series of polypeptides with sizes up to that of topoisomerase II, and these high molecular weight polypeptides can be immuno- precipitated with topoisomerase II antibodies. (3) We have inserted the top2 full-length cDNA sequence into plasmid expression vector systems under the control of inducible promoter AP, or the trp/lac hybrid promoter. Under the appropriate inducing conditions, we can detect the synthesis of antigenic molecules in E. coli cells. The sizes of these polypeptides correspond to that of topoisomerase II (Y. Chu, M. Lee, and T. Hsieh, unpublished results). (4) The Northern blot-hybridization experiment showed that the top2 mRNA in Drosophila cells is approximately 5.1 kb (kilobases) in length. This size is what is expected for an mRNA coding for a polypeptide with the size of topoisom- erase II (MWr 170,000). CYTOGENETIC LOCATION OF TOP2 We have mapped the chromosomal location of zop2 by in situ hybridization to polytene chromosomes from the larval salivary glands. It is located in the left arm of chromosome 2 at a position 37D (Fig. 1). To further refine FIGURE 1.—In situ hybridization of top2 probe to polytene chromosomes. Biotinylated fop2 cDNA probe was hybridized to 3rd instar larval salivary glands and detected by the color reaction from the peroxidase conjugated to avidin. From (23). NCI MONOGRAPHS, NUMBER 4, 1987 the mapped position, we took advantage of the available deficiency strains with varying deletions in this chro- mosomal region. We have the top2 location determined with respect to various deletion end points by both in situ hybridization and genomic blot-hybridization experiments. From these data, top? is localized in a region of approxi- mately 100 kb corresponding to the cytogenetic position 37D2-6. Furthermore, these mapping results also indicated that the orientation of the top2 gene with respect to chro- mosome 2 is such that the 5’ end of the gene, compared with its 3’ end, is proximal to the centromere (or distal to the telomere). We do not yet know the location of top2 with respect to the known lethal complementation groups in this chro- mosomal region. However, it is interesting to note that a meiotic drive mutation Sd (segregation distorter) is also mapped to this region (26). The phenotypic definition of Sd is that males heterozygous for Sd (Sd-bearing chromosome 2/ Sd*-normal chromosome 2) produce functional gametes almost all of which carry Sd-chromosome 2 (27). The molecular basis of Sd and its possible relationship to top2 is an interesting question and has yet to be answered. STRUCTURAL ANALYSIS OF TOP2 Using the cloned top2 cDNA as probes we have isolated genomic DNA sequences containing and surrounding top2 locus in Drosophila. By comparing the cloned cDNA and genomic DNA sequences and from S1 nuclease digestion experiments, we determined the intron/exon structure of top2 (Fig. 2). There are 5 exons encoding topoisomerase 11 and the largest intron is about 1 kb in length and located near the 5’ end of the gene while the other three are all less than 100 bp in length. The major site of transcription initiation has been determined by both the primer extension and S1 nuclease digestion experiments (Nolan J, et al., unpublished data). There is no clear homology to the consensus sequences of TATA box, CAAT box, and GC box in the sequences up- stream from transcription initiation. The polyadenylation/ cleavage site was mapped by determining the nucleotide sequences at the 3’ ends of the cDNA clones. The poly- adenylation/ cleavage consensus sequence AATAAA is lo- cated 27 nucleotides from the polyadenylation site. The complete nucleotide sequence of zop2 is determined (Wyckoff E, et al., unpublished data). There is a long open reading frame in the DNA sequence, which can produce a polypeptide composed of 1447 amino acid residues. The calculated molecular weight of this polypeptide is about 164,000, in excellent agreement with the measured size of purified topoisomerase II (28,29). The deduced amino acid sequence indicates that this protein is highly basic with a calculated pl of 9.2. However, toward the carboxyl terminal of the protein, it has a segment rich in the acidic residues. COMPARISON WITH OTHER TYPE Il DNA TOPOISOMERASES The Drosophila topoisomerase II shares striking sequence homology with other type II topoisomerases, especially those from eucaryotic sources. The amino acid sequence of the Drosophila enzyme can be aligned to give significant homology with those from Bacillus subtilis (30), bacterio- phage T4 (31,32), Saccharomyces cerevisiae (33), and DNA TOPOISOMERASES IN CANCER THERAPY 1Kb a rw ~N ob Fo No Fw Lo Fon Fo Ly AFcl § EE—r——— A FIGURE 2.—Restriction map of top2 locus in Drosophila. The structure of top2 is shown by the open boxes standing for the exons and by the restriction enzyme cleavage sites. The full-length cDNA clone (AFcl) is also shown under the genomic map. The restriction sites are: Sa/l (S), Pstl (P), Mul (M), Bglll (Bg), BamHI (B), and EcoRI (R). From 23). Schizosaccharomyces pombe (34). Homology comparison with bacterial DNA gyrase sequence clearly indicates that the amino terminal half of the Drosophila enzyme is closely related to the gyrB subunit (approximately 26% in homol- ogy). While the sequence at the carboxy terminal half diverges from the gyrA sequence, they still share significant homology, especially around the region where the active site tyrosine residue is inferred (35). The sequence homology between the yeast and Drosophila enzymes is much more extensive. Comparing the sequences between Saccharo- myces and Drosophila enzymes, the overall homology is approximately 46%, which is comparable to the extent of homology (approximately 49%) between the enzymes from budding and fission yeasts (34). The extensive sequence homology observed for all the DNA topoisomerases II ranging from bacteriophage to eucaryotes suggests that these enzymes are fairly well conserved during the course of evolution. The similarity in the physical and enzymatic properties for all the eucaryotic DNA topoisomerases 11 also strongly supports this notion. The Drosophila topoisomerase 11, similar to the mammalian enzymes, is sensitive to inhibition by a variety of antineo- plastic agents. For instance, Drosophila enzyme is very sensitive to the epipodophyllotoxin drug, VM26 (36,37), while other drugs like adriamycin, ellipticine, 4’~(9-acri- dinylamino) methanesulfon-m-anisidide and actinomycin D are less potent in promoting the topoisomerase-mediated DNA cleavage reaction (M. Sander and T. Hsieh, unpub- lished results). CONCLUDING REMARKS In an effort to establish a genetic system to thoroughly analyze the functions of eucaryotic topoisomerase II, we have isolated and characterized the DNA sequences encod- ing the Drosophila enzyme. The location of this gene in the Drosophila chromosome has been mapped both at the cytogenic and molecular genetic level. We are currently in the process of testing the functions of this gene in the growth and development of Drosophila cells. The similarity in the biochemical properties among the eucaryotic enzymes and their extensive sequence homology suggest that the Drosophila system might be a useful one in establishing the functions of this enzyme in eucaryotic cells. It might also provide a system in analyzing the mechanism of action of some antitumor drugs and in studying the subsequent cellular events in repairing the damages incurred by these drugs. REFERENCES (I) GELLERT M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981. (2) WANG JC: DNA topoisomerases. Annu Rev Biochem 54: 665-695, 1985. (3) CozzARELLI NR: DNA topoisomerases. Cell 22:327-328, 1980. (4) Liu LF: DNA topoisomerases—Enzymes that catalyze the breaking and rejoining of DNA. Crit Rev Biochem 15:1-24, 1983. (5) WANG JC: Interaction between DNA and an Escherichia coli protein w. J Mol Biol 55:523-533, 1971. (6) GELLERT M, MizuucHI K, O'DEA MH, et al: DNA gyrase: An enzyme that introduces superhelical turns into DNA. Proc Natl Acad Sci USA 73:3872-3876, 1976. (7) DINARDO S, VOELKEL K, STERNGLANZ R: DNA topo- isomerase II mutant of Saccharomyces cerevisiae: Topo- isomerase II is required for segregation of daughter mole- cules at the termination of DNA replication. Proc Natl Acad Sci USA 81:2616-2620, 1984. (8) Goto T, WANG JC: Yeast DNA topoisomerase II is encoded by a single-copy, essential gene. Cell 36:1073-1080, 1985. (9) THRASH C, VOELKEL K, DINARDO S, et al: Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisom- erase I activity. J Biol Chem 259:1375-1377, 1984. (10) Goto T, WANG JC: Cloning of yeast top 1, the gene encoding DNA topoisomerase I, and construction of mutants defective in both DNA topoisomerase I and DNA topoisomerase II. Proc Natl Acad Sci USA 82:7178-7182, 1985. (11) UEMURA T, YANAGIDA M: Isolation of type I and II DNA topoisomerase mutants from fission yeast: Single and double mutants show different phenotype in cell growth and chromatin organization. EMBO J 3:1737-1744, 1984. (12) HoLM C, Goto T, WANG JC, et al: DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553-563, 1985. (13) UEMURA T, YANAGIDA M: Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J 5:1003-1010, 1986. (14) BERRIOS M, OSHEROFF N, FISHER PA: In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci USA 82:4142-4146, 1985. (15) EARNSHAW WC, HALLIGAN B, COOKE CA, et al: Topoisom- erase II is a structural component of mitotic chromosome scaffolds. J Cell Biol 100:1706-1715, 1985. (16) GASSER SM, LAROCHE T, FALQUET J, et al: Metaphase chromosome structure: Involvement of topoisomerase II. J Mol Biol 188:613-629, 1986. (17) CocKERILL PN, GARRARD WT: Chromosomal loop an- chorage of kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44:273-282, 1986. (18) GASSER SM, LAEMMLI UK: The organization of chro- matin loops: Characterization of a scaffold attachment site. EMBO J 5:511-518, 1986. (19) LUCHNIK AN, BAKAYEV VV, ZBARSKY IB, et al: Elastic torsional strain in DNA within a fraction of SV40 minichromosomes: Relation to transcriptionally active chromatin. EMBO J 1:1353-1358, 1982. 10 (20) Ryos1 M, WORCEL A: Chromatin assembly in Xenopus oocytes: In vivo studies. Cell 37:21-32, 1984. (21) WEINTRAUB H: Assembly and propagation of repressed and derepressed chromosomal states. Cell 42:705-711, 1985. (22) SINDEN RR, CARLSON JO, PETTUOHN DE: Torsional tension in the double helix measured with trimethyl- psoralen in living E. coli cells: Analogous measurements in insect and human cells. Cell 21:713-783, 1980. (23) NoLAN JM, LEE MP, WYCKOFF E, et al: Isolation and characterization of the genes encoding Drosophila DNA topoisomerase II. Proc Natl Acad Sci USA 83:3664-3668, 1986. (24) SANDER M, HSIEH T: Double strand DNA cleavage by type II DNA topoisomerase from Drosophila melano- gaster. J Biol Chem 258:8421-8428, 1983. (25) YouNG RA, Davis RW: Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 80: 1194-1198, 1983. (26) BRITTNACHER JG, GANETZKY B: On the components of segregation distortion in Drosophila melanogaster. 11. Deletion mapping and dosage analysis of the Sd locus. Genetics 103:659-673, 1983. (27) SANDLER L, HIRAIZUMI Y, SANDLER I: Meiotic drive in nat- ural populations of Drosophila melanogaster. 1. The cytogenic basis of segregation-distortion. Genetics 44: 223-250, 1959. (28) SHELTON ER, OSHEROFF N, BRUTLAG D: DNA topoisom- erase II from Drosophila melanogaster: Purification and physical characterization. J Biol Chem 258:9530-9535, 1983. (29) HSIEH T: Purification and properties of type II DNA topo- isomerase from embryos of Drosophila melanogaster. Methods Enzymol 100:161-170, 1983. (30) MORIGA S, OGASAWARA N, YOSHIKAWA H: Structure and function of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res 13:2251-2265, 1985. (31) HUANG W-M: Nucleotide sequence of a type II DNA topoisomerase II gene. Bacteriophage T4 gene 39. Nucleic Acids Res 14:7751-7766, 1986. (32) HUANG W-M: The 52-protein subunit of T4 DNA topo- isomerase is homologous to the gyrA-protein of gyrase. Nucleic Acids Res 14:7379-7390, 1986. (33) GIAVER G, LYNN R, Goro T, et al: The complete nucleotide sequence of the structural gene TOP2 of yeast DNA topoisomerase II. J Biol Chem 261:12448-12454, 1986. (34) UEMURA T, MORIKAWA K, YANAGIDA M: Nucleotide sequence determination of fission yeast DNA topoisom- erase II gene: Structural and functional relations to other DNA topoisomerases. EMBO J 5:2355-2361, 1986. (35) LYNN R, GIAEVER G, SWANBERG SL, et al: Tandem regions of yeast DNA topoisomerase II share homology with different subunits of bacterial gyrase. Science 233: 647-649, 1986. (36) Rowe TC, WANG JC, Liu LF: In vivo localization of DNA topoisomerase II cleavage sites on Drosophila heat shock chromatin. Mol Cell Biol 6:985-992, 1986. (37) UDVARDY A, SCHEDL P, SANDER M, et al: Topoisomerase II cleavage in chromatin. J Mol Biol 191:231-246, 1986. DNA Topoisomerase Activity Is Required as a Swivel for DNA Replication and for Ribosomal RNA Transcription! Steven J. Brill,? Stephen DiNardo, ?? Karen Voelkel-Meiman,? and Rolf Sternglanz* * ABSTRACT —Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously. The topoisomerase II mutants (Zop2) are conditional-lethal, temperature-sensitive mutants defective in the termination of DNA replication and the segregation of daughter chromosomes. The topoisomerase I mutants (top), including strains with null mutations, are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top! top2 double mutants grow poorly at the permissive temperature and stop DNA and ribosomal RNA synthesis at the restrictive temperature. Transfer RNA synthesis remains relatively normal. The rate of polyA” RNA synthesis is down about 3-fold in the double mutant at the non-permissive temperature but the synthe- sis of three specific RNA polymerase II transcripts is unaffected. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topo- isomerase can perform the required function (except for termina- tion of DNA replication where topoisomerase II is required).— NCI Monogr 4:11-15, 1987. Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both S. cerevisiae (1-3) and S. pombe (4). The topoisom- erase II mutants (top2) are conditional-lethal, tempera- ture-sensitive (Ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes (2-5), but otherwise appear to replicate and transcribe DNA normally. Topoisomerase 1 mutants (top!) including strains with null mutations are viable and exhibit no obvious growth defects, demon- strating that DNA topoisomerase I is not essential for viability in yeast (/,4,6,7). In contrast to the single mutants, topl top2 Ts double mutants from both S. pombe and S. cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature (4,7). Here we describe further properties of S. cerevisiae topl top2 Ts mutants including significant defects in cell growth and RNA synthesis. ISupported by Public Health Service grant GM-28220 from the National Institute of General Medical Sciences, National Institutes of Health, Department of Health and Human Services. 2 Department of Biochemistry, State University of New York, Stony Brook. 3 Present address: Department of Biochemistry and Biophysics, Univer- sity of California Medical Center, San Francisco. 4 We thank P. J. G. Butler for an important suggestion and Allan Miller for help with the experiment on MA Tal and SIR3 mRNAs. * Reprint requests: Rolf Sternglanz, Ph.D., Department of Biochem- istry, State University of New York, Stony Brook, NY 11794. MATERIALS AND METHODS Strains and growth media.—Yeast strains used in this study are members of a single tetrad with the following genotypes: SD116 Mata ade? ura3 his3 his7 trpl canl topl-1; SD117 MATa ade2 ura3 his3 trpl leu2 canl top2-1; SD118 MATa ade2 ura3 his3 trpl leu2 canl; and SD119 MATa ade2 ura3 his3 his7 trpl topl-1 top2-1. Cells were grown in YPD or synthetic complete medium with 2% lactate (8) or Y minimal medium supplemented with glucose, yeast extract, and amino acids as described (9). RNA synthesis.—Cells were grown at 25°C in Y medium to a density of 2X 107 cells/ml. The culture was then split into two parts, one at 25°C and the other at 37°C. At various times 1 ml of each culture was removed and incubated with 5 uCi [5,6]-*H-uracil for 5 min at the respective temperature. The reaction was stopped by the addition of 2 ml 15% TCA solution. The sample was pelleted, the pellet resuspended in 2 ml fresh 15% TCA and kept on ice for 30 min. The sample was then filtered and radioactivity determined. Preparation of poly(A)" and poly(A)" RNA.—Cells were grown and labeled as above except that 3-ml aliquots were labeled with 5 pCi/ml [5,6]-*H-uracil for 10 min. Total RNA was isolated and resuspended in 0.5 ml of 10 mM Tris, 1 mM EDTA. One-tenth of the RNA was taken for TCA precipitation and determination of the rate of total RNA synthesis. The remainder of the RNA was applied to an oligo (dT)-cellulose (Collaborative Research, type 3) column according to manufacturer’s instructions. Frac- tions were precipitated with 3 volumes of cold 15% TCA solution and kept on ice for 30 min. Samples were filtered and radioactivity determined as above. Galactokinase assays.—Cells were grown at 25°C in synthetic complete medium with 2% lactate to mid-log phase, and then split into two parts, one at 25°C and the other at 37°C. After 15 min, cells were induced by the addition of galactose (0.5% final concentration). At various times after induction, aliquots (5 X 10° cells) were removed and assayed for galactokinase activity essentially as de- scribed (10,11). RESULTS A topl top2 double mutant was constructed by crossing a topl mutant which totally lacks topoisomerase I activity with a top2 temperature-sensitive mutant. The resulting double mutant is temperature-sensitive for viability and grows noticeably more slowly than either single mutant at the permissive temperature, 25°C (Table 1). In fact, the topl top2 double mutant grows so poorly on agar that this 11 TABLE 1.—Doubling Times of Topoisomerase Mutants Strain Relevant genotype Doubling time (min) SD118 T0P* 132 SDI117 top2-1 138 SDI116 topl-1 169 SDI119 topl-1 top2-1 200 4 The doubling time was determined in liquid YPD medium at 25°C. Each value is the mean of at least 4 experiments. property can be used to select plasmid clones carrying the wild-type TOPI or TOP? gene simply by selecting for more rapidly growing colonies after transformation with a gene library [(6), unpublished observations]. These transformants have properties of the appropriate single mutant. The results reported below were obtained with double mutant strain SD119 carrying the topI-1 allele, a mutation originally called makI-1 and isolated after chemical muta- genesis (12). Strains with this mutation have no detectable topoisomerase I activity (/). Double mutants with the top 1- 6, topl-7 and topl-8 null mutations (6) were also con- structed. Their growth properties and defects in RNA synthesis (see below) were indistinguishable from those of strain SD119. The ability of the top] top2 double mutant to traverse the cell cycle at the non-permissive temperature, 37°C, was compared with that of the single mutants. When exponen- tially growing double mutant cells are shifted to 37°C no single terminal morphology is observed, a result also seen by others (4,7). A different result is obtained when G, cells are used as the starting population. In such an experiment, cells are grown to stationary phase, Go, on agar plates at 25°C (13), resuspended in liquid medium at 37°C, and examined in the microscope as a function of time. Initially all the cells appear as single unbudded cells. Normal growth leads to enlargement of the single cells during G1, followed by the appearance of small buds at about the start of S phase (DNA synthesis). These buds grow progressively during S phase and G2 until cytokinesis leads to two cells, a mother cell and a daughter cell derived from the bud. Figure 1 summarizes the results for this experiment; it shows the proportion of small and large single cells, small and large buds, and aberrant multiply-budded forms for the various strains. It is clear that the wild-type strain and the top! mutant can progress through the cell cycle normally, although the appearance of buds in the topl strain is delayed. The top2 mutant shows the terminal phenotype described previously, the accumulation of large budded cells arrested at nuclear division, and large un- Top * OF TOTAL % top N A RRS top! AMNION AER ER ER NY N top! top2 TIME (HR) FIGURE 1.—Cell morphology of topoisomerase mutants at 37°C upon outgrowth from Gy. Cells were grown to confluence on YPD plates at 25°C, suspended in YPD broth, and diluted into 37°C YPD broth at zero time. At the indicated times, aliquots were removed and fixed with an equal volume of 3.7% formaldehyde, 150 mM NaCl. Cells were observed by phase contrast light microscopy and scored for small single cells (black bars), large single cells (hatched bars), cells with small buds (open bars), cells with large buds (stippled bars), and cells with aberrant shapes (striped bars). Buds whose diameter was greater than one-half the mother’s diameter were considered large. Aberrant forms were typically cells with multiple buds. Each time point represents the scoring of approximately 150 cells. 12 NCI MONOGRAPHS, NUMBER 4, 1987 budded cells (2,3). The latter arise from the former as a result of an aberrant mitosis. They are not viable cells (3) and are therefore probably aneuploid. In contrast to the other strains, the topl top2 double mutant cells show a striking phenotype; they grow in size but do not bud, apparently arresting in G1 with this protocol (Figure 1). To understand the inability of the double mutant to traverse the cell cycle, we investigated the effect of eliminating topoisomerase activity on DNA replication and RNA transcription. The results described below show that both processes are affected in a topl top2 mutant. We had shown previously that the ztop2 Ts mutant is capable of undergoing one complete round of DNA replication at the non-permissive temperature (2). A top! mutant grows normally, having no obvious defect in DNA replication (6,7). In contrast, DNA replication is severely affected in the topl top2 double mutant. As we have published elsewhere (/4), double mutant cells synchronized in the G1 phase of the cell cycle with the yeast pheromone, a-factor, and then released from the a-factor block at the non-permissive temperature, 37°C, do not synthesize DNA. This does not by itself indicate a specific block in DNA replication, since the double mutant arrests in G1 with this protocol (just as it did in the experiment described in Figure 1). However, a similar result is found when cells are shifted to 37°C just at the beginning of S phase. Moreover, cultures shifted to 37°C at any time during S phase cease DNA synthesis rapidly (/4). The results suggest that topoisomerase activity is required continuously for ongoing DNA replication and that either topoisomerase can perform the necessary function. Only when the activity of both enzymes is abolished, in the double mutant at 37°C, does DNA synthesis stop. A defect in DNA replication cannot be the only pheno- type of the topl top2 mutant since exponentially growing cells arrest heterogeneously rather than with a single termi- nal morphology. We therefore considered the possibility that the double mutant was also defective in RNA synthesis. The rate of RNA synthesis was compared for a wild-type strain, the topoisomerase single mutants and the double mutant. Figure 2 shows that all four strains exhibit a rapid decrease in the rate of RNA synthesis after a shift to 37°C. This inhibition is transient and due to heat shock. The wild-type strain and the top! mutant recover from heat shock in about one hour and resume normal RNA synthe- sis. The top2 Ts mutant also recovers from heat shock, but after two hours at 37°C it shows another reduction in the rate of RNA synthesis. This occurs at the time cells begin to die as they attempt to segregate their intertwined chromo- somes at mitosis (2,3). The topl top2 double mutant, on the other hand, never resumes a normal rate of RNA synthesis, leveling off at 10% of the initial rate (Figure 2). As an initial attempt to characterize which transcription units were most affected in the double mutant, RNA pulse- labeled with *H-uracil was fractionated into polyA™ and polyA* pools on an oligo(dT)-cellulose column. The polyA* pool represents mostly RNA polymerase II tran- scripts while the polyA™ pool contains RNA polymerase I transcripts (25S and 18S ribosomal RNA) and RNA poly- merase III transcripts (tRNA and 5S RNA). Figure 3 com- pares the rate of RNA synthesis for these two types of RNAs. It can be seen that the rate of polyA™ RNA syn- thesis is more severely affected than is polyA* RNA syn- DNA TOPOISOMERASES IN CANCER THERAPY RATE OF RNA SYNTHESIS I 30 60 90 Lr) 1 1 L 0 30 60 90 120 150 180 O 120 150 180 TIME (MIN) FIGURE 2.—Rates of RNA synthesis in topoisomerase mutants. Cultures growing at 25°C were split in half at zero time, one-half kept at 25°C and the other shifted to 37°C. At intervals aliquots were pulse-labeled with [5,6]-3H-uracil at the respective temperature for 5 min. The rate of RNA synthesis is the total TCA precipitable radioactivity incorporated in the 5-min pulse. Rates are normalized relative to the 25°C zero time value for each strain. thesis. After 100 min at 37°C, the rate of polyA™ RNA synthesis is 15% of the initial rate while the rate of polyA™ RNA synthesis is about 30%. These results suggested that ribosomal and/or tRNA synthesis might be most affected in the double mutant. The synthesis of these transcripts was specifically analyzed by pulse-labeling RNA with *H-uracil and subjecting the RNA to gel electrophoresis and visualizing it by fluorography. As published elsewhere (14), a drastic inhibition of ribosomal RNA synthesis is observed, particularly of the 25S and 18S rRNAs, whose rate of synthesis drops to 10% of the initial rate. On the other hand, tRNA synthesis is virtually unaf- fected in the double mutant. Although ribosomal RNA synthesis is most affected in the topoisomerase double mutant, the synthesis of polyA* RNA also is inhibited to some extent (Figure 3B). We there- tore examined the effect on a specific RNA polymerase II transcription unit, the tightly regulated GALI gene, which codes for galactokinase. There is very little transcription of this gene in the absence of galactose and high expression after galactose induction. Wild-type and top! top2 mutant cells were grown at 25°C in a non-inducing carbon source, shifted to 37°C and induced with galactose 15 min after the temperature shift. Control cultures were induced at 25°C. 13 TOP* top! top2 N oO = w x — z > ~ wn < Zz x 02— At 37° = 01 A370 = 0.05 — 0.02 1 1 1 1 1 1 1 1 1 1 0 30 60 90 120 150 0 30 60 90 120 150 TIME (MIN) FIGURE 3.—Rates of poly(A)" and poly(A)~ RNA synthesis. Cultures growing at 25°C were split in half at zero time, one-half kept at 25°C and the other shifted to 37°C. At intervals aliquots were pulse-labeled for 10 min at the respective temperature by incubation with [5,6]-H-uracil. Total labeled RNA was isolated and fractionated into poly(A)" and poly(A)~ fractions on an oligo (dT)-cellulose column. The rate of RNA synthesis is the TCA precipitable radioactivity in each fraction. Rates are normal- ized relative to the 25°C zero time value. At various times after induction, samples were removed and assayed for galactokinase, as a measure of GALI expression. This protocol allows us to ask if the normal kinetics of induction are observed after both topoisomer- ases have been inactivated. Table 2 shows galactokinase enzymatic activity in cells 60 min after induction. The wild- type strain has about 2.5 times as much galactokinase activ- ity as does the double mutant, but this difference is seen at both 25°C and 37°C. Kinetic studies show that there is no hint of slower induction of GALI at 37°C in the double mutant (data not shown), suggesting that even after the inactivation of both topoisomerases efficient synthesis of GALI mRNA can take place. The amount of two other mRNAs, the products of the MATal gene and the SIR3 gene, was also measured in the topl top2 double mutant 60 min after shifting a culture from 25°C to 37°C. Both of these mRNAs are synthesized TABLE 2.—Galactokinase Activity” Strain Relevant genotype 25°C 37°C 25°C/37°C SD118 Top* 227 20.9 L1 SD119 topl top2 9.0 7.9 1.1 4 Cultures were induced for 60 min at 25°C or 37°C and then assayed for galactokinase activity as described in Materials and Methods. The values shown are the nmoles of galactose-1-phosphate produced in a 45-min assay. 14 constitutively at a relatively low level. The primary trans- cript from MATal is approximately 0.5 kb and contains two small introns. The STR3 message is about 3.1 kb. The steady-state amounts of both mRNAs as measured by nuclease S1 protection were the same in the double mutant compared to wild-type 60 min after a shift from 25°C to 37°C (data not shown). Since the MA Tal mRNA has a half-life of about 3 min (15), the experiment demonstrates that this transcript can be synthesized efficiently in the absence of topoisomerase activity. Although the half-life of the S/R3 message is unknown, if it is typical of most yeast mRNA, it is likely to be much less than 60 min (16), and therefore the same conclusion can be drawn for this RNA polymerase II transcription unit. DISCUSSION In this report, and elsewhere (1/4), we have presented evidence that DNA topoisomerases are required for DNA replication and ribosomal RNA transcription in yeast. Surprisingly, topoisomerase I and topoisomerase II can substitute for each other in these processes. Saavedra and Huberman have shown that either enzyme can also substi- tute for the other in the in vivo relaxation of torsionally strained 2-micron plasmid DNA (17). The only essential function known in which one topoisomerase cannot substi- tute for the other is in the segregation of sister chromatids at mitosis. This is probably due to the fact that only topo- isomerase II is able to decatenate intact DNA molecules. NCI MONOGRAPHS, NUMBER 4, 1987 On the other hand, both enzymes can efficiently relax nega- tively and positively supercoiled DNA. We view their abil- ity to substitute for one another as a reflection of this sim- ilarity. Accordingly, either enzyme seems to be capable of carrying out a role as a swivel, using different strand pass- ing mechanisms to relax the torsional strain that arises dur- ing replication, and, perhaps surprisingly, during transcrip- tion at the rDNA loci. Our data suggest an extreme effect on ribosomal RNA synthesis, a moderate effect on RNA polymerase II (mRNA) transcription and virtually no effect on tRNA synthesis. Is topoisomerase activity required for initiation of transcription (i.e., transcriptional activation) or for the elongation phase? The striking effect on rRNA expression suggests that elongation is affected. There are about 120 copies of the ribosomal RNA genes arranged as a tandem array on chromosome XII. Within each copy there is diver- gent transcription of the 5S RNA (by RNA polymerase III) and the 35S RNA precursor of the 25S and 18S ribosomal RNAs (by RNA polymerase I). This arrangement leads to convergent transcription between adjacent members of the array. The details of nucleolar architecture and the mecha- nism of initiation and termination of ribosomal transcrip- tion are still unclear in yeast. Nevertheless, it is clear that the DNA unwinding events required during transcription of the long tandem array of highly active genes would give rise to substantial torsional strain, which might make rRNA synthesis uniquely sensitive to topoisomerase deple- tion. The alternative possibility, that there is a specific association of a topoisomerase with polymerase I, or with its initiation factors, seems unlikely since either topoisom- erase can act in rRNA synthesis. Nevertheless, this possibil- ity can be readily tested in yeast through the introduction of a single copy of a genetically marked rDNA gene (18) into another chromosomal location. We find that expression of the GALI, SIR3, and MATal loci does not seem to require topoisomerase action, whereas the decrease in the rate of polyA" RNA synthesis indicates that some polymerase II units may be affected. It is possible that the reduction that we see is due to a strong effect on a small number of genes that code for relatively abundant mRNA species where topological unwinding problems might be unusually severe. In fact, recent experiments have shown that the expression of several ribosomal protein genes is drastically reduced in the double mutant (J. Warner, personal communication). However, since rRNA expression stops immediately in the absence of topoisom- erase activity, ribosomal protein gene transcription may be affected only secondarily, as a result of tight coordinate control over the synthesis of various ribosome components. It is also possible that the polyA" RNA which we have purified is somewhat contaminated with rRNA and that this leads to an overestimate of the effect on polyA" RNA synthesis. Very recently it has been reported that a fission yeast topl top2 Ts mutant is also defective in DNA and RNA synthesis (5). Thus, the biological roles of topoisomerases in two very different yeast species appear to be similar. It is not clear whether the situation in yeast can be generalized to higher eukaryotes. While the roles that have been uncov- DNA TOPOISOMERASES IN CANCER THERAPY ered for the topoisomerases in yeast should be conserved, it is possible that additional or more specialized roles have been added during the evolution of multicellular organisms. REFERENCES (I) THRASH C, VOELKEL K, DINARDO S, et al: Identification of Saccharomyces cerevisiae mutants deficient in DNA topoisomerase I activity. J Biol Chem 259:1375-1377, 1984. (2) DINARDO S, VOELKEL K, STERNGLANZ R: DNA topoisom- erase II mutant of Saccharomyces cerevisiae: Topoisom- erase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 81:2616-2620, 1984. (3) HoLM C, GoTo T, WANG JC, et al: DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553-563, 1985. (4) UEMURA T, YANAGIDA M: Isolation of type I and II DNA topoisomerase mutants from fission yeast: Single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J 3:1737-1744, 1984. (5) UEMURA T, YANAGIDA M: Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J 5:1003-1010, 1986. (6) THRASH C, BANKIER AT, BARRELL BG, et al: Cloning, char- acterization, and sequence of the yeast DNA topoisom- erase I gene. Proc Natl Acad Sci USA 82:4374-4378, 1985. (7) Goto T, WANG JC: Cloning of yeast TOPI, the gene encod- ing topoisomerase I, and the construction of mutants de- fective in both DNA topoisomerase I and DNA topoisom- erase II. Proc Natl Acad Sci USA 82:7178-7182, 1985. (8) SHERMAN F, FINK GR, LAWRENCE C: Methods in Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1979. (9) ZAKIAN VA, BREWER BJ, FANGMAN WL: Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell 17:923-934, 1979. (10) ADAMS BG: Induction of galactokinase in Saccharomyces cerevisiae: Kinetics of induction and glucose effects. J Bac- teriol 111:308-315, 1972. (11) JouNsTON LH: Rapid detection of allelic recombination at the gall locus in yeast by assay of the recombinant gene product. Genet Res 39:85-97, 1982. (12) WICKNER RB, LEIBOWITZ MJ: Chromosomal genes essential for replication of a double-stranded RNA plasmid of Sac- charomyces cerevisiae: The killer character of yeast. J Mol Biol 105:427-443, 1976. (13) NASMYTH K: Molecular analysis of a cell lineage. Nature 302:670-676, 1983. (14) BRILL SJ, DINARDO S, VOELKEL-MEIMAN K, et al: Need for DNA topoisomerase activity as a swivel for DNA replica- tion and for transcription of ribosomal RNA. Nature. In press. (15) MILLER AM: The yeast MATal gene contains two introns. EMBO J 3:1061-1065, 1984. (16) CHIA L-L, MCLAUGHLIN C: The half-life of mRNA in Sac- charomyces cerevisiae. MGG 170:137-144, 1979. (17) SAAVEDRA RA, HUBERMAN JA: Both DNA topoisomerases I and II relax 2 um plasmid DNA in living yeast cells. Cell 45:65-70, 1986. (18) ELION EA, WARNER JR: The major promoter element of rRNA transcription in yeast lies 2 kb upstream. Cell 39: 663-673, 1984. 15 & ue J . le el pit ae leh he loi i a i. a4 Sra rl Sa EL LE 7 ar FIL, 3 lt apd oH od yd HE “hse FR 5 Lat vais o # El A? Bh = Fame 2 J oll wl Lg Asin } et TN (1 = at ts By aE J . ell Te rts qe : mE, a INA 2 ie Sak fA Ee 0 fe fi uy par wn Sul RT } ar “Lip Ea di Lanai kul & Association of Topoisomerase | With Transcriptionally Active Loci in Drosophila David S. Gilmour* and Sarah C. R. Elgin’ ABSTRACT —Immunofluorescence staining of the polytene chromosomes of Drosophila shows high levels of topoisomerase I associated with transcriptionally active regions. A photocrosslink- ing technique demonstrates the presence of topoisomerase I in the region of transcription of the active heat-shock genes. Campto- thecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, topoisomerase I is found to interact with the transcriptionally active genes hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes prior to heat shock. The interaction occurs predominantly within the transcribed region, with specific sites observed on both the transcribed and nontranscribed strands of the DNA. Little inter- action is seen with nontranscribed flanking sequences. Campto- thecin only partially inhibits transcription of the hsp28 gene dur- ing heat shock, causing a reduced level of transcripts which are nonetheless full length. The results point to a dynamic set of inter- actions at the active locus.—NCI Monogr 4:17-21, 1987. Numerous studies have shown that changes in chromatin structure accompany gene activation and transcription. In general, an “unfolding” of the chromatin is thought to occur, observed by greater sensitivity to nucleases and other probes of chromatin structure (/-5). Changes in DNA topology could be an important parameter in this process as well as in transcription. We have studied the distribution of topoisomerase I, a protein which facilitates such topo- logical changes, in the chromatin of Drosophila. Topo- isomerase I is recognized as a major chromosomal protein; it relaxes both positively and negatively supercoiled DNA by introducing transient single-stranded nicks in the DNA [for reviews, see (6-8)]. A variety of experimental approaches indicate that topoisomerase I interacts intimately with transcribed regions of the genome. Topoisomerase I has been found to be enriched in fractions of nucleosomes derived from tran- scribed regions and nucleoli (9,10). Specific sites of topo- isomerase I binding have been detected near the 5” and 3’ ends of the Tetrahymena rDNA transcription unit (/1,12). The distribution of topoisomerase I on Drosophila poly- tene chromosomes can be observed by immunofluorescence staining with affinity-purified antibodies (13,/4). The enzyme is localized to discrete sites, including the visible puffs (sites of intense RNA synthesis). Figure 1 shows that topoisomerase I is present at the heat-shock loci following transcriptional activation of these genes; it is not detected I Department of Biology, Washington University, St. Louis, MO. * Reprint requests: Dr. David S. Gilmour, Department of Biology, Washington University, St. Louis, MO 63130. at these loci prior to heat shock. Intense staining of the nucleoli has also been observed, suggesting an interaction between topoisomerase I and the RNA polymerase I-tran- scribed rRNA genes. Comparison of the distribution pat- terns of topoisomerase I and RNA polymerase II reveals considerable similarity, but not coincidence (13). The immunofluorescence staining method provides an overview of the chromosomal distribution of a protein but is limited in resolution to 30-100 kb regions of DNA. More precise mapping is provided by an in vivo protein-DNA crosslinking method (7/5). Protein-DNA adducts can be generated by irradiating intact cells with UV light. Follow- ing removal of proteins not covalently bound to the DNA, specific proteins are immunoprecipitated; characterization of the coprecipitating DNA (using recombinant DNA probes) reveals the in vivo distribution of the protein on the DNA. Like RNA polymerase II, topoisomerase I can be shown to be recruited to heat-shock genes during heat shock. Topoisomerase I is crosslinked to transcribed regions of the Asp26 heat-shock gene but not to nontran- scribed flanking sequences. Topoisomerase I apparently can interact independently of RNA polymerase II; different ratios of topoisomerase I and RNA polymerase II are cross- linked to the highly transcribed Asp70 heat-shock gene and the moderately transcribed copia genes. The sites of topoisomerase I cleavage in the DNA can be mapped in vivo using a specific inhibitor of topoisomerase I, camptothecin. Camptothecin inhibits topoisomerase I activity by stabilizing the covalent protein-DNA interme- diate (16). When the reaction complex is treated with SDS, topoisomerase I is recovered covalently coupled to the DNA. The nick produced by topoisomerase I remains when the protein moiety is removed with proteinase K; the posi- tion of the nick can be mapped using an indirect end-label- ling technique (17,18). We have examined the camptothecin-induced cleavage events in the region containing the small heat-shock genes hsp26, hsp23, and hsp28, using Drosophila cultured cells (19). These genes from the locus 67B have been previously cloned and mapped (20-22); they are activated by a shift of the culture temperature from 25°C to 36°C. Figure 2 shows that the region of chromatin containing these three genes is extensively cut in heat-shocked, camptothecin-treated cells. In contrast, this region is not extensively cut in noninduced cells nor in cells that have been induced but not treated with camptothecin. The very weak cuts that are detected in non- induced cells occur at positions similar to those in heat- shocked cells, suggesting that they may originate from cells that have been inadvertently stressed. Both single- and double-stranded cuts are observed pre- dominantly in the transcribed regions of the heat-shock 17 18 63BC G4EF 67B 68C ’ 85F 87A 87C T4EF 798 95D NCI MONOGRAPHS, NUMBER 4, 1987 i Alkaline Gel Single Stranded Cuts FIGURE 2.—Camptothecin induces both single- and double-stranded cuts in the vicinity of the small heat-shock genes in induced but not in noninduced cells. The region containing the small heat-shock genes (hsp26, hsp23, and hsp28) was analyzed in genomic DNA from Schneider 2 cultured cells maintained under control (25°C) or heat- shock conditions (36°C), treated with 0, 20, or 100 uM camptothecin (dissolved in DMSO) for 20 minutes. Each DNA sample was cut with BamHI and a portion was run on either a 1% neutral or a 1% alkaline agarose gel. Following transfer of the DNA to nitrocellulose, the region containing the small heat-shock genes was detected with the nick- translated, BamHI1/ Hind 111 fragment isolated from clone 88.1 (20). BamHI digestion produces a major band of 12 kb corresponding to the intact genomic DNA fragment. Camptothecin induces extensive double- and single-stranded cuts which result in a complex pattern of lower molecular weight fragments. These cuts are localized to the heat-shock gene transcription units depicted here by the black arrows; gene 1 is a developmentally regulated gene that is not expressed in these cells. The origin of the band below the parental band and present in all lanes is unknown. The ethidium bromide stained gel showed that less DNA was loaded in the HS, 20 uM camptothecin lane than in other lanes. Repro- duced with permission from (19). genes. However, the intensities of the high-molecular- weight bands on the alkaline gel appear weaker than those on the neutral gel, suggesting that there are more single- stranded than double-stranded breaks. Single- and double- stranded cuts are both observed over a wide range of drug treatments, the lowest tested being 10 nM camptothecin for a five-minute period (data not shown). The positions of the camptothecin-induced cuts on the individual strands of DNA (separated on alkaline gels) have been assessed using strand-specific probes prepared from inserts in M13 phage DNA. Figures 3A and 3B (lanes 4) show that several camptothecin-induced cuts occur within Asp28 and hsp23 in heat-shocked cells; each strand displays its own unique pattern of cuts. Some cleavage sites, most notably the strong cut near the 3’ end of Asp28, occur in both strands. These coincident cleavage events no drug 10uM no drug 10uM ime | Me a si | A 1 2 34586 1 2 34586 -- hsp28 Nontranscribed A B Transcribed FIGURE 3.—Camptothecin-induced cuts near the Asp28 and hsp23 genes. Heat-shocked cells were treated with either 10 pM camptothecin or an equivalent volume of DMSO for 3 minutes at 36°C, and then collected by a 5-minute spin in a clinical centrifuge. Cells were lysed in 1% SDS and a portion of the lysate was treated immediately with proteinase K, phenol/ chloroform extracted, and ethanol precipitated. The remaining DNA was not treated with protease but extracted several times with chloroform followed by ethanol precipitation. The DNA was cut with EcoRI in the presence of 0.5 mM phenylmethylsulfonyl fluoride and then ethanol precipitated. Precipitates were dissolved in 0.5% SDS/50 mM Tris chloride pH 8.0/10 mM EDTA and half was treated with proteinase K. Both samples were again ethanol precipitated and then run on an alkaline gel, along with the sample treated immediately with proteinase K. Cuts on specific strands were detected with strand-specific probes homologous to a region downstream of hsp28. Panel A shows the results for the nontranscribed strand. Samples in lanes 1, 2, and 3 are from cells not treated with camptothecin and those in lanes 4, 5, and 6 are from cells treated with camptothecin. Lanes 1 and 4 are samples that were treated with proteinase K prior to the organic extraction. Lanes 2 and 5 are samples that were never treated with proteinase K. Lanes 3 and 6 are samples that were treated with proteinase K after restriction digestion, just prior to electrophoresis. This probe detects cleavage fragments whose 5’ ends were generated with camptothecin. Panel B is the same blot as in panel A hybridized with a probe specific for the transcribed strand. This probe detects cleavage fragments whose 3’ ends were generated with camptothecin. The results show that cutting is due to an enzyme that binds to the 3’ end of the cleavage sites (i.e., to topoisomerase I), and that interaction with topoisomerase I occurs on both strands, primarily in the region of transcription. Reproduced with permission from (19). probably account for the double-stranded cutting observed using neutral gels. The double-stranded cuts induced by camptothecin are more suggestive of a topoisomerase II activity than a topoi- somerase I activity. Therefore, the products of the campto- thecin-induced cuts have been characterized in more detail using a procedure which preserves the protein-DNA inter- mediate (/1). Topoisomerase I forms a covalent linkage with the 3’ end of the nicked DNA, while topoisomerase II forms a covalent linkage with the 5’ end of the cleaved DNA (8). To determine to which end of the DNA the — FIGURE 1.—Distribution pattern of topoisomerase I on polytene chromosome arms 3L (a—c) and 3R (df). Topoisomerase I was visualized by the method of indirect immunofluorescence. Chromosomes were obtained from third instar larvae of Drosophila melanogaster grown at 25°C (a and d) and from larvae heat shocked at 37° C for 20 min prior to dissection (b, c, €, and f). (a, b, d, and e) fluorescent images; (c and f) phase contrast images of b and e. Reproduced with permission from (13). DNA TOPOISOMERASES IN CANCER THERAPY 19 camptothecin-induced protein complex is attached, the mobilities of specific single-stranded DNA fragments before and after protease treatment were determined on DNA denaturing gels. The presence of protein will slow the mobility of a fragment while protease treatment should return the mobility to that of naked DNA. Figure 3B (lanes 5 and 6) shows that fragments whose 3’ ends are generated by camptothecin treatment are retarded before but not after protease treatment. In contrast, Figure 3A (lanes 5 and 6) shows that fragments whose 5” ends are generated by camptothecin are not retarded in the absence of protease treatment. As shown in Figure 3, topoisomerase I interacts primar- ily with the DNA of the transcribed regions of the active heat-shock genes. It might be anticipated that campto- thecin would inhibit transcription, especially if the drug lengthens the lifetime of the protein-DNA intermediate. Figure 4 (lanes 4 and 5) shows that camptothecin only par- tially inhibits transcription of Asp28 in heat-shocked cells. The effect is seen regardless of whether the drug is adminis- tered just before or just after heat shock. Similar levels of transcriptional inhibition were also seen with 0.1 uM and 1 uM camptothecin (data not shown). The transcripts pro- duced appear to be of normal size (even on longer auto- radiographic exposures), indicating that most of the tran- scripts are completed. Figure 4 (lane 2) shows that camptothecin does not by itself induce transcription of this heat-shock gene. The in vivo analyses described above show that topo- isomerase I interacts with specific sites on each strand of the DNA within the transcribed region; very little interac- tion is observed outside of the transcription unit. It appears NHS jr po 9 HS ee 4 5 FIGURE 4.—Camptothecin partially inhibits transcription of hsp28. RNA purified from control and heat-shocked cells was size-separated on an agarose gel; the level of Asp28 mRNA was determined from the North- ern blot of the gel. Cells were treated in the following ways: lane 1, RNA from noninduced cells treated with 0.29% DMSO; lane 2, RNA from noninduced cells treated with 10 uM camptothecin for 40 minutes; lane 3, RNA from cells heat shocked for 40 minutes; lane 4, RNA from cells treated for 5 minutes with 10 pM camptothecin at room temperature followed by a 40-minute heat shock; lane 5, RNA from cells heat shocked for 5 minutes and then treated with 10 uM camptothecin for 35 minutes at 36°C. The ethidium bromide stained gel indicated that each lane contained comparable amounts of total RNA. The RNA blot was analyzed using the Hind I11/ Pst I fragment from 88.1 (16) which hybrid- izes to hsp28 and approximately 400 base pairs flanking each side of the gene. Reproduced with permission from (19). 20 that high levels of topoisomerase I are associated with the heat-shock genes in vivo. Immunofluorescence microscopy reveals intense staining of heat shock loci using antibody to topoisomerase I [(13), Figure 1]. Crosslinking studies show that only slightly less topoisomerase I than RNA polymer- ase II is crosslinked to the heat-shock genes (15); high levels of RNA polymerase II are known to interact with these active loci. Finally, the “parental” restriction frag- ments containing the heat-shock genes at 67B are greatly depleted following camptothecin treatment (Figures 2 and 3). The region of interaction of topoisomerase I with DNA corresponds very closely with the region of transcription. Despite this close correlation with transcription, campto- thecin only partially inhibited transcription of Asp28. We suggest that the drug merely lengthens the lifetime of the protein-DNA intermediate, slowing but not continuously blocking transcription; these intermediates are readily reversible in vitro (16). Normally, the interaction of topo- isomerase I with the gene must be transient if RNA poly- merase is to traverse the DNA coding strand. A comparison of the distribution of topoisomerase I cuts on several genes reveals only limited similarity. All the genes analyzed have conspicuous cuts near the 3’ ends of the transcription units, which could be related to transcrip- tion termination. In addition, cuts of varying intensity occur near the transcription initiation sites. The presence of cleavage sites within the intron of the Asp83 heat-shock gene (23) indicates a general distribution of topoisomerase I across the transcribed region (data not shown). Since no common pattern in the cutting is detected, we suggest that topoisomerase I interacts with the DNA in part in a manner dependent on the local DNA sequence. The observation that topoisomerase I cuts naked DNA at specific sites in the presence of camptothecin supports this (16). Based on the cutting patterns induced in vitro, potential consensus sequences have been identified (12,24). However, a compu- ter search for these consensus sequences has failed to reveal an absolute correlation between the consensus sequences and the camptothecin-induced cleavages observed here. Some of the preferred sites detected here are also preferred cutting sites for calf thymus topoisomerase I on the purified DNA (data not shown). The abundance of cutting sites suggests that this “recognition site” occurs quite frequently. However, the mere presence of a binding site is not suffi- cient for the topoisomerase I to interact with the DNA as indicated by its absence from the inactive genes. The sequences must also be accessible to the enzyme. Measure- ments with various DNA cleavage reagents indicate that actively transcribed genes are markedly more accessible than the inactive genes in isolated nuclei (3-5). The role of topoisomerase I in the transcription of chro- matin remains unclear. If active chromatin is to be main- tained in a configuration under torsional strain as has been suggested (25-28), it would seem necessary to require a very stringent regulation of the abundant topoisomerase I activity. However, Kmiec et al. (29) recently reported that while torsional strain occurs during assembly of a transcrip- tionally active complex in the Xenopus system, it is not required during transcription. One major role of topo- isomerase I during transcription may be a relatively passive one. Topoisomerase I, which relaxes both positively and negatively supercoiled DNA in vitro (30,31), could fulfill the role of a general swivel to alleviate any topological NCI MONOGRAPHS, NUMBER 4, 1987 constraints generated during transcription. The lack of a highly specific role is inferred from the finding that yeast can survive without topoisomerase I; apparently, topo- isomerase II can fulfill the role of topoisomerase I in this organism (32-34). Whether or not topoisomerase I also plays a more specific role in chromatin unfolding and gene activation in other eukaryotes remains to be seen. REFERENCES (I) SIMON JA, SUTTON CA, LOBELL RB, et al: Determinants of heat shock-induced chromosome puffing. Cell 40:805-817, 1985. (2) DANEHOLT B, ANDERSSON K, BJORKROTH B, et al: Visuali- zation of active 75 S RNA genes in the Balbiani rings of Chironomus tentans. Eur J Cell Biol 26:325-332, 1982. (3) WEINTRAUB H, GROUDINE M: Chromosomal subunits in active genes have an altered conformation. Science 193: 848-856, 1976. (4) CARTWRIGHT IL, ELGIN SCR: Nucleosomal instability and induction of new upstream protein-DNA associations accompany activation of four small heat shock protein genes in Drosophila melanogaster. Mol Cell Biol 6: 779-791, 1986. (5) Wu C, WONG Y-C, ELGIN SCR: The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity. Cell 16:807-814, 1979. (6) VOSBERG P-H: DNA topoisomerases: Enzymes that control DNA conformation. Curr Top Microbiol Immunol 114:19-102, 1985. (7) FisHER LM: DNA supercoiling and gene expression. Nature 307:686-687, 1984. (8) WANG JC: Topoisomerase. Annu Rev Genet 54:665-698, 1985. (9) WEISBROD S: Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography. Nucleic Acids Res 10:2017-2042, 1983. (10) HIGASHINAKAGAWA T, WAHN H, REEDER RH: Isolation of ribosomal gene chromatin. Dev Biol 55:375-386, 1977. (11) GOCKE E, BONVEN BJ, WESTERGAARD O: A site and strand specific nuclease activity with analogies to topoisomerase I frames the rRNA gene of Tetrahymena. Nucleic Acids Res 11:7661-7678, 1983. (12) BONVEN JB, GOCKE E, and WESTERGAARD O: A high affin- ity topoisomerase I binding sequence is clustered at DNase I hypersensitive sites in Tetrahymena R-chromatin. Cell 41:541-551, 1985. (13) FLEISCHMANN G, PFLUGFELDER G, STEINER EK, et al: Dro- sophila DNA topoisomerase I is associated with transcrip- tionally active regions of the genome. Proc Natl Acad Sci USA 81:6958-6962, 1984. (14) STEINER EK, EISSENBERG JC, ELGIN SCR: A cytological approach to the ordering of events in gene activation using the Sgs-4 locus of Drosophila melanogaster. J Cell Biol 99:233-238, 1984. (15) GILMOUR DS, PFLUGFELDER G, WANG JC, et al: Topo- isomerase I interacts with transcribed regions in Droso- phila cells. Cell 44:401-407, 1986. (16) HSIANG Y-H, HERTZBERG R, HECHT S, et al: Camptothecin DNA TOPOISOMERASES IN CANCER THERAPY induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873-14878, 1985. (17) NEDOSPASOV SA, GEORGIEV GP: Non-random cleavage of SV40 DNA in the compact minichromosome and free in solution by micrococcal nuclease. Biochem Biophys Res Commun 92:532-539, 1980. (18) Wu C: The 5’ ends of Drosophila heat shock genes in chro- matin are hypersensitive to DNase I. Nature 286:854-860, 1980. (19) GILMOUR DS, ELGIN SCR: Localization of specific topo- isomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor campto- thecin. Mol Cell Biol. In press. (20) CORCES V, HOLMGREN R, FREUND R, et al: Four heat shock proteins of Drosophila melanogaster coded within a 12- kilobase region in chromosome subdivision 67B. Proc Natl Acad Sci USA 77:5390-5393, 1980. (21) CrAIG EA, MCCARTHY BJ: Four Drosophila heat shock genes at 67B: Characterization of recombinant plasmids. Nucleic Acids Res 8:4441-4457, 1980. (22) SIROTKIN K, DAVIDSON N: Developmentally regulated transcription from Drosophila melanogaster chromosome site 67B. Dev Biol 89:196-210, 1982. (23) O’CoNNOR D, Lis JT: Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation. Nucleic Acids Res 9:5075-5092, 1981. (24) BuLLock P, CHAMPOUX JJ, BOTCHAN M: Association of crossover points with topoisomerase I cleavage sites: A model for nonhomologous recombination. Science 230: 954-958, 1985. (25) Ryost M, WORCEL A: Chromatin assembly in Xenopus oocytes: In vivo studies. Cell 37:21-32, 1984. (26) VILLEPONTEAU B, LANDELL M, MARTINSON H: Torsional stress promotes the DNAase I sensitivity of active genes. Cell 39:469-478, 1984. (27) LUCHNIK AN, BAKAYEV VV, ZBARSKY IB, et al: Elastic tor- sional strain in DNA within a fraction of SV40 mini- chromosomes: Relation to transcriptionally active chro- matin. EMBO J 1:1353-1358, 1982. (28) HARLAND RM, WEINTRAUB H, MCKNIGHT SL: Transcrip- tion of DNA injected into Xenopus oocytes is influenced by template topology. Nature 302:38-43, 1983. (29) KMIEC EB, MASARU R, WORCEL A: Gyration is required for 5S RNA transcription from a chromatin template. Proc Natl Acad Sci USA 83:1305-1309, 1986. (30) CHAMPOUX JJ, DULBECCO R: An activity from mammalian cells that untwists supercoiled DNA—A possible swivel for DNA replication. Proc Natl Acad Sci USA 69:143-146, 1972. (31) BAASE WA, and WANG JC: An w protein from Drosophila melanogaster. Biochemistry 13:4299-4303, 1974. (32) Goto T, WANG JC: Cloning of yeast TOPI, the gene encod- ing topoisomerase I, and the construction of mutants defective in both DNA topoisomerase I and DNA topoi- somerase II. Proc Natl Acad Sci USA 82:7178-7182, 1985. (33) SAAVEDRA RA, HUBERMAN RH: Both DNA topoisomerase I and II relax 2 um plasmid DNA in living yeast cells. Cell 45:65-70, 1986. (34) UEMURA T, YANAGIDA M: Isolation of type I and II DNA topoisomerase mutants from fission yeast: Single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J 3:1737-1744, 1984. 21 nN pny .- nn a h a iE f= Bo Fl ys A he . A hye FO ai DETICr [a } La A ee? ht. PRET IL I Ha a i gry, CF Lo TA i wi [dT A w=, wm i HEE fra, : rE On peng NE A 2 = Bh, Bg REA 4. 2 Ta u iL i = J6 Foal 35 5 ely Loh Te =a el hl _« ples ” hE akan NE MT a ate - i 1 : pe Lh He Hn ou TT CEA qe Real, dl ow J ud Ts = of LT - Ws ty « } | h ye 5 Ha = we oo a or : } i } - a 1] el pn, i gid a le "vy 22a = 0 i " a fT pn NT So : A, Mma gi er IR Sami i yA Ey A = Hl = on SRT 1 Rae md 10 Ty 1h AR id Fo La Ark g sr vd . Ln Ba fi meg a tid ah. 7 ot Wp o EifeYEgdte n 4, onl EO an RE i v dua fig J agi SY i iw AE ly rit uf i 1 . oh Sil 4 Ras RA do HRT Ae nom, 3 Fat Cr nig re, i dh i re enue = Sr dle diy . | ibe ok LE: Sh h 0 - ne i Sel eh I : ay a) ¢ bE: | 8 0 Ee § jo al pe ir he It t gar, oe oe Pile | a SE sn Sige Augie re Hine ES Hint lin FEEL ir , } sehen pl i A HRS Ee Eee aan B Fut] Sif a eal Ree ne na i Te eT i By go BS Ry : las : wl Fat het vin berg i Gogh Ad, Bat A it del Be va fe atm: } TREE Bh Ea ' ar 3 fie B x eid i ’ F EN = FET fis . ES } lao " pi ¥ . or Fr » Ln — t o = FL rape lf, - 4 | 1 wiih J - ot ny “ i 1 3 . CT - b= B i . = —_) i ' [a ITE EPL iw i 9, Fh oe El i jiSY ; i 3 { ea ) ER y i A - : FEE KE a® ud : } ) ted nT As gl J i rh i . E 0 . . Yow i 3 F j EL ole fh hd B [i i p . 1: - 5 I hit 2 f u 2 , 4 T 1 4 i i JE . HK bh RIE Jib = + . " bl ’ : W 1 i R in ) - BE } Cour Rain = Ey ad rl i i . : Ta gon taife on Th z I - i A a = | i= A k Saleh LAs - ' dh X ar oa 18 AE ee fi LY i i url ald 2 5 4 : i 1 i 5 » Bilt inl fails 2 . ' Ce = . = + pv eye we iY pA AL i ry - wand NEGR Cage eal | 3 - 3 i i. aie cae ak M it g te 2 } } 7 = rl! Spd gl i & n k =i% * r 5 1 - |] - oo } ' : V a 1B pi i N a 5 h A £ BE | 5 3 . = 3 fi } - ie TEE BRUTY orf Ee i > EE 2 ) AL 3. Sed ER +E Fe F no Fad “I : hk a i ¥ == 5 El 2.x A hi P= 5 - fe heal BDL Bh i B = k 1 Be “ hr - Wr LIE a ] Tal } " ihn 2 Lr En SiS Cc 1 ol BEI I~ LF oh Au ein. i ) : Ehsi (01 ) Cnda] # Structural Aspects of Mammalian DNA Replication: Topoisomerase 11 William G. Nelson2* and Donald S. Coffey?? ABSTRACT —The dynamic biology of mammalian DNA requires structural complexity. Previous studies on the structure and function of mammalian DNA have suggested that mammal- ian cell nuclei contain DNA organized into loop-domains by a nuclear structural subcomponent termed the nuclear matrix. The loop-domains, functioning as replicons during DNA synthesis, appear to be replicated by biosynthetic complexes located at fixed sites within the nuclear matrix milieu. Recently, we have found the mammalian DNA topoisomerase II enzyme to be associated with newly replicated DNA, and we speculate that the enzyme may be strategically positioned to untangle topologically inter- twined daughter loop-domains before mitotic segregation. Further studies demonstrating growth-related elevations of DNA topo- isomerase II levels in rat prostatic adenocarcinoma tissues support a role for the enzyme in cellular proliferation in vivo. The possible participation of DNA topoisomerase II in mammalian DNA replication and the proliferation-dependent appearance of the enzyme in neoplastic tissues may have important implications for therapeutic strategies directed at DNA topoisomerase II.—NCI Monogr 4:23-29, 1987. INTRODUCTION The mammalian genome exhibits a structural complexity commensurate with its functional diversity. The conforma- tion of DNA within a mammalian cell nucleus must both permit functional independence of genetic subunits in pro- cesses, such as transcription and replication, and satisfy limits of scale: the mammalian cell contains a total length of nearly 1.8 M of DNA packed into a roughly spherical nucleus of 10 um diameter. Furthermore, active genetic processes requiring unraveling of the DNA duplex for tem- plate activity introduce additional problems in DNA topology. DNA topoisomerases, enzymes capable of cata- Supported by Public Health Service grants 84-CA-22 and CA-15416 (National Cancer Institute) and AM-22000 (National Institute of Arthritis and Musculoskeletal and Skin Diseases) from the National Institutes of Health, Department of Health and Human Services. W. G. Nelson is the recipient of Medical Scientist Training Program Award T532-GM-07309. 2The Oncology Center and Departments of Urology and Pharmacology, The Johns Hopkins University School of Medicine, and The James Buchanan Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, MD. 3 We thank Ruth Middleton for assistance in the preparation of this manuscript. * Reprint requests: Dr. William G. Nelson, Department of Urology, The Johns Hopkins Hospital, 123 Marburg Bldg., 600 N. Wolfe St., Baltimore, MD 21205. lyzing changes in the topology of DNA substrate mole- cules, have been proposed to function in a variety of such processes [reviewed in (/-3)]. Precisely how these enzymes function to resolve DNA topology problems encountered by the mammalian genome is not well understood. We have been studying the structure of replicating DNA within the mammalian cell nucleus. The nucleus directs the replication of the entire genome in discrete subunits termed “replicons” (4). Replicons containing an average of 50-100 kilobase pairs of DNA reel through replicating sites in 30 minutes, unwinding at speeds greater than 100 rpm, to produce untangled daughter DNA molecules which segre- gate at mitosis. Previous studies (5, 6) have revealed that the mammalian nucleus organizes DNA into loop-domains which serve as replicons during DNA replication. We have found that the mammalian DNA topoisomerase II enzyme, an enzyme capable of resolving topological problems encountered during DNA replication, is associated with newly replicated daughter DNA molecules (7). Addition- ally, we have found increased levels of the topoisomerase 11 enzyme in neoplastic tissues correlated with growth in vivo (8). Here, we review several features of the organization of DNA within the mammalian nucleus and of mammalian DNA replication, and we describe recent data implicating topoisomerase II in mammalian DNA replication. ORGANIZATION OF THE GENOME INTO LOOP-DOMAINS Several studies [(6,9); for review, see (/0)] have indicated that the nuclear matrix, a structural subcomponent of the mammalian cell nucleus, orders DNA into loop-domains. The chromosome scaffold, an analogous structural sub- component of the mitotic chromosome, preserves the loop- domain conformation through mitosis (//). The nuclear matrix can be readily isolated by removing histones and other soluble proteins from cell nuclei; the resultant structure contains a fraction (approximately 10%) of the total nuclear protein (12,13). Loop-domains of DNA, containing 90 kilobase pairs of DNA, have been visualized in the presence of ethidium bromide as a fluorescent halo surrounding the nuclear matrix (6). Ethidium bromide titration experiments have revealed that the loop-domains contained negatively supercoiled DNA and that elements within the nuclear matrix interact with DNA at the base of the loop-domains in such a way that each loop-domain is topologically independent (6). Interestingly, DNA topo- 23 isomerase II may be well positioned to manipulate the topology of DNA loop-domains. The enzyme has been found to be a component of both the nuclear matrix (14) and the chromosome scaffold (75,16). Pienta and Coffey (17) have proposed a new model for chromosome structure featuring the organization of mam- malian DNA into loop-domains. Their model predicts that DNA can be very tightly packed (with a packing ratio approaching 1:12,000) into a mitotic chromatid with 60- kilobase pair DNA loop-domains radially distributed, 18 loops per turn, along the shaft of the chromatid. Clearly, the organization of the mammalian DNA into discrete topologically independent units, the loop-domains, provides structural basis for both the functional heterogeneity and the spatial economy exhibited by the mammalian genome. DNA LOOP-DOMAINS REPLICATE AT A FIXED SITE IN THE NUCLEUS The demonstration that the nuclear matrix organizes the genome into DNA loop-domains has facilitated the study of mammalian DNA replication. Berezney and Coffey (18) first reported an association of newly replicated DNA with nuclear matrix structures isolated from rat livers regener- ating after partial hepatectomy. Pardoll, Vogelstein, and Coffey (5) observed an enrichment for newly replicated DNA among DNA remaining associated with the nuclear matrix of proliferating cells following nuclease digestion and suggested that eucaryotic DNA might be replicated at a fixed site located on the nuclear matrix. In further studies, Vogelstein, Pardoll, and Coffey (6) directly observed the replication of DNA loop-domains at a fixed site on the nuclear matrix by monitoring the progression of auto- radiographic grains from a [*H]thymidine pulse label into the loop-domain halo surrounding the nuclear matrix. Supporting this model, Smith and Berezney (19,20) have identified DNA polymerase a associated with the nuclear matrix of replicating cells and have found that nuclear matrix structures competently continue to synthesize DNA after isolation. DNA TOPOISOMERASE Il IS ASSOCIATED WITH NEWLY REPLICATED DNA In a recent study (7), we sought to determine whether DNA topoisomerase II might function in mammalian DNA replication by assessing the proximity of the enzyme to newly replicated DNA in cultured rat prostatic adeno- carcinoma cells. The antineoplastic agent teniposide, an epipodophyllotoxin, interacts with mammalian DNA topo- isomerase II in such a way that the enzyme can be isolated in covalent linkage with DNA following treatment with sodium dodecyl sulfate (SDS) (27-25). In our study, we exploited this interaction to trap topoisomerase II on cellular DNA in covalent complexes assayable by a potassium-sodium dodecyl sulfate (K-SDS) precipitation procedure specific for protein-linked DNA (7,26). Cultured cells that had been pulse labelled with [H]thymidine for 90 seconds were permeabilized at 4°C in order to terminate the pulse label. When these cells were treated with teniposide, lysed with SDS and subjected to the K-SDS precipitation assay, pulse-labelled DNA was recovered with the K-SDS precipitate (Figure 1, bar B). Very little pulse- labelled DNA was detected in the K-SDS precipitate when 24 Bm + teniposide 0.6 = Nn - teniposide << x 2 = 0 a i 403 0.4 w= 5 2 22 “ ce E ‘c @ gS T 202 8 5 o c Oo £ © = SE > 3 % BS 2 E = a + a 5 ‘ ! Oo A B C D E F FIGURE 1.—Teniposide treatment results in covalent linkage of pulse- labelled DNA to protein (topoisomerase II) in cultured rat prostatic adenocarcinoma cells. The fraction of pulse-labelled [2 H]JDNA linked to protein (protein-linked [>’H]DNA / total [°’H]DNA) was determined for (A) control, lysates from pulse-labelled cells that had been permeabilized and incubated in the absence of teniposide; (B) teniposide, lysates from cells incubated with 20 uM teniposide; (C) + proteinase K, lysates from teniposide-treated cells digested with proteinase K; (D) + novobiocin, lysates from cells incubated with teniposide and 2 mM novobiocin; (E) — ATP, lysates from cells incubated with teniposide but without ATP; (F) — MgCl, lysates from cells incubated with teniposide but without MgCl,. Protein-linked [’H]DNA fractions are expressed as means + SEM. Reproduced with permission from (7). lysates were digested with proteinase K, confirming the specificity of the assay for protein-linked DNA (Figure 1, bar C). Teniposide-induced trapping of covalent complexes between topoisomerase II and pulse-labelled DNA was markedly reduced in the presence of novobiocin and in the absence of ATP or magnesium (Figure 1, bars D-F). Pulse and pulse-chase labelling experiments revealed that tenipo- side treatment resulted in trapping of topoisomerase II-DNA complexes near the site of DNA replication. When SDS lysates from teniposide-treated pulse and pulse-chase protein - linked 3H-DNA (relative specific activity) 0 0.2 0.4 0.6 0.8 I 14C - DNA remaining protein - linked after sonication (relative to unsonicated) FIGURE 2.—Enrichment of newly replicated DNA with covalent topo- isomerase II-DNA complexes. Cultured rat prostatic adenocarcinoma cells that had been labelled for 72 hours with [!4C]thymidine were incubated with [3H]thymidine for 90 seconds (pulse) or exposed to [*H]thymidine for 90 seconds and then incubated in excess unlabelled thymidine for 45 minutes (pulse-chase). After labelling, cells were permeabilized, treated with teniposide, and lysed in an SDS Lysis buffer. Then, in order to progressively reduce the size of DNA remaining linked to topoisomerase II, the lysates were sonicated for 0, 5, 10, 15, 20, and 30 seconds before being subjected to the K-SDS assay. The specific activity of [PH]DNA in protein-linked DNA relative to the specific activity of [’HJDNA in total DNA was determined and plotted as a function of ['*C]JDNA remaining protein linked after sonication (normalized to the unsonicated protein-linked[ 'C]DNA value). Repro- duced with permission from (7). NCI MONOGRAPHS, NUMBER 4, 1987 labelled cells were first sonicated in order to reduce the average fragment size of protein-linked DNA and then subsequently subjected to the K-SDS assay, the [*H]thy- midine label was found to be enriched among protein- linked DNA (Figure 2). No enrichment was observed among sonicated protein-linked DNA from teniposide- treated, pulse-chase labelled cells (Figure 2). In order to determine whether teniposide induced the trapping of topoisomerase II-DNA complexes directly with newly replicated daughter DNA strands, we subjected sonicated lysates from teniposide-treated, pulse-labelled cells to the K-SDS assay run under denaturing conditions [100°C; see (7)]. We found an enrichment for pulse-labelled DNA among protein-linked single-stranded DNA (13.7- = 1.3-fold) equal to the enrichment found for pulse-labelled DNA among protein-linked double-stranded DNA (13.4- + 1.3-fold). Alkaline sucrose sedimentation studies pro- vided additional evidence for a direct association of topoisomerase II with newly replicated daughter DNA molecules. After teniposide treatment, protein-linked DNA from uniformly labelled cells sedimented to a size 25-358, while protein-linked DNA from pulse-labelled cells sedi- mented to a size <12S characteristic of nascent DNA chains (Figure 3). DNA TOPOISOMERASE Il FUNCTION DURING MAMMALIAN REPLICATION: A MODEL The necessity for unraveling each DNA strand in a parental DNA duplex to expose complimentary DNA Ol 25 0.08 0.06 0.04 0.02 top bottom 0.3 fraction of protein - linked 3H-DNA 0.2 Oo. 0 0 4 8 12 16 20 24 top bottom gradient fraction number FIGURE 3.—Nascent DNA strands can be trapped in direct covalent linkage with topoisomerase II by teniposide. Topoisomerase II-linked total (A, 72-hour label) and newly synthesized (B, 90-second label) [PH]DNA were analyzed by alkaline sucrose gradient centrifugation. Arrows in (A) denote the position of 2S (28 bases) and 13S (2657 bases) internal markers. Reproduced with permission from (7). DNA TOPOISOMERASES IN CANCER THERAPY § <=— nonreplicated DNA loops tangling of replicated DNA loops fixed site of DNA synthesis Ry —topoisomerase Il $ 2% RN topoisomerase II DNA loops by topoisomerase II FIGURE 4.—A model for DNA topoisomerase II function during mammalian DNA replication. In (A), loop-domains of DNA replicate at fixed sites (on the nuclear matrix). Without topoisomerase II in (B), daughter DNA loop-domains become hopelessly entangled during replication fork progression. Topoisomerase II, interacting with newly synthesized DNA near replicating sites, acts to untangle daughter DNA loop-domains in (C) before mitotic segregation. templates for semiconservative DNA replication constitutes a major topological challenge for mammalian cells. Cairns (27) recognized the need for an effective swivel in his early studies of the replication of covalently closed circular DNA molecules. Several recent studies (28-33) have suggested that topoisomerase II may be required to resolve intertwined products of eucaryotic DNA replication before mitotic segregation. Sundin and Varshavsky (28,29) have proposed that intertwining of replicated SV40 daughter molecules occurs during bidirectional replication as replication forks converge within 100-200 base pairs of the replication terminus. We speculate that parental topological turns may be passed continuously by the replication apparatus through to daughter strands during semidiscontinuous fork elonga- tion. Perhaps one parental topological turn may accumulate among the daughter strands with each semidiscontinuous replication step. The mammalian topoisomerase II enzyme, associated with daughter DNA molecules near the repli- cating fork, would be well positioned to remove parental topological turns from double-stranded daughter DNA molecules (Figure 4). ELEVATED TOPOISOMERASE Il LEVELS IN GROWING NEOPLASTIC TISSUES After finding an association between DNA topoisomerase II and newly replicated DNA in cultured prostatic adeno- carcinoma cells, we suspected that topoisomerase II might be characteristically present in growing mammalian cells. Elevated topoisomerase II levels have been detected 25 during rat liver regeneration (34), after growth factor stimu- lation (35), and during proliferation in culture (36). We assessed enzyme levels in the rat dorsal prostate and in two rat dorsal prostate tumors with differing growth rates in vivo (8). The Dunning R3327-H and R3327-G tumor sub- lines were derived from a tumor which arose spontaneously in the dorsal prostate of a rat in 1961 (37). Maintained by serial passage subcutaneously in the flanks of rats in our laboratory for over a decade, each Dunning tumor subline has a stable and distinct growth phenotype in vivo: the R3327-H tumor is a well-differentiated, androgen-depen- dent adenocarcinoma which doubles in 21 days; the R3327- G tumor has an anaplastic appearance and a 4-day doubling time (38). We evaluated topoisomerase II levels in each tissue by an enzyme activity assay and by using anti-topoisomerase II antisera in immunoblot and immunohistochemical analyses. We assayed for enzyme activity in nuclear extracts prepared from each tissue using a P4-unknotting assay (39) specific for type II topoisomerases. The R3327-G tumor nuclear extract contained an activity capable of resolving topologi- cal knots in knotted substrate P4 DNA molecules (Figure 5, lane 2). The unknotting activity required magnesium and ATP and was inhibited by novobiocin, teniposide, and amsacrine (Figure 5, lanes 3-7). When serial dilutions of equivalent nuclear extracts prepared from each tissue were assayed for topoisomerase II activity, a greater level of activity was detected in the R3327-G tumor extract than in extracts from either the R3327-H tumor or the dorsal prostate, and the R3327-H tumor extract contained a greater level of enzyme activity than the extract from the dorsal prostate (Figure 6). Immunoblot analysis using anti- topoisomerase II antisera for the detection of topoisomerase II polypeptides (40) revealed higher levels of enzyme polypeptides in protein-containing extracts prepared from the R3327-G tumor than in extracts prepared from either DNA Topoisomerase IL Activity in Dunning R3327-G Tumor Nuclear Extract Cc 9 a wr 5 2 £88 tS Sef 3 x a g 2255 E papNg oc ER EEE < —==8 ZZ J 1 I + + + unknotted linear = —— knotted Z 34 85767 FIGURE 5.—Nuclei isolated from the Dunning R3327-G rat prostatic adenocarcinoma contain a topoisomerase II unknotting activity. The ability for a nuclear extract prepared from the Dunning R3327-G tumor to catalyze the conversion of knotted P4 DNA substrate molecules to unknotted P4 DNA product circles was assessed for: (1) no extract, an assay mixture that did not contain the nuclear extract; (2) complete, an assay mixture containing the R3327-G tumor nuclear extract; (3) — ATP, an assay mixture without ATP; (4) — MgCl,, an assay mixture without MgCly; (5) +novobiocin, a complete assay mixture supple- mented with 1.0 mM novobiocin; (6) + teniposide, an assay mixture with 20 uM teniposide (VM-26); and (7) + amsacrine, an assay mixture with 20 uM amsacrine. Reproduced with permission from (8). | 26 Nuclear Extract (ng DNA Equivalents) I P4 DNA I oOo 125625313156 78 unknotted, linears knotted i] 2 3 4 5 6 O = 125 62.5 31.3 15.6 7.8 unknotted linear = knotted 2 3 4 8 6 I 1 0 125 62.5 31.3 |156 7.8 unknotted linear = knotted | 2 FIGURE 6.—Nuclear extracts from Dunning R3327-H and R3327-G tumors contain greater levels of topoisomerase II unknotting activity than nuclear extracts from rat dorsal prostate. A series of equivalent nuclear extracts (lanes 2-6) derived from the dorsal prostate (A), the R3327-H tumor (B), and the R3327-G tumor (C) were analyzed for P4 DNA unknotting activity. Reproduced with permission from (8). 34 5 6 the R3327-H tumor or the dorsal prostate (Figure 7). In fact, topoisomerase II polypeptides were barely detectable in normal dorsal prostate extracts. Immunohistochemical staining of histologic sections prepared from each tissue using anti-topoisomerase II antisera demonstrated the presence of the enzyme in cell nuclei for each tissue; however, many nuclei from each tissue did not appear to contain detectable topoisomerase II (Figure 8). Further- more, a greater fraction of nuclei from the R3327-G tumor exhibited positive immunohistochemical staining for topo- isomerase II than from either the R3327-H tumor or the dorsal prostate, and the R3327-H tumor contained a greater fraction of stained nuclei than the dorsal prostate (Figure 8 and Figure 9, bar D). When topoisomerase II unknotting activity levels, topoisomerase II polypeptide NCI MONOGRAPHS, NUMBER 4, 1987 A Immunodetection of DNA Topoisomerase II Absorbance —= SDS - PAGE — B Quantitation of Immunoreactive DNA Topoisomerase IL | 08 06 04 0.2 i 1 A J % 2 4 6 8 10 G Tumor Tissue Extract (ug DNA) DNA Topoisomerase IL (170K Peak Areaq, Relative) C. DNA Topoisomerase II Tissue Levels H © | 3 E Pa > OB gz €2 © os 220 27 oa gE: a 2 o2 © gd « Dorsal H Tumor G Tumor Prostate FIGURE 7.—Immunoblot analysis of normal and neoplastic rat prostate tissue extracts using anti-topoisomerase II antiserum. Displayed are: (A) an autoradiograph and densitometry scan from the immunoblot analysis of an extract prepared from an R3327-G tumor tissue homogenate containing 10 ug DNA; (B) the dependence of immuno- reactive topoisomerase II densitometry peak area on G tumor extract quantity; and (C) relative DNA topoisomerase II polypeptide levels detected in dorsal prostate, H tumor, and G tumor tissue extracts. Reproduced with permission from (8). levels, and the fraction of nuclei containing topoisomerase II were compared to known growth parameters for each of the tissues [(38); John T. Isaacs, personal communication], a strong correlation between topoisomerase II expression and cell proliferation in vivo was found (Figure 9). CONCLUDING REMARKS The mammalian nucleus maintains a complex DNA conformation, and DNA replication presents mammalian DNA TOPOISOMERASES IN CANCER THERAPY nuclei with formidable structural problems. We believe that the demonstration of an association between topoisomerase IT and newly replicated DNA and the finding of elevated topoisomerase II levels correlated with tissue growth in vivo strongly support a role for topoisomerase II in resolving structural problems arising during DNA repli- cation. Our findings have important implications for antineo- plastic chemotherapy strategies directed at topoisomerase II. Tumor cell kill in vivo by topoisomerase II-targeted agents might be limited to the fraction of cells containing the enzyme. In the rat Dunning R3327-H prostatic adeno- carcinoma, a slow growing tumor, only a small fraction of FIGURE 8.—Anti-topoisomerase II immunohistochemical staining of tissue sections from (A) dorsal prostate, (B) R3327-H tumor, and (C) R3327-G tumor. Arrow denotes an anti-topoisomerase II antiserum- stained nucleus; bar corresponds to 50 microns. Reproduced with permission from (8). 27 B A. Tissue Growth Rate O B. Rate of DNA Synthesis - v7 C. Topoisomerase II Te en Level 0.8F = D. Fraction of Nuclei Containing Topoisomerase Il E. Topoisomerase Il Unknotting Activity 0.6 RON) XX RXR 0.4 Relative to G Tumor T 0.2 ABCDE ABCDE Dorsal H Tumor G Tumor Prostate FIGURE 9.—Comparison of topoisomerase II levels and tissue growth parameters for dorsal prostate, the R3327-H tumor, and the R3327-G tumor. Plotted relative to the R3327-G tumor are: (A) tissue growth rate, the reciprocal of the doubling time in days (38); (B) rate of DNA synthesis, assessed by [H]thymidine incorporation (J. T. Isaacs, personal communication); (C) topoisomerase II polypeptide level (8); (D) fraction of nuclei containing topoisomerase II (8); and (E) topoisomerase II unknotting activity, estimated from densitometric analysis of ethidium bromide-stained agarose gels containing P4- unknotting assays by the reduction of knotted P4 DNA fluorescence catalyzed by topoisomerase II present in extracts prepared from 31.3 ng DNA equivalents of nuclei (see Fig. 6). Reproduced with permission from (8). the cells contained topoisomerase II (Figures 8 and 9). Similar slow growing tumors encountered in clinical oncology might be difficult to eradicate using enzyme- targeted agents. Furthermore, factors other than enzyme levels might also affect the efficacy of topoisomerase 1I- directed therapy. In our laboratory, we have recently found that the biological response modifier tumor necrosis factor enhances the cytotoxicity of chemotherapeutic drugs tar- geted at topoisomerase II without apparently increasing enzyme-mediated DNA damage (47). Hopefully, future studies on the biology of DNA topoisomerases and on the pharmacology of topoisomerase-mediated cytotoxicity will provide a basis for the rational design of novel antineoplas- tic regimens featuring topoisomerase-targeted agents. REFERENCES (I) GELLERT M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981. (2) Liv LF: DNA topoisomerases—Enzymes that catalyze the breaking and rejoining of DNA. CRC Crit Rev Biochem 15:1-24, 1981. (3) WANG JC: DNA topoisomerases. Annu Rev Biochem 54:665-697, 1985. (4) HUBERMAN JA, RIGGS AD: On the mechanism of DNA replication in mammalian chromosomes. J Mol Biol 32:327-341, 1968. (5) PARDOLL DM, VOGELSTEIN B, COFFEY DS: A fixed site of DNA replication in eucaryotic cells. Cell 19:527-536, 1980. (6) VOGELSTEIN B, PARDOLL DM, COFFEY DS: Supercoiled loops and eucaryotic DNA replication. Cell 22:79-85, 1980. (7) NELSON WG, L1u LF, Correy DS: Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells. Nature 322:187-189, 1986. 28 (8) NELSON WG, CHO KR, HSIANG Y-H, et al: Growth-related elevations of DNA topoisomerase II levels are found in Dunning R3327 rat prostatic adenocarcinomas. Cancer Res 47:3246-3250, 1987. (9) Cook PR, BRAZELL IA, JOST E: Characterization of nuclear structures containing superhelical DNA. J Cell Sci 22: 303-324, 1976. (10) NELSON WG, PIENTA KJ, BARRACK ER, et al: The role of the nuclear matrix in the organization and function of DNA. Annu Rev Biophys Biophys Chem 15:457-475, 1986. (11) PAuLsoN JR, LAEMMLI UK: The structure of histone- depleted metaphase chromosomes. Cell 12:817-828, 1977. (12) BEREZNEY R, COFFEY DS: Identification of a nuclear protein matrix. Biochem Biophys Res Commun 60:1410-1417, 1974. (13) BEREZNEY R, COFFEY DS: Isolation and characterization of a framework structure from rat liver nuclei. J Cell Biol 73:616-637, 1977. (14) BERRIOS M, OSHEROFF N, FISHER PA: In situ localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci USA 82:4142-4146, 1985. (15) EARNSHAW WC, HALLIGAN B, COOKE CA, et al: Topoisom- erase II is a structural component of mitotic chromosome scaffolds. J Cell Biol 100:1706-1715, 1985. (16) EARNSHAW WC, HECK MMS: Localization of topoisomerase II in mitotic chromosomes. J Cell Biol 100:1716-1725, 1985. (17) PIENTA KJ, COFFEY DS: A structural analysis of the role of the nuclear matrix and DNA loops in the organization of the nucleus and chromosome. J Cell Sci Suppl 1:123-135, 1984. (18) BEREZNEY R, COFFEY DS: Nuclear protein matrix: Associa- tion with newly synthesized DNA. Science 189:291-293, 1975. (19) SMITH HC, BEREZNEY R: DNA polymerase « is tightly bound to the nuclear matrix of actively replicating liver. Biochem Biophys Res Commun 97:1541-1547, 1980. (20) SMITH HC, BEREZNEY R: Nuclear matrix-bound deoxyribo- nucleic acid synthesis: An in vitro system. Biochemistry 21:6751-6761, 1982. (21) Ross W, ROWE T, GLISSON B, et al: Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Res 44:5857-5860, 1984. (22) GL1SSON BS, SMALLWOOD SE, Ross WE: Characterization of VP-16-induced damage in isolated nuclei from L1210 cells. Biochim Biophys Acta 783:74-79, 1984. (23) LoNG BH, MUSIAL ST, BRATTAIN MG: Single- and double- strand DNA breakage and repair in human lung adenocar- cinoma cells exposed to etoposide and teniposide. Cancer Res 45:3106-3112, 1985. (24) YANG L, Rowe TC, Liu LF: Identification of DNA topoisomerase II as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. Cancer Res 45:5872-6876, 1985. (25) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian topoisomerase II. J Biol Chem 259:13560-13566. (26) RowE TC, CHEN GL, HSIANG Y-H, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (27) CAIRNS J: The chromosome of Escherichia coli. Cold Spring Harbor Symp Quant Biol 28:43-46, 1963. (28) SUNDIN O, VARSHAVSKY A: Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers. Cell 21:103-114, 1980. (29) SUNDIN O, VARSHAVSKY A: Arrest of segregation leads to NCI MONOGRAPHS, NUMBER 4, 1987 accumulation of highly intertwined catenated dimers: Dissection of the final stages of SV40 DNA replication. Cell 25:659-669, 1981. (30) DINARDO S, VOELKEL K, STERNGLANZ R: DNA topoisom- erase II mutant of Saccharomyces cerevisiae: Topoisom- erase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 81:2616-2620, 1984. (31) UEMURA T, YANAGIDA M: Isolation of type I and II DNA topoisomerase mutants from fission yeast: Single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J 3:1737-1744, 1984. (32) UEMARA T, YANAGIDA M: Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J 5:1003-1010, 1986. (33) HoLM CH, GoTo T, WANG JC, et al: DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553-563, 1984. (34) DUGUET M, LAVENOT C, HARPER F, et al: DNA topoisom- erases from rat liver: Physiologic variations. Nucleic Acids Res 11:1059-1075, 1983. DNA TOPOISOMERASES IN CANCER THERAPY (35) MiskIMINS R, MIsSkKIMINS WK, BERNSTEIN H, et al: Epidermal growth factor-induced topoisomerase(s), intra- cellular translocation and relation to DNA synthesis. Exp Cell Res 146:53-62, 1983. (36) SULLIVAN DM, GLISSON BS, HODGES PK, et al: Prolifera- tion dependence of topoisomerase II mediated drug action. Biochemistry 25:2248-2256, 1986. (37) DUNNING WEF: Prostate cancer in the rat. Natl Cancer Inst Monogr 12:351-369, 1963. (38) IsAaAcs JT, COFrey DS: Model systems for the study of prostate cancer. Clin Oncol 2:479-498, 1983. (39) Liu LF, Davis LF, CALENDER R: Novel topologically knotted DNA from bacteriophage P4 capsids: Studies with DNA topoisomerases. Nucleic Acids Res 9:3979- 3989, 1981. (40) HALLIGAN BD, EDWARDS KA, Liu LF: Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. J Biol Chem 260:2475-2482, 1985. (41) ALEXANDER RB, NELSON WG, COFFEY DS: Tumor necrosis factor enhances the cytotoxicity of chemotherapeutic drugs targeted at DNA topoisomerase II. Cancer Res 47: 2403-2406, 1987. 29 A Li fad tt 0 Hh \ a he a im HH {= t (ER fT Tet op ike i oo a! ali it hi ae Pa i het se Fin} is EE ; + A, . 1 in Te B la Baal a aa i R E Engg dial fie Bit 2 AT wb 3 eh F of 3 RS Fatt oC Eat al B ag SECON 14 btm | : i jis 5 A) rl] RT| Er Fa le EE I 5 i Ww wprdot ite asl x Regulation of DNA Topoisomerases During Cellular Differentiation Annette L. Bodley, Hai-Young Wu, and Leroy F. Liu?* ABSTRACT—DNA topoisomerase II has been shown to be a nuclear marker for cell proliferation and a therapeutic target in cancer chemotherapy. In order to study the regulation of DNA topoisomerases during cellular differentiation, MELC were in- duced to differentiate by treatment with 5 mM HMBA. At day five, approximately 95% of MELC had reproducibly undergone differentiation. The level of topoisomerase II, as measured by immunoblotting with anti-topoisomerase II antisera, showed a parallel decrease to approximately 5-10% of the control level by day five. Activity measurements showed a similar decrease during the time course of MELC differentiation. Inmunofluorescence studies at day five showed that only about 5% of the MELC had strong nuclear immunofluorescence. These results indicate that the level and activity of DNA topoisomerase II are significantly lower in differentiated versus undifferentiated cells. We also observed that the level and activity of topoisomerase II dropped twofold as cells grew to high cell densities in the absence of HMBA. In contrast, the topoisomerase I levels in MELC remained relatively constant throughout growth and differentia- tion.—NCI Monogr 4:31-35, 1987. INTRODUCTION It has recently been shown that mammalian DNA topoisomerases I and II are the targets for many antineo- plastic drugs (/-9). In cultured mammalian cells, the addition of camptothecin, a topoisomerase I targeting drug, or VM-26 or amsacrine, topoisomerase II targeting drugs, produces reversible protein linked DNA breaks [reviewed in (/,10)]. Studies in which the purified DNA topoisomerases were used have suggested that these drugs may inhibit the DNA rejoining reaction inherent in the mechanism of both DNA topoisomerases. The abortive complex formed in the presence of these antitumor drugs has been termed the cleavable complex. Exposure of the cleavable complex to strong protein denaturants results in single- and double-strand breaks in the DNA with the covalent linking of a topoisomerase polypeptide to a specific terminus of each broken DNA strand. All evidence ABBREVIATIONS: MELC = murine erythroleukemia cells; HMBA = hexamethylene bisacetamide; BSA =bovine serum albumin; PBS = phosphate-buffered saline. I'Supported in part by Public Health Service grants CA-39962 from the National Cancer Institute, and GM-27731 from the National Institute of General Medical Sciences, National Institutes of Health, Department of Health and Human Services. 2 Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD. * Reprint requests: Leroy F. Liu, Ph.D., Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. indicates that the formation of drug-induced cleavable complexes is responsible for most if not all of the drug effects, including cell death (1-10). Based on the mechanism of the interaction between topoisomerases, DNA, and the topoisomerase targeting drugs, we might predict that one factor determining the cytotoxicity of these particular antitumor drugs should be the cellular level of the topoisomerase in question. Differing from many other cytotoxic drugs, a high cellular level of topoisomerase should correlate with greater cytotoxicity for the topoisomerase targeting drugs. This suggests that the cellular regulation of topoisomerases, the drug target, might be an important factor in the efficacy of a drug treatment. Earlier studies have already shown that the activity of DNA topoisomerase II is significantly affected by the growth state of the cell (17-15). The mouse erythroleukemia cell appears to be a pro- erythroblast which has been transformed by the Friend virus complex (16-18). When various types of chemical agents are added, MELC can be induced to undergo a program of differentiation which is similar in many ways to normal erythropoiesis (/9-22). Using the MELC as a model system, we have studied the effect of cellular growth and differentiation upon the level and activity of DNA topoisomerases I and II. MATERIALS AND METHODS Cells and culture techniques.—MELC, clone 745 of C. Friend (purchased from N.I.G.M.S., #GM0086D) were cultured in MEM medium, supplemented with 2 mM glutamine, antibiotics, and 15% heat-inactivated fetal calf serum, at 37°C. To induce differentiation, log phase cells were pelleted and resuspended in MEM medium containing 2 mM glutamine, 15% heat-inactivated fetal calf serum, and 5 mM HMBA at a cell concentration of 1.5X 10° cells/ml. The progress of differentiation was monitored using a benzidine staining assay (21). To monitor growth, live cells (determined by trypan blue exclusion) were carefully counted using a hemacytometer. Extract preparation.—Cells (107) were centrifuged at 200 X g and washed twice with PBS. The cell pellet was resuspended in 188 ul cold, low salt buffer (20 mM Tris, pH 8.0, 5 mM KCl, | mM MgCl, | mM DTT, 1 mM PMSF, 20 mM NaHSOs, 0.5 pg aprotinin) and incubated on ice for ten minutes. The cells were homogenized using fifteen strokes with a tight fitting Dounce homogenizer. The nuclei and cytosol were incubated thirty minutes at 0°C, then centrifuged for three minutes at 15,000 X g. The supernatant was saved. The nuclei were resuspended in 188 ul of cold, high salt buffer (low salt buffer containing 0.35 M NaCl) and incubated for 80 minutes at 0°C. The nuclei were 31 centrifuged for 5 minutes at 15,000X g. The supernatant was saved. The cell extract used in immunoblots and enzyme assays was the combined cytosolic and nuclear supernatants. The extracted nuclei contained less than 5% of the total topoisomerase protein as measured by immuno- blotting. Immunoblotting.— Immunoblotting was done as de- scribed previously (23). P4 Unknotting assay.—The catalytic activity of topo- isomerase II was monitored using the P4 unknotting assay (24). Reactions (20 ul each) containing 50 mM Tris, pH 7.5, 100 mM KCl, 10 mM MgCl,, 1 mM ATP, 0.5 mM DTT, 0.5 mM EDTA, 30 ug/ml BSA, 0.5 ug knotted P4 DNA, and 0.05, 0.2, 0.5, 1.0, or 2.0 ul of cell extract (serial dilutions) were incubated at 37°C for thirty minutes. The reaction was stopped by the addition of 5 ul of 5% sarkosy, 50 mM EDTA, 25% sucrose, and 0.05% bromophenol blue. Reaction products were analyzed by electrophoresis, using a 0.79% agarose gel run in TBE buffer. Plasmid relaxation assay.—The catalytic activity of topoisomerase I was monitored using a plasmid relaxation assay. Reactions (20 ul each) containing 50 mM Tris, pH 7.5, 100 mM KCl, 10 mM MgCl, 0.5 mM DTT, 0.5 mM EDTA, 30 ug/ml BSA, 0.5 ug supercoiled plasmid DNA, and 0.0004, 0.002, 0.01, 0.05, or 0.2 ul of cell extract (serial dilutions) were incubated at 37°C for thirty minutes. The reaction was stopped by the addition of 5 ul of 5% sarkosyl, 50 mM EDTA, 25% sucrose, and 0.05% bromophenol blue. Reaction products were analyzed by electrophoresis using a 19% agarose gel run in TBE buffer. Immunofluorescence.—MELC (2.5X 10° cells/sample) were pelleted and washed twice with PBS. Cells were fixed in 39% formaldehyde at room temperature for fifteen minutes, then washed three times with cold PBS. Perme- abilization was accomplished in 0.25% NP40, 0.25% Triton ABCDE FGHIJK LMN FIGURE 1.—Immunoblot of cell extracts from HMBA induced MELC using anti-topoisomerase II antisera. MELC were induced to differen- tiate by the addition of 5 mM HMBA. 107 cells were removed daily for extract preparation as described in Materials and Methods. Extracts (from 5.3 X 103 cells for each sample) from day 1 through day 5, lanes A-E, respectively, and lanes G-K, respectively, were electrophoresed on a 7.5% SDS gel. Lane F: the extract from log phase, control cells. Lanes L-N: 100, 200, and 400 ng of purified HeLa topoisomerase II, respectively. Electrophoretic transfer and immunoblotting, using rabbit anti-topoisomerase II IgG, were done as described previously (25). The medium (including 5 mM HMBA) was changed every 24 hours, from day 2-5, in the cell sample shown in lanes G-K, whereas the medium was not changed in the cell sample used in lanes A-E. 32 9 Ul ‘|oAeT |] 0dO] 10 8AlISO4 dulpizuag Cell Growth (cell / ml X 109%) Days FIGURE 2.—The change in topoisomerase II levels during MELC differentiation. MELC were induced to differentiate as described in Figure 1. The cell concentration (dotted line) and degree of differentia- tion (solid line) were determined daily as described in Materials and Methods. The level of topoisomerase II (dashed line) was determined from densitometry tracings of lanes A-F of the immunoblot from Figure 1. X-100 for five minutes at room temperature. Following two washes in cold PBS containing 10 mM glycine (pH 7.2), the first antibody, rabbit anti-topisomerase II, was added at 30 ug/ml in PBS containing 0.1% Triton X-100, 10 mM glycine, 10 mM BSA. After a fifteen-minute incubation at room temperature, the cells were washed three times in cold PBS containing 10 mM glycine. Incubation with rhodamine conjugated goat anti-rabbit IgG (30 ug/ml) was done for fifteen minutes at room temperature in PBS, 0.25% Triton X-100, 10 mM glycine, and 10 mM BSA. After three washes with cold PBS containing 10 mM glycine, the cells were observed under the microscope. RESULTS Murine erythroleukemia cells, in log phase growth (1.5 X 10° cells/ ml), were induced to differentiate by the addition of 5 mM HMBA. Sixteen hours after the addition of HMBA, and every twenty-four hours thereafter, cell sam- ples were removed. At each time point, the cell number and the degree of differentiation were determined. In addition, a cell extract was made at each time point. The cell extracts were used for topoisomerase I and II activity assays and for immunoblot analysis. During the differentiation of MELC, the level of topoisomerase II dropped to 5-10% of the original level observed in control, log phase, undifferentiated MELC (Figure 1). This drop is shown along with the change in cell concentration and % differentiation in Figure 2. Concomi- tantly, there was a greater than ten fold drop in the topoisomerase II catalytic activity observed in the whole cell extracts (Figure 3). Topoisomerase II activity was monitored in an assay which measures the ability to convert knotted P4 DNA circles into simple monomers. In these experiments, 95% of the cells underwent terminal differentiation. Since the level of topoisomerase II protein and activity dropped by approximately the same amount, we wished to determine if the remaining topoisom- erase II was localized in those cells which remained NCI MONOGRAPHS, NUMBER 4, 1987 CONTROL DAY | DAY 2 DAY 3 ABCDEABCDEABCDE ABCDE LAL Aad LEE al EL LR 1 1 P4 circle P4 knots DAY 4 DAY 5 ry ABCDE ABCDE F P4 circle P4 knots FIGURE 3.—The unknotting activity of DNA topoisomerase II decreases during MELC differentiation. MELC were induced to differentiate and cell extracts were made as described in Figure 1. The extracts, made from unfed cells, were assayed using the P4 unknotting assay described in Materials and Methods. Lanes A-E: serial dilutions of the extracts (made from each day) with final concentrations of 2.0, 1.0, 0.5, 0.1, and 0.05 ul extract per 20-ul reaction, respectively. Control extract was made from log phase, undifferentiated cells. Lane F is knotted P4 DNA without extract. undifferentiated. Indirect immunofluorescence, using rho- damine conjugated goat anti-rabbit IgG, was done on differentiating MELC. The immunofluorescence of day five differentiated MELC, shown in Figure 4D, is obviously quite diminished relative to the control, undifferentiated, log phase cells, shown in 4B. Although not observable in Figure 4, due to the small size of the photographed field, in FIGURE 4.—Indirect immunofluorescence of MELC during differentiation. MELC were induced to differentiate as described in Figure 1. Cells were removed daily and prepared for viewing by indirect immunofluorescence, using rabbit anti-topoisomerase 11 IgG, as described in Materials and Methods. Anti-topoisomerase II immunofluorescence of control, log-phase cells is shown in 4B; day five differentiated cells in 4D; and their corresponding bright fields in 4A and 4C, respectively. DNA TOPOISOMERASES IN CANCER THERAPY addition to the lowered immunofluorescence of the general population on day five of differentiation, there remained about 59% of the cells which retained their bright topoisom- erase II immunofluorescence in the nuclei. This result suggests that the small amount of topoisomerase II protein and activity remaining in the population after differentia- tion may be attributed to those cells which remained undifferentiated and that the level of topoisomerase II in a differentiated cell may be significantly lower than 109% of its control level. The level of topoisomerase 1 was measured simultane- ously in the same experiments. The immunoblot (Figure 5) showed a small drop, 20%-30%, in the overall level of topoisomerase I, which correlates with the simultaneous drop in cell volume that occurred as MELC were induced to differentiate. The small change in topoisomerase I catalytic activity agreed with the Western blot analysis (data not shown). To determine if the large change in topoisomerase II level which occurred during MELC differentiation was the result of cellular differentiation or was merely a reflection of the change in the growth state of the cells, we monitored the level and activity of topoisomerases I and II during the growth of undifferentiated MELC. As the cells approached stationary phase, the topoisomerase I level remained constant while the topoisomerase II level dropped about two fold (Figure 6). Topoisomerase 1 and II activity measurements, determined concomitantly, agreed with the protein level results (data not shown). Topoisomerase II immunofluorescence showed little change between growing, 33 log phase cells and stationary phase cells (data not shown). These results indicate that a portion of the large drop in the topoisomerase II level observed during MELC differentia- tion may be due to the change in the cells’ growth state, but that much of the drop is a function of the process of differentiation. DISCUSSION DNA topoisomerase II is an abundant nuclear protein in proliferating cells. Based on immunological crossreactivity of human and mouse topoisomerase II, we established that approximately one million topoisomerase II molecules were present in a mouse erythroleukemia cell or HeLa cell in logarithmic phase of growth. Previous studies and unpublished results from our laboratory have shown that topoisomerase II is a nuclear marker for cell proliferation. The level of topoisomerase II in nontumorigenic cells is particularly sensitive to mitogen stimulation, serum stimu- lation, and contact inhibition [(//-15); unpublished results]. Our present study has demonstrated that the topoisomerase II level is also sensitive to the process of cellular differentia- tion. The immunoblotting experiment showed that the topoisomerase II level decreased gradually during the time course of MELC differentiation. At day five, only 5-109 of the topoisomerase II remained. Since 5% of the MELC remained undifferentiated at day five, the residual topo- isomerase II may be present mostly in the undifferentiated population. Studies using indirect immunofluorescence have shown that approximately 5% of the MELC showed strong topoisomerase II immunofluorescence in the nucleus at day five. It thus seems possible that the level of topoisomerase II in differentiated MELC is much less than 5% of its control level. The decrease in topoisomerase II level may either be a required step during MELC differentiation or be the result of differentiation. Although we could not distinguish between these two possibilities, we have noticed from the immunofluorescence study that the topoisomerase II im- munofluorescence in cells which were induced to differen- tiate dropped gradually. Thus, if the decrease in topo- isomerase II level is a necessary step during MELC differentiation, the decrease does not have to be extensive. ABCDEF GHI 100 K 66 K a i FIGURE 5.—Immunoblot of topoisomerase I in cell extracts from HMBA induced MELC. The cell extracts used in lanes A-F of Figure 1 were immunoblotted with rabbit anti-topoisomerase I IgG. Lane A: control, log phase cells; lanes B-F: cells from day 1-5 of differentiation, respectively; lanes G-K: 100, 200, and 400 ng of purified HeLa topoisomerase I, respectively (both 100K and proteolyzed 66K forms are present). 34 T T T T T 3 po < —100 © \, Oo [To lL \, ® — 0 N\, ue \, .* ~ > \ or ob 2 CHE Ls | E ~% = 20 A | 2 Ko) ° “a, 60 oO . vu — = y ., 3 3 “. w 5 3 o =) = 2 10+ 2 = Oo . 2 =z Ff 20 @ O .® i oe" 5 1 i I 1 ° 0 7 2 3 4 = Days FIGURE 6.—Topoisomerase levels during MELC growth. MELC were seeded in MEM complete medium at 1.5 X 10° cells/ml. Cell number (dotted line) was determined and cell extracts were prepared daily as described in Methods. Anti-topoisomerase I and II immunoblots were done as described in Materials and Methods. Densitometry tracings of these immunoblots were used to determine the levels (in percent) of topoisomerase I (solid line) and topoisomerase 11 (dashed line). The levels of topoisomerases I and II were the same for day 0-1. During this study, we have also observed that the level of DNA topoisomerase II in uninduced MELC decreased twofold during the five-day period of growth. The decrease could be due to either the increase in cell density or the exhaustion of the nutrients in the growth medium. Since the level of topoisomerase II still dropped twofold with daily changes of the growth medium, it seemed possible that the high cell density was responsible for the drop in the topoisomerase II level. In contrast, DNA topoisomerase I is largely unaffected by either the growth state or the differentiation status of MELC. Our present study suggests that DNA topoisom- erase II is a specific nuclear marker for cell proliferation and differentiation. The exact role of topoisomerase II during growth and differentiation awaits further studies. REFERENCES (I) HSIANG YH, HERTZBERG R, HECHT S, et al: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem 260:14873-14878, 1985. (2) NELSON EM, TEWEY KM, Liu LF: Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomerase II on DNA by 4’-(9-acridinylamino)-methanesulfon-m- anisidide. Proc Natl Acad Sci USA 81:1361-1365, 1984. (3) TEWEY KM, CHEN GL, NELSON EM, et al: Intercalative antitumor drugs interfere with the breakage/reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:9182-9187, 1984. (4) TEWEY KM, ROWE TC, CHEN GL, et al: Adriamycin- induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466-468, 1984. (5) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative antitumor drugs interfere with the breakage/reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1984. (6) Ross W, ROWE TC, GLISSON B, et al: Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleav- age. Cancer Res 44:5837-5860, 1984. (7) YANG L, ROWE TC, NELSON EM, et al: In vivo mapping of NCI MONOGRAPHS, NUMBER 4, 1987 DNA topoisomerase II-specific cleavage sites on SV40 chromatin. Cell 41:127-132, 1985. (8) YANG L, Rowe TC, Liu LF: Identification of DNA topoisomerase as an intracellular target of antitumor epipodophyllotoxins in simian virus 40-infected monkey cells. Cancer Res 45:5872-5876, 1985. (9) Rowe TC, CHEN GL, HSIANG YH, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (10) POMMIER Y, SWARTZ RE, KOHN KW, et al: Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic inter- calating agents. Biochemistry 23:3194-3201, 1984. (11) TAuDOU G, MIRAMBEAU G, LAVENOT C, et al: DNA topoisomerase activities in concanavalin A-stimulated lymphocytes. FEBS Lett 126:431-435, 1984. (12) Tricor! JV, SAHAI BM, McCorMICK PJ, et al: DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells. Exp Cell Res 158:1-14, 1985. (13) MiSKIMINS R, MISKIMINS WK, BERNSTEIN H, et al: Epidermal growth factor-induced topoisomerase(s). Exp Cell Res 146:53-62, 1983. (14) GALLO RC, WHANG-PENG J, ADAMSON RH: Studies on the antitumor activity, mechanism of action, and cell cycle effects of camptothecin. J Natl Cancer Inst 46:789-795, 1971. (15) DUGUET M, LAVENOT C, HARPER F, et al: DNA topoisom- erases from rat liver: Physiological variations. Nucleic Acids Res 11:1059-1075, 1983. DNA TOPOISOMERASES IN CANCER THERAPY (16) FRIEND C, PATULEIA MC, DEHARVAN E: Erythrocyte maturation in vitro of murine (Friend) virus-induced leukemia cells. Natl Cancer Inst Monogr 22:505-520, 1966. (17) TAMBOURIN PE, WENDLING F: Malignant transformation and erythroid differentiation by polycythaemia inducing Friend virus. Nature (New Biol) 234:230-233, 1971. (18) TAMBOURIN PE, WENDLING F: Target cell for oncogenic action of polycythaemia-inducing Friend virus. Nature 256:320-322, 1975. (19) RIFKIND RA, MARKS PA: Erythroleukemic differentiation. Annu Rev Biochem 47:419-448, 1978. (20) FRIEND C, SCHER W, HOLLAND JG, et al: Hemoglobin synthesis in murine virus-induced leukemic cells in vitro: Stimulation of erythroid differentiation by dimethyl sulf- oxide. Proc Natl Acad Sci USA 68:378-382, 1971. (21) ScHER W, FRIEND C: Breakage of DNA and alterations in folded genomes by inducers of differentiation in Friend erythroleukemic cells. Cancer Res 38:841-849, 1978. (22) REUBEN RC, WIFE RL, BRESLOW R, et al: A new group of potent inducers of differentiation in murine erythroleu- kemia cells. Proc Natl Acad Sci USA 73:862-866, 1976. (23) HALLIGAN BD, EDWARDS KA, Liu LF: Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. J Biol Chem 260:2475-2482, 1985. (24) Liu LF, Davis JL, CALENDAR R: Novel topologically knotted DNA from bacteriophage P4 capsids: Studies with DNA topoisomerases. Nucleic Acids Res 9:3979- 3989, 1981. 35 givgd 101 Tt i 3 Si jana ie al Eder ead _ bt : 3 MLN Be Te i re iE a gt aC mgm Lo! a al jg iy ides «i ii ir of of yt RR fs er Na pus fs Ag ar Lyall 1% ’ D - Ne dbs an i aie marly Joe) yt yar hat . lla ich Je Tis er by al i bi ‘ as B iy =f ng 5 » Eh. 3 : Hi i ih bin lg Zh k Taha ia oa fi ) 3 EF pT Cad ae. «Ff pid Lh of 51 rE aps io pt yg fo ntuts SA v 0 sd i Ny $= 1 wk A: 3 3 wu Hr ar Hh ARH a pi ee . SECT Hy th a } hota ; Thay fn ro iq ik, Spd EE : me ra or gl TH {od an Sedrgld Is Co = ol Cy B dl SEL Fri oe En a rs tay 1b” 5 SH i pt x 1G dn 5 she K 43 ey JAAR EE tHE hid ary i Selly Die, = opt fe Ly wh Le Crs fs 3 Wy : al. a) Ee a. 3 = Ie a " aw i, gl Se ~ i ak se ray 5d . as 5 gt he 2 = AR at R x i ihn hint Emr as : Ja a i I" 2 hy - r= at oe fi bp ie iit i 2 te ul Elly ne i fi [a hs Ag i = = Toe ei Re fil Waar Sn 8 gti EE 2 3 an Tye - J Cab Ei kf A apt #1 E Li RETR o I ie IP i ot i) : Ei wu & Si i Me on bf S10 1 eis . oR a iu hil ll eb Ned o ew He ody Ly i ph i i + Thal BLT Samiti = yi i a defn a EO pet seh ] PE - B RES AF yt E i Ee n nl a Boma Ev JR he pal Lg Wet 3 ne PEE ey fo = Pe h Hay fF - f= B Wh oo n'y Sin LE a, ¥ i et CS NA Aoi Lh de) er 5) 3 Erie re iy = ES 3 He RED es ei i & i pS Fk yr I 48 - att of lat rd ks ¥ = i ) = wi Ci EH i riipagh ii Jy ta alta, fry He hy LE ly. os = Se bh J | By a = n a 3 Fe ag tox . ft FS ag A _ Hi ST MEL nto il ETRE 1s aul 4 tn 1 " a Share . x = 7 fs : . bt Flair Rie oT Ak ok in ath a La pr oy Ha he is hi og A. i Bk ae fd . ns rh i is a ey ire RA ho off i : is fe al i ie a a i, shied ticks Ty. j Tididgtan Sal £5 BT foe Tiny Tay } Ye ¥ i ate PAE ne “ie, 1 Fi fren ; = Bf gion mac Fel ki : aa Es Lal ats a Bll ilo vider i Sel. Lali 6m ie IF i ing + i = h El § Involvement of Intracellular ATP in Cytotoxicity of Topoisomerase ll-targetting Antitumor Drugs Gary Kupfer, Annette L. Bodley, and Leroy F. Liu?* ABSTRACT —The effect of the ATP pool on the cytotoxic action of teniposide (VM-26) has been studied in mouse leukemia cells (L1210). L1210 cells in tissue culture were treated with VM-26 (10 uM) in the presence of DNP, an uncoupler of oxidative phosphorylation. The simultaneous treatment of DNP (1 mM) increased cell survival 100-200 fold. Pre- or post-treatment with DNP had little effect on cell survival. Other uncouplers and in- hibitors of ATP synthesis had effects similar to DNP. The interfer- ence of DNP with the cytotoxic action of VM-26 was also seen with another topoisomerase II-targetting drug, m-AMSA, but not with the topoisomerase I-targetting drug camptothecin. Studies using either purified topoisomerase II or cultured mammalian cells had shown that DNP had little effect on the amount of cleav- able complexes induced by VM-26. We propose that an ATP re- quiring process(es) which occurs subsequent to the formation of the cleavable complexes is involved in the cytotoxic action of topoisomerase II—targetting drugs.—NCI Monogr 4:37-40, 1987. INTRODUCTION DNA topoisomerase II has recently been identified as an intracellular target for a number of clinically important antitumor drugs such as adriamycin, m-AMSA, ellipticine, actinomycin D, mitoxantrone, bisantrene, and epipodo- phyllotoxins (VP-16 and VM-26) [reviewed in (/)]. These drugs affect the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing an abortive topoisom- erase II-DNA cleavable complex (2-6). The cleavable complexes are reversible. However, the exposure of the cleavable complexes to strong protein denaturants results in protein-linked DNA breaks, in which a topoisomerase II subunit is linked to each 5’-phosphoryl end of the broken DNA strand via a tyrosyl phosphate bond (6). While all evidence indicates that drug-induced cleavable complexes ABBREVIATIONS: VM-26 (teniposide)=4’-demethylepipodophyl- lotoxin thenylidine-B-D-glucoside; m-AMSA =4'-(9-acridinyl- amino)-methanesulfon-m-anisidide; DNP = dinitrophenol; FCCP =carbonyl cyanide (trifluoromethoxy)phenylhydrazone; DBP= dibromophenol; CCCP=meta-chloro carbonyl cyanide phenyl- hydrazone; SDS =sodium dodecyl sulfate. I'Supported by Public Health Service grants CA-39962 and CA-40884 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. L. F. Liu is a recipient of the American Cancer Society Faculty Research Award. 2 Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD. * Reprint requests: Leroy F. Liu, Ph.D., Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. are the primary lesion responsible for drug cytotoxicity and the antitumor activity, little is known about the cell killing mechanism associated with the formation of the reversible cleavable complexes. The bactericidal action of the antibiotic, nalidixic acid, and its quinolone analogs serves as a prototype for the study of the cell killing action of topoisomerase II-targetting antitumor drugs. Nalidixic acid targets at the GyrA subunit of E. coli DNA topoisomerase II (DNA gyrase) and inter- feres with the breakage-reunion reaction of DNA topo- isomerase II by stabilizing an abortive enzyme-DNA cleav- able complex [reviewed in (7)]. Genetic and biochemical studies have indicated that the nalidixic acid-induced cleavable complex is responsible for the bactericidal action of nalidixic acid [reviewed (8,9)]. Nalidixic acid selectively kills growing bacterial cells, and its bactericidal action requires RNA and protein synthesis (10). The metabolic inhibitor, DNP, also effectively abolishes the bactericidal action of nalidixic acid (10). It appears that active cell metabolism is necessary for the bactericidal action of nali- dixic acid. To test whether active cell metabolism is sim- ilarly required for the cytotoxic action of topoisomerase II-targetting antitumor drugs, we have investigated the role of the ATP pool in the cytotoxicity of VM-26. MATERIALS AND METHODS Enzymes and drugs.—HelLa DNA topoisomerase II was purified to homogeneity by a modification of the previously published procedure (11). VM-26 (teniposide) was a gift from Bristol-Myers Co. m-AMSA (NSC 249992) and camptothecin (NSC 94600) were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. DNP, FCCP, and firefly luciferin-luciferase were purchased from Sigma Co. Cell culture and SV40 infection.—1L1210 cells were main- tained in Fischer’s medium supplemented with 10% fetal calf serum and antibiotics. Cell survival was assayed by colony formation in soft agar. BSC-1 cells were maintained in MEM supplemented with 10% fetal calf serum and anti- biotics. BSC-1 cells were infected with SV40 (strain 776) at an m.o.i. of 10 p.f.u/cell. Total DNA was isolated by SDS lysis followed by proteinase K treatment as described ©). Measurement of intracellular ATP concentration.—The luciferin-luciferase assay was used to measure the ATP concentration in L1210 cells (12). RESULTS To test whether DNP can protect cells from VM-26 cyto- toxicity, L1210 cells were treated with VM-26 (10 uM, one 37 00 La tith oo £ > 0.00 E = Oo = —] a A © =~ 3 © = oO § 0.0001 2 Qo od [re 0.00001 © Uglied bl 250 500 1000 DNP Dose (um) FIGURE 1.—Dinitrophenol co-treatment reduces VM-26 cytotoxicity in L1210 cells. L1210 cells were treated with 10 uM VM-26 for one hour in the presence of various amounts of DNP. After treatment, cells were washed, serially diluted, and seeded into soft agar. Visible colonies were counted two weeks later. Surviving fraction of cells were calculated relative to respective controls with DNP alone. The plating efficiencies of the control cells treated with DNP only were: no DNP, 50%; 250 uM DNP, 57%; 500 uM DNP, 69%; 1 mM DNP, 52%). hour) in the presence of various concentrations of DNP (Fig. 1). Dose dependent increase of cell survival as moni- tored by colony formation was observed. At | mM DNP, there was a 180-fold increase of cell survival. The effect of DNP was also observed with another topoisomerase II- targetting drug, m-AMSA. DNP (1 mM) increased cell survival 80-fold in the presence of 10 uM m-AMSA. The effect of DNP appeared to be specific for DNA topoisom- erase II-targetting drugs: Cell survival of camptothecin (a topoisomerase I-targetting drug)-treated L1210 cells was unaffected by the simultaneous treatment with DNP (Fig. 2). To investigate whether the reduction of the intracellular ATP pool is responsible for the observed protective effect of DNP, another uncoupler of oxidative phosphorylation, FCCP, an inhibitor of oxidative phosphorylation, sodium cyanide, and an inhibitor of glycolysis, 2-deoxyglucose, 1.0 -© o on Surviving Fraction Cells 0.l I T 10 oO Camptothecin (uM) FIGURE 2.—Dinitrophenol co-treatment does not alter camptothecin cyto- toxicity in L1210 cells. L1210 cells were treated with 1 or 10 uM camp- tothecin (lactone form) and 1 mM DNP as described in Fig. 1 legend. Solid circle: camptothecin + DNP. Open circle: camptothecin only. 38 TABLE 1.—Metabolic inhibitors reduce VM-26 cytotoxicity in L1210 cells? DNP FCCP 2-Deoxyglucose NaCN — Inhibitors (1 mM) (100 pM) (20 mM) (1 mM) Relative survival 183 59 29 74 1 4 L1210 cells were treated with 10 uM VM-26 in the presence of various metabolic inhibitors. The relative survival of VM-26 treated L1210 cells in the absence of any metabolic inhibitors was taken as 1. were also tested for their protective effect against VM-26 cytotoxicity. As shown in Table 1, DNP (1 mM), FCCP (100 uM), 2-deoxyglucose (20 mM), and sodium cyanide (1 mM) reduced VM-26 cytotoxicity 183, 59, 29, and 74- fold, respectively. The effect of DNP on the intracellular ATP level in L1210 cells was also measured by the luciferin- luciferase assay. | mM DNP reduced the intracellular ATP pool approximately threefold. It has recently been demonstrated that heat-shock can protect cells from VM-26 cytotoxicity (G. Li, personal communication). Since DNP is known to be a heat-shock inducer (3), the protective effect of DNP might be the result of a heat-shock response. In order to test this hypothesis, L1210 cells were preincubated with DNP for various times and then tested for VM-26 cytotoxicity. As shown in Table 2, preincubation of L1210 cells with DNP had little effect on VM-26 cytotoxicity. Neither did incuba- tion of L1210 cells with DNP after VM-26 treatment have any protective effect on VM-26 cytotoxicity (data not shown). The fact that simultaneous treatment with DNP can maximally protect cells from VM-26 cytotoxicity ar- gued against heat-shock induction as the basis for the ob- served protective effect. Since the cleavable complex is most likely the critical lesion responsible for VM-26 cytotoxicity, the possibility that DNP might affect the formation of topoisomerase II- DNA cleavable complexes induced by VM-26 was tested both in cultured L1210 cells and SV40 virus-infected BSC-1 cells. In cultured L1210 cells, DNP (1 mM) had less than a two-fold effect on the number of protein-linked breaks as measured by K-SDS precipitation method (data not shown). In SV40 virus-infected BSC-1 cells, DNP had no detectable effect on the amount of SV40 form III (linear) DNA induced by VM-26 (Fig. 3). These experiments thus suggest that DNP neither prevents VM-26 from reaching the target enzyme, DNA topoisomerase II, nor interferes with the formation of abortive VM-26-topoisomerase II-DNA cleavable complexes. TABLE 2.—Preincubation with DNP does not protect L1210 cells from VM-26 cytotoxicity? Post-incubation time 0 hr 1 hr 4 hrs Relative survival 1 1 9 4 L1210 cells were treated with 1 mM DNP for one hour and then post-incubated in drug-free medium for 0, 1, and 4 hours before the addition of 10 uM VM-26 (one hour). Cell survival was assayed by colony formation in soft agar. Relative survival of VM-26 treated L1210 cells (no DNP) was taken as 1. NCI MONOGRAPHS, NUMBER 4, 1987 DISCUSSION Our present results suggest that the protective effect of DNP with respect to VM-26 cytotoxicity is most likely due to a reduction of the intracellular pool of ATP. It is in- teresting that a two- to three-fold reduction of the intra- cellular ATP pool can lead to more than one hundred-fold increase in cell survival. The ATP-dependent process(es) that is affected by DNP treatment must be highly sensitive to changes in the intracellular ATP pool. The protective effect of DNP with respect to drug cyto- toxicity has been reported in both prokaryotic and eukary- otic systems. The protective effect of DNP against the bac- tericidal effect of nalidixic acid is probably specific for E. coli B strain and Lon deficient K strains. E. coli B strain (naturally deficient in Lon protease) rapidly loses its viabil- ity upon exposure to nalidixic acid. The rapid cell killing by nalidixic acid is primarily due to S.O.S. induction in the ab- sence of Lon protease [reviewed in (/4)]. Lon protease nor- mally controls SulA, an inducible S.0.S. protein which in- teracts with SulB protein to inhibit normal cell division (/4). In the absence of Lon protease, S.0O.S. induction leads to sustained accumulation of SulA, which causes filamentous growth and rapid cell death (/4). The protective effect of DNP against the bactericidal action of nalidixic acid in E. coli B strain may be due to a block in S.0.S. induction, which requires a notably high ATP pool (15). The protective effect of DNP in X-irradiated mamma- lian cells has also been reported. Uncouplers of oxidative phosphorylation such as DNP, DBP, and CCCP, at doses that decrease the intracellular ATP level, can increase the survival of X-irradiated mammalian cells (16,17). How- ever, the mechanism of this protective effect is not known. Heat-shock proteins are known to increase the survival of VM-26-treated CHO cells (G. Li, personal communica- tion). Since DNP is also known to be an inducer of the heat-shock response, the protective effect of DNP against VM-26 cytotoxicity might be due to an induced heat-shock response. The fact that maximal protection of cells from VM-26 requires co-treatment with DNP argues against heat-shock induction being responsible for the observed protection. Pre-treatment of cells with DNP for up to four hours had much less effect than the co-treatment. We suggest that DNP may affect an ATP-requiring process(es) ABCDEFGH WT] MY Cellular DNA SV40 DNA(I) " (1 2 1h) FIGURE 3.—Dinitrophenol does not alter the amounts of VM-26 induced linear SV40 DNA in SV40 virus-infected BSC-1 cells. SV40 virus- infected BSC-1 cells were treated with 100 uM VM-26 in the presence of 250, 500, and 1 mM DNP. SV40 DNA was isolated and loaded onto an 0.89% agarose gel. Lanes A-D: controls with 0, 250 uM, 500 uM, and 1 mM DNP, respectively. Lanes E-H: same as lanes A-D, respectively, except that 10 uM VM-26 was present in each sample. DNA TOPOISOMERASES IN CANCER THERAPY CELL DEATH FIGURE 4.—A working model for the possible involvement of ATP in the cytotoxic action of topoisomerase II-targetting antitumor drugs. which directly or indirectly mediates the cell killing signal initially produced by the reversible cleavable complexes. A schematic diagram of our working model is shown in Fig. 4. Further studies are necessary to delineate steps sub- sequent to the drug-induced, reversible topoisomerase II-DNA cleavable complex which is the key lesion induced by many topoisomerase [I-targetting antitumor drugs. REFERENCES (I) CHEN GL, Liu LF: DNA topoisomerases as therapeutic targets in cancer chemotherapy. Annu Rep Med Chem 21:257-262, 1986. (2) NELSON EM, TEWEY KM, Liu LF: Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisom- erase II on DNA by 4’-(9-acridinylamino)-methanesulfon- m-anisidide. Proc Natl Acad Sci USA 81:1361-1365, 1984. (3) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative anti- tumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1984. (4) YANG L, NELSON EM, Liu LF: In vivo mapping of DNA topoisomerase Il-specific cleavage sites on SV40 chro- matin. Cell 41:127-132, 1985. (5) YANG L, Rowe TC, Liu LF: Identification of DNA topo- isomerase II as an intracellular target of antitumor epipo- dophyllotoxins in simian virus 40 infected-monkey cells. Cancer Res 45:5872-5876, 1985. (6) Rowe TC, CHEN GL, HSIANG YH, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (7) GELLERT M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981. (8) Goss WA, Cook TM: Nalidixic acid—mode of action. In Antibiotics, vol 3. Berlin: Springer-Verlag, 1975, pp 174-196. (9) PEDRINI AM: Nalidixic acid. In Antibiotics, vol 5. Berlin: Springer-Verlag, 1979, pp 154-175. (10) DEeitz WH, Cook TM, Goss WA: Mechanism of action of nalidixic acid on Escherichia coli. 111. Conditions required for lethality. J Bacteriol 91:768-773, 1966. (11) HALLIGAN BD, EDWARDS KA, Liu LF: Purification and characterization of a type II DNA topoisomerase from bovine calf thymus. J Biol Chem 260:2475-2482, 1985. (12) LEASTERS JJ, HACKENBROCK CR: Firefly luciferase assay for ATP production by mitochondria. In Methods in Enzymology (DeLuca MA, ed), vol 57. New York: Aca- demic Press, 1978, pp 36-50. 39 (13) RiTOSSA F: A new puffing pattern induced by heat shock and DNP in Drosophila. Experientia 18:571-573, 1962. (14) DRLICA K: Biology of bacterial deoxyribonucleic acid topo- isomerases. Microbiol Rev 48:273-289, 1984. (15) BARBE J, VILLAVERDE A, CAIRO J, et al: ATP hydrolysis during SOS induction in Escherichia coli. J Bacteriol 167:1055-1057, 1986. 40 (16) DALRYMPLE GV, SANDERS JL, BAKER ML: Dinitrophenol decreases the radiation sensitivity of L cells. Nature 216:708-709, 1967. (17) LAVAL F, LITTLE JB: Enhancement of survival of X-irradi- ated mammalian cells by the uncoupler of oxidative phosphorylation, m-chloro carbonyl cyanide phenylhy- drazone. Radiat Res 71:571-578, 1977. In Vivo and In Vitro Stimulation by Antitumor Drugs of the Topoisomerase ll-induced Cleavage Sites in c-myc Proto-oncogene Jean-Francois Riou,’ Marie-José Vilarem,? Christian J. Larsen, ? Eric Multon,"* and Guy F. Riou"* ABSTRACT —Stimulation of topoisomerase II cleavage activ- ity by antitumor drugs with or without DNA intercalative ability has been tested in vivo on the c-myc proto-oncogene. Two human tumor cell lines (N417 and HL60 cells) were treated with mAMSA, OH-9-ellipticine, VM26, and BD-40 (an aza-ellipticine analog), and DNA breaks were mapped in the gene locus by Southern blot hybridization with c-myc probes. Most of the major cleavage sites induced in vivo by drugs in the 5’ end of c-myc were also observed in vitro using purified topoisomerase II and a c-myc gene DNA insert. Moreover, they closely mapped to some DNAse I hypersensitive sites, the presence of which reflects gene activity. DNA from drug treated cells probed with a human 3; globin pseudogene, and the c-mos proto-oncogene did not reveal topoi- somerase II cleavage bands, suggesting that topoisomerase II- mediated drug activity may correlate with gene activity.—NCI Monogr 4:41-47, 1987. INTRODUCTION A number of DNA intercalating agents currently used in cancer chemotherapy induce protein-linked single-strand and double-strand DNA breaks (/). Topoisomerase II (Topo II) has been demonstrated to be one major target of these drugs (2), which interfere with the Topo II-catalyzed breakage-reunion reaction, resulting in the blockage of the enzyme-DNA complex in its putative cleavage step. Sub- sequent treatment of such a complex with protein denatur- ing agents results in DNA strand breaks and in the covalent linking of one Topo II subunit to each 5’ phosphoryl end of the broken DNA strand (3). Other antitumor agents such as epipodophyllotoxins (VM26 and VP16), which do not intercalate DNA, have been shown to act by a similar mechanism (4). ABBREVIATIONS: Topo II=DNA topoisomerase II; DHS= DNAse 1 hypersensitive site; SDS=sodium dodecyl sulfate; EDTA =ethylenediaminetetraacetic acid; BSA=bovine serum albumin; mAMSA =4'-(9-acridinylamino)-methanesulfon-m-ani- sidide; 0 AMSA =4'-(9-acridinylamino)-methanesulfon-o-anisidide; VM26 =4'-demethyl epipodophyllotoxin tenylidene-B-D-gluco- side; NM HE = 2-methyl-9-hydroxyellipticinium; OH-9-E1=9 hy- droxyellipticine; BD-40 = 10-diethyl-aminopropylamino-6-meth- yl-5H pyrido 3’,4":4,5 pyrrolo 2,3-g isoquinoline. I Laboratoire de Pharmacologie Clinique et Moléculaire, Institut Gustave Roussy, Villejuif, France. 2U301 INSERM, Hopital Saint Louis, Paris, France. 3 Centre de Recherches du Service de Santé des Armées, Clamart, France. * Reprint requests: Dr. Guy F. Riou, Laboratoire de Pharmacologie Clinique et Moléculaire, Institut Gustave Roussy, 94800 Villejuif, France. Most of the studies aimed at the characterization of the protein-linked DNA lesions referred to the alkaline elution (1). While this method provides a convenient means for quantitative evaluation of lesions, it does not give any information on their precise location in genes. This objec- tive can be approached by use of Southern blot hybridiza- tion technique with DNA probes representing specific parts of the genes to be studied. In this work, we have studied the stimulation of Topo II by DNA intercalative or noninter- calative antitumor drugs on proto-oncogene c-myc locus, which plays important functions in the control of cell pro- liferation (5). Two human tumor cell lines containing an amplified c-myc gene were studied, and the in vivo results were compared to data from in vitro experiments with puri- fied Topo II and DNA substrate containing a complete c-myc gene. MATERIALS AND METHODS Cell lines and drugs.—Small cell lung carcinoma cell line NCI-N417 (6) and human promyelocytic leukemia cell line HL60 were grown in stationary suspension. mAMSA and oAMSA (Dr. B. Baguley, New Zealand), VM26 (Sandoz laboratory) were dissolved in dimethyl sulfoxide, OH-9-E1 (Dr. N. Dat Xuong) in 0.1 N HCI, BD-40 [Dr. Bisagni (7)], and NMHE (Dr. J. B. Le Pecq) in distilled water. In vivo drug activity.—Cells in exponential growth were exposed to drugs for 3 hours at 37°C in fresh medium without fetal calf serum. Cells were washed with 50 mM Tris, pH 7.9, | mM EDTA, and immediately lysed with 2% SDS, 50 mM Tris, pH 7.9, 20 mM EDTA. Proteinase K was added to final concentration of 1 mg/ml for 4 hours at 50°C. DNA preparation was performed as previously reported (8). Procedure for cell nuclei isolation and treat- ment with pancreatic DNAse I have been already detailed by Dyson et al (9). Cell DNA samples (2.5-10 ug) were digested to completion by Xbal or EcoRI restriction endo- nucleases and electrophoresed in horizontal 1.2% agarose slab gel as previously described (8). In vitro drug assay.— A \ phage clone (AK76) (10) con- taining a 15-kb fragment encompassing the whole human c-myc locus was used as a DNA substrate. The purification of calf thymus Topo II has been already described (11). The reaction mixture (20 ul) containing 20 mM Tris HCI, pH 7.9, 50 mM KCl, 10 mM MgCl,, 0.5 mM ATP, 0.5 mM EDTA, 0.5 mM dithiothreitol, 15 ug/ml of BSA, and 25 ng of AK76 DNA digested by either EcoRI or Xbal and Topo II (100 ng) was incubated at 37°C for 10 min. with drugs at various concentrations. The reaction was stopped by SDS- proteinase K. Hybridization and probes.—DNAs were analyzed by 41 PROBE B PROBE A : : 7: FIGURE l.—Map of the human c-myc : : : oncogene. The restriction map cor- : : i responds to the c-myc primary i : : structure determined by Gazin et : i ‘ al. (20). The three c-myc exons ; q ’ : tee T] ; OE Fl : were positioned in boxes 1, 2, and E X H cl NY ity X hp E 3. The probes A and B used for Gl hybridization are indicated at the Ch: Clal top of the figure. For clarity, not E: EcoRI 1Kb all the known restriction sites are H: Hind Ill er shown in the diagram. Pi Pvull X: Xbal Southern blot hybridization with human c-myc specific probes (figure 1). Probe A is the Clal- EcoRI fragment cor- responding to the third exon, and probe B is the Pvull fragment corresponding to the first exon. Procedures for 32P-labeling of probes (specific activity: 2-4 X 10% cpm/ug DNA), hybridization under stringent conditions and auto- radiography were as described by Maniatis et al. (12). RESULTS mAMSA and VM26 Induce in Vivo Topo Il Mediated DNA Breaks in the c-myc Proto-oncogene Locus of Small Cell Lung Carcinoma Line (NCI-N417) Cells in exponential phase of growth were treated with mAMSA and VM26 at doses previously shown to induce the formation of double-stranded DNA breaks in vitro and in vivo in c-myc proto-oncogene (8,11). Purified DNA preparations were digested to completion with Xbal endo- nuclease and analyzed by Southern blot hybridization using a c-myc probe encompassing the Ist exon (probe B, figure 1). Genomic DNA pattern from N417 cells revealed the presence of a germline 7.2-kb band (figure 2A, lane 1) containing the 1st exon and 5’ flanking sequences (see re- striction map of c-myc gene in figure 1). In contrast, DNAs from cells treated with VM26 (lanes 2-4) or mAMSA (lanes 5-7) presented several hybridization bands of smaller sizes reflecting Topo II cleavage reaction as previously shown with EcoRI restriction enzyme in c-myc gene (8). The intensity of these bands was dependent on drug concentra- tion (figure 2A) and/or duration of drug incubation (see FIGURE 2.—A) Cleavage sites induced in vivo by antitumor drugs in the amplified c-myc gene of N417 cells. DNA preparations (5 ug) from drug-treated N417 cells (3 hours) were digested with Xbal and analyzed by Southern blot hybridization using a 32P-labeled c-myc probe B (Ist exon). I, untreated cells; 2-4, VM26 treated cells (0.5, 2.5, and 5 pM); 5-7, mAMSA (0.5, 5, 20 uM); 8, 0AMSA (5 uM); 9, OH-9-El (10 uM). B) DNAse I hypersensitive sites determined in the c-myc gene of N417 isolated nuclei. DNA preparations (2.5 ug) were digested with Xbal and analyzed by Southern blot hybridization using 32P-labeled c-myc probe B (Ist exon). /, control nuclei; 2-4, nuclei treated for 5S min. at 37°C with 0.2, 0.5, and 1 ug/ml of DNAse I. DHS d is localized in c-myc Ist exon and generates two cleavage bands. DNA fragment sizes are in kilobase pairs (kb). The blots were exposed to Kodak XARS film for 3 days. 42 NCI MONOGRAPHS, NUMBER 4, 1987 © FT fritid mAMSA P X 1 L -3J y YMze FIGURE 3.—Mapping of the Topo II cleav- age sites and DHS in the 5’ end of c-myc locus. The first c-myc exon is positioned in box 1. The probe B used for hybridiza- tion is indicated at the top of the diagram. 7} TOPO I] A) Position of cleavage sites induced in | — ’ vivo by mAMSA in N417 cells. B) Posi- } tion of cleavage sites induced in vivo by VM26 in N417 cells. C) Position of cleav- age sites induced in vitro by calf thymus Topo II without any drug (11). D) Posi- OO F— DNase | tion of the DHS in N417 cell DNA. Site 4 N 417 lettered e (dotted arrow) has been deduced : from previous experiments (8). E) Posi- tion of the DHS determined in HL60 cell Iv In, Mn, MN, o : Pvu ll : Xball x figure 6A), indicating that stabilization of cleavable com- plexes was more efficient. Interestingly, oAMSA, an mAMSA isomer reported to be biologically inactive (13), did not induce as many bands when used at the same con- centration as mAMSA (compare lane 6 and 8 in figure 2A). This suggests that the biological activity of these drugs is indeed mediated by Topo II. The cleavage patterns obtained with mAMSA (figure 2A, lanes 5-7) and VM26 (figure 2A, lanes 2-4) revealed noticeable differences. On the other hand, most of the cleavage sites generated by both drugs were clustered in a DNA fragment located at 2 kb from the 5” end of the Ist c-myc exon (figure 3A and B). The use of Xbal restriction enzyme allowed us to distinguish three major cleavage sites. Due to the poor resolution of the gel, these sites could not be individualized in the EcoRI-DNA pattern [probe A, figure 1, and see (8)]. No cleavage site was detected in the Ist exon. DNA Breaks Mediated in Vivo by mAMSA and VM26 in the c-myc Gene of N417 Cells Correspond to a Subset of the Topo Il DNA Breaks Induced in Vitro Purified Topo II induces double-stranded DNA breaks in a complete human c-myc gene inserted in a A phage vector (11). mAMSA considerably stimulates the cleavage DNA TOPOISOMERASES IN CANCER THERAPY 8 DNA by Dyson et al. (9). 1} DNase | 11 HL 60 i, 1 Kb of sites located in the 5’ part of the Ist exon but does not qualitatively modify the pattern of cleavage. Comparison of in vivo and in vitro generated cleavage patterns (figure 3A, B, and C) shows that four of the cleavage sites induced in vivo by drugs map at the same (or very similar) sites as those obtained in vitro. The presence of additional sites in the in vitro pattern has been related to loss of chromatin structure that unmasks new DNA Topo II sites (11). OH-9-El and BD-40 (an Aza-ellipticine Analog) Do Not Induce c-myc Cleavage in N417 Cells Experiments were repeated using ellipticine derivative OH-9-El, NMHE, and an aza-ellipticine analog BD-40. All these drugs exhibit antitumor activity. No band was detected in the OH-9-El pattern (figure 2A, lane 9) as well as in the NMHE and BD-40 pattern (not shown). In a few cases, c-myc gene cleavage was detected but at a very low level as compared to that of mAMSA or VM26. Thus, these drugs were apparently inactive in N417 cells. In con- trast, OH-9-El appears to be active in vitro (figure 4). Some differences in the activities of OH-9-El and mAMSA can be detected: OH-9-El (figure 4A, lane 4) did not stimulate the cleavage of a site in the first intron (arrow 7) 43 and stimulated a site located in the 2nd exon (arrow 5, compare with the result obtained with mAMSA, lane 3). Finally in vitro cleavage patterns generated by NMHE and BD-40 were very similar to that of OH-9-El (data not shown). Topo Il Cleavage Sites Map Close to DNAse | Hypersensitive Sites in the c-myc Locus of N417 Cells DNAse I hypersensitive sites (DHS) in c-myc gene locus were determined by treatment of N417 cell nuclei with DNAse 1[(8) and this report, figure 2B]. Four major sites were identified (figure 3D) corresponding to previously reported DHS, with the exception of site e, which could not be detected using our mapping strategy. Drug stimulated Topo II cleavage sites and DHS clearly appear to map within the same regions of the c-myc 5’ end (compare maps A, B, and D in figure 3). In contrast, the DHS d mapped in exon 1, close to the two promoters P1 and P2 of the c-myc gene (14), has no Topo II cleavage site equivalent. HL60 and N417 Cells Have Identical mMAMSA Mediated Cleavage Sites and DNAse | Hypersensitive Sites in c-myc Gene To determine whether these findings could be repro- duced in other tumor cells of human origin, we submitted HL60 cells to mAMSA treatment. DNA preparations were digested to completion with EcoRI restriction endonu- clease and analyzed by Southern blot hybridization, using human c-myc probe A (figure SA). In addition to the 13-kb germline c-myc DNA band (lanes 1 and 4), DNA patterns from mAMSA treated cells exhibited identical cleavage bands in N417 cells and HL60 (lanes 2 and 3). Consistent with this result, the position of DHS in c-myc gene of HL60 cells [as determined by Dyson et al. (9) and reproduced in figure 3E] is very similar to that of N417 cells (figure 3D). Kb 23 - 65 ~ 43 - 20 - 13 ~- 1.0 10 - 08 - 08 - 44 BD-40 Stimulates the Topo Il Mediated in Vivo Cleavage of the c-myc Oncogene Locus in HL60 Cells In contrast to its apparent lack of activity in N417 cells, BD-40 stimulates Topo II activity in the c-myc locus of HL60 cells (figure 5B). The cleavage pattern was not qual- itatively different from that of mAMSA-treated cells. Topo Il Mediated Cleavage Is Not Stimulated by Drugs in Nonactively Transcribed Genes of N417 Cells In order to determine whether the transcriptional activity of genes could be related to in vivo Topo II activity, DNA from mAMSA-treated N417 cells was successively hybrid- ized with two probes detecting DNA fragments from human S, globin pseudogene and human c-mos proto-onco- gene, which are weakly or not expressed in these cells as judged by Northern blot (data not shown). It is interesting to note that no cleavage band was detected (figure 6) even after a much longer exposure time of X-ray films (7 days instead of 24 hours). Moreover, no DHS could be detected in c-mos gene of N417 cells (data not shown). DISCUSSION This work was aimed at accurately localizing, in c-myc gene, lesions induced by antitumoral agents whose DNA activity is mediated by Topo II. The c-myc proto-oncogene is assumed to play critical functions in the control of cell proliferation and differentiation; a knowledge of the con- trol of its activity by some regulatory elements is emerging [for example, see (15)]. Two different human cell lines bear- ing a c-myc amplification were chosen in order to increase the sensitivity of the detection of bands corresponding to cleavage sites. Given the choice of the restriction enzyme and the c-myc exon 1-specific probe, the detected fragments FIGURE 4.—Stimulation by mAMSA and OH-9-El of in vitro Topo II cleavage products of the c-myc gene. Restriction enzyme digest of AK76 DNA (25 ng) was incubated with Topo II for 10 min., the reaction was stopped with SDS- proteinase K treatment and the samples were analyzed by electrophoresis and xga) Southern blot hybridization using probe A or B as described in Materials and Methods. A) EcoRI digest of AK76 DNA, using probe A. /, control DNA (no Topo II); 2, + Topo II (100 ng); 3, same as 2+ mAMSA | uM; 4, same as 2+ OH-9-El 1 uM. B) Xbal digest of AK76 DNA, using probe B. /, control DNA (no Topo II); 2, + Topo II (100 ng); 3, same as 2, + mAMSA 1 uM; 4, same as 2 + OH-9-El 1 uM. The arrows indicate the cleavage products generated by Topo II alone or by Topo II and drugs. DNA length fragments are given in kilobase pairs (kb). Sizes of exons 1, 2, and 3 have been standardized to gel migration scale. NCI MONOGRAPHS, NUMBER 4, 1987 13 Kb c-myc By globin ¢ DNA TOPOISOMERASES IN CANCER THERAPY FIGURE 5.—Cleavage sites induced in vivo by mAMSA in the amplified c-myc gene of N417 and HL60 cells. Exponentially growing cells (3-4 days of culture) were exposed to mAMSA for 3 hours or to BD-40 for 1 hour at 37°C in fresh medium. Drug action was stopped by lysis of cells with SDS and proteinase K. DNA preparations from drug treated cells were digested with EcoRI and analyzed by Southern blot hybridization using the 32P-labeled probe A of the c-myc gene. A) 1, DNA (5 ug) from untreated N417 cells. 2, DNA (5 ug) from mAMSA (5 uM) treated N417 cells. 3, DNA (10 pg) from mAMSA (5 uM) treated HL60 cells. 4, DNA (10 pg) from untreated HL60 cells. B) I, DNA (5 ug) from untreated HL60 cells; 2-3, DNA (5 pg) from BD-40 (0.05-0.5 uM) treated HL60 cells. A similar cleavage pattern in c-myc gene was observed for N417 and HL60 treated cells (compare lanes 2 and 3). The blots were exposed to Kodak XARS film for 24 hours. Gel migration conditions were different in A and B. - FIGURE 6.—In vivo Topo II-induced cleavage sites in c-myc, c-mos, and pseudo B; globin genes. Exponentially growing cells were exposed to mAMSA (5 pM) for 1-2 hours at 37°C in fresh medium. Drug action was stopped by lysis of cells with SDS and proteinase K. DNA preparations (2.5 pg) from drug treated cells were digested with EcoRI and analyzed by Southern blot hybridization using 32P_labeled probes of: c-myc gene 2.5 Kb- iD wh wn (probe A) (A), B; globin pseudogene (B) (21) and c-mos gene (C) (22). 1, untreated cells. 2, mAMSA-treated cells (5 uM, 1 hour). 3, mAMSA- treated cells (5 uM, 2 hours). The blots were exposed to Kodak XARS film for 24 hours (c-myc) and 7 days (B; globin and c-mos). c-mos 45 represent the Topo II cleavage sites status in a region encompassing the exon 1 and 6.0 kb of the 5 flanking sequences (see map in figure 1). Our data show that mAMSA, BD-40, and VM26 stimulate, with individual variations, the occurrence of breaks all located in the 2.5-kb segment immediately close to the exon 1. Importantly, these sites coincide with those resulting from hybridization of EcoRI digested N417 cell DNA with an exon 3-specific probe (8). The use of Xbal and an exon 1 probe resulted in a more acute mapping and explains why certain sites detected in this work had previously escaped detection. An important point of our results concerns the identity of sites induced by mAMSA in N417 and HL60 cells. Moreover, BD-40 generates in HL60 cells a cleavage pat- tern identical to that of mAMSA. Assuming that the struc- ture of chromatin is identical in both cells, it can be con- cluded that the interaction between DNA, Topo II, and drugs is the same in this portion of the c-myc locus. Because of a greater precision than in our previous report (8), Topo II cleavage patterns generated by mAMSA and VM26 in N417 cell DNA were found to differ by a single site (figure 3A and B). Besides, BD-40 and mAMSA induce in HL60 cells cleavage patterns identical to those of N417 cells, implying that both drugs stimulate Topo II activity at the same sites. The question arises as to whether this difference between mAMSA (or BD-40) and VM26 is due to their DNA intercalative or nonintercalative capacity or to a differential ability to bind to the DNA-Topo II complex. It is interesting to compare cleavage sites generated in vivo in c-myc to those resulting from an in vitro assay (figure 3A, B, and C). All in vivo sites have an in vitro counterpart at very similar if not identical positions. This implies that specific Topo II recognition sequences exist in these regions. The presence of such Topo II sequences has been already reported in certain genes including proto- oncogene c-fos (16). The difference between in vivo and in vitro cleavage sites is readily explained by assuming a differential accessibility to them, due to the chromatin con- formation. With regard to this point, OH-9-El, a potent in vitro Topo II stimulatory drug, is inactive in N417 cells. Although this can reflect the incapacity of the drug to pene- trate the nucleus, it is important to emphasize the apparent correlation that exists between the absence of drug cyto- toxicity (E. Multon, unpublished results) and the lack of DNA breaks at the concentrations used. In an effort to understand the biological significance of these results, we have mapped the DHS in the 5” end of the c-myc locus (figure 3D). Most of them are identical or map very closely in N417 cells and HL60 cells, confirming that the functional structure of the gene is identical in both cells. Some of them strikingly coincide with Topo II cleavage sites: the site a (the major DHS as judged by the intensity of the bands) is located in the same region as three major Topo II sites, presumably because of an open structure of the surrounding chromatin. The region containing the site b and the corresponding Topo II site is also of interest as a third promoter of c-myc transcription [designated P,, see (14)] has been located close to these sites. In the same region is located an element which negatively controls transcription of c-myc in human as well as in mouse cells (15). Furthermore, the DHS b has been shown to disappear during the late differentiation stage of HL60 cells (14). 46 Finally, the absence of any Topo II site symmetrical of the DHS d in the exon 1 certainly reflects the absence of a Topo II recognition sequence in this region and is in agreement with other data which failed to reveal the pres- ence of such sites in coding sequences (for example, c-fos). The absence of DHS and Topo II cleavage sites in the vicinity of two genes (c-mos and 8; globin pseudogene) which are not or weakly transcribed may be of significance in terms of the possible involvement of Topo II in the transcription process. However, our results are not com- pletely conclusive as their negativity (verified after an over- exposure of the blots to films) could be due to a lack of sensitivity of the method. Should it be confirmed, this result would mean that Topo II-mediated drugs selectively impair active genes. Finally, some recent data seem to indicate that the acti- vation of c-myc through an amplification process or a chromosome rearrangement is not an initial step of tumori- genesis but rather occurs during tumor progression (17,18). In that respect, it will be important to look for Topo II cleavage sites in the 3’ end of c-myc, which also has some regulatory activity, and to extend these studies to other genes (N-ras, N-myc), which are also implicated in tumor progression (19). REFERENCES (1) Ross AE, GLAUBIGER DL, KoHN KW: Protein associated DNA breaks in cells treated with Adriamycin or ellipticin. Biochim Biophys Acta 519:23-30, 1978. (2) NELSON EM, TEWEY KM, Liu LF: Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomer- ase II on DNA by mAMSA. Proc Natl Acad Sci USA 81:1361-1365, 1984. (3) Liu LF, Rowe TC, YANG L, et al: Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258: 15365-15370, 1983. (4) CHEN GL, YANG L, Rowe TC, et al: Nonintercalative antitumor drugs interfere with the breakage-reunion reac- tion of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1984. (5) KLEIN G, KLEIN E: Conditioned tumorigenicity of activa- ted oncogenes. Cancer Res 46:3211-3224, 1986. (6) CARNEY DN, GAZDAR AF, BEPLER G, et al: Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res 45:2913-2923, 1985. (7) DucrocQ C, WENDLING F, TEURLEZ-PERRIN M, et al: Structure-activity relationship in a series of newly synthe- sized 1-amino-substituted ellipticine derivatives. J Med Chem 23:1212-1216, 1980. (8) Riou JF, MuLTON E, VILAREM MJ, et al: In vivo stimula- tion by antitumor drugs of the topoisomerase II induced cleavage sites in c-myc protooncogene. Biochem Biophys Res Commun 137:154-160, 1986. (9) DYSON PJ, LITTLEWOOD TD, FORSTER A, et al: Chromatin structure of transcriptionally active and inactive human c-myc alleles. EMBO J 4:2885-2891, 1985. (10) MATHIEU-MAHUL D, CAUBET JF, BERNHEIM A, et al: Molecular cloning of a DNA fragment from human chromosome 14(14q11) involved in T-cell malignancies. EMBO J 4:3427-3433, 1985. (11) Riou JF, VILAREM MJ, LARSEN CJ, et al: Characterization of the topoisomerase II-induced cleavage sites in the c-myc protooncogene: In vitro stimulation by the antitumoral NCI MONOGRAPHS, NUMBER 4, 1987 intercalating drug mAMSA. Biochem Pharmacol 35: 4409-4413, 1986. (12) MANIATIS T, FRITSCH EF, SAMBROOK J: Molecular Clon- ing, a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. (13) WILSON WR, BAGULEY BC, WAKELIN LPG, et al: Interac- tion of the antitumor drug mAMSA and related acridines with nucleic acids. Mol Pharmacol 20:404-414, 1981. (14) BENTLEY DL, GROUDINE M: A block to elongation is largely responsible for decreased transcription of c-myc in differ- entiated HL60 cells. Nature 321:702-706, 1986. (15) CHUNG J, SINN E, REED RR, et al: Trans-acting elements modulate expression of the human c-myc gene in Burkitt lymphoma cells. Proc Natl Acad Sci USA 83:7918-7922, 1986. (16) DARBY MK, HERRERA RE, VOSBERG HP, et al: DNA topo- isomerase II cleaves at specific sites in the 5’ flanking region of c-fos protooncogenes in vitro. EMBO J 5:2257- 2265, 1986. (17) TAYAY, TERADA M, SUGIMURA T: Role of oncogene ampli- fication in tumor progression. In Advances in Viral DNA TOPOISOMERASES IN CANCER THERAPY Oncology (Klein G, ed), vol 7. New York: Raven Press, 1987, pp 141-154. (18) MATHIEU-MAHUL D, Si1GAUX F, ZHU C, et al: A t(8,14) (q24,q11) translocation in a T cell leukemia (L1.ALL) with c-myc and TcR-alpha chain locus rearrangements. Int J Cancer 38:835-840, 1986. (19) ALITALO K, SCHWAB M: Oncogene amplification in tumor cells. In Advances in Cancer Research (Klein G, Wein- house S, eds). New York: Academic Press, 1986, pp 189- 234. (20) GAzIN C, DUPONT DE DINECHIN S, HAMPE A, et al: Nucleo- tide sequence of the human c-myc locus: Provocative open reading frame within the first exon. EMBO J 3:383-387, 1984. (21) FritscH EF, LAWN RM, MANIATIS T: Molecular cloning and characterization of the human fg; like globin gene clus- ter. Cell 19:959-972, 1980. (22) WATSON R, OSHARSSON M, VANDE WOUDE GF: Human DNA sequences homologous to the transforming gene (mos) of Moloney murine sarcoma virus. 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Camptothecin Inhibits Hsp 70 Heat-Shock Transcription and Induces DNA Strand Breaks in hsp 70 Genes in Drosophila Thomas C. Rowe, * Elsa Couto, and David J. Kroll’ ABSTRACT —Camptothecin is a cytotoxic drug which inhibits cellular nucleic acid synthesis. Associated with this inhibition is the induction of protein-linked DNA strand breaks. Recent stud- ies have demonstrated that camptothecin interferes with the DNA breakage and rejoining activity of the enzyme DNA topoisomerase I and stabilizes a cleavable complex between this enzyme and DNA. Treatment of this complex with a protein denaturant results in DNA strand breaks and the covalent attachment of topoisomerase to the 3’-end of the DNA breaks. In this paper we have mapped the camptothecin-induced DNA breaks on the hsp 70 heat-shock gene in cultured Drosophila cells. Drug-induced breaks are present primarily within the coding region of this gene and occur only when transcription of this gene is activated by heat. Camptothecin (20 uM) was also observed to inhibit heat-induced hsp 70 transcription greater than 70%. The possible role of topoisomerase I in hsp 70 heat-shock gene transcription is discussed. —NCI Monogr 4:49-53, 1987. INTRODUCTION Camptothecin is a cytotoxic alkaloid which has strong antitumor activity against mouse L1210 and Walker 256 rat carcinosarcoma (/-3). Camptothecin is a potent inhibitor of cellular RNA and DNA synthesis but not protein syn- thesis (4). Inhibition of heterogeneous nuclear (hnRNA) and nucleolar RNA synthesis by camptothecin results in shortened RNA molecules (5). This inhibition is rapidly reversed upon drug removal (6). Drug inhibition of cellular DNA synthesis, however, is only partially reversible with the extent of reversibility being dependent on the duration of drug exposure. Camptothecin does not affect the enzy- matic activities of purified DNA or RNA polymerases, sug- gesting that some other target(s) is (are) responsible for in- hibition of nucleic acid synthesis (6). Another significant effect of camptothecin is the rapid induction of single-stranded protein-linked DNA breaks in drug-treated cells. Upon drug removal these breaks are rapidly resealed (6,7). Recently, Hsiang et al. (7) have shown that camptothecin stimulates single-strand DNA breakage by purified mammalian DNA topoisomerase I. Drug-induced DNA breakage by topoisomerase I is imme- diate and these breaks are readily rejoined if 0.5 M salt is subsequently added to the reaction. Immunoblotting stud- ies have shown that treatment of cells with camptothecin I Department of Pharmacology and Therapeutics, College of Medicine, The J. Hillis Miller Health Center, University of Florida, Gainesville. * Reprint requests: Thomas C. Rowe, Ph.D., Department of Pharma- cology and Therapeutics, College of Medicine, Box J-267, The J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610. results in the selective loss of more than 90% of topoisomer- ase I from the DNA-free fraction of cell lysates, suggesting that this enzyme is responsible for the camptothecin- induced protein-linked DNA breaks in cells (Hsiang Y-H, and Liu LF, personal communication). Topoisomerase I is a ubiquitous enzyme which catalyzes changes in the topological structure of DNA by breaking and rejoining the DNA phosphodiester backbone (8,9). The transient, enzyme-mediated single-strand DNA break has been shown to result in the formation of a covalent phosphotyrosine bond between the enzyme and the 3’-end of the broken DNA strand (8,9). The energy conserved in this covalent intermediate is thought to drive the rejoining of the transiently broken DNA strand. It has been pro- posed that camptothecin inhibits topoisomerase I by inter- fering with the DNA rejoining reaction of this enzyme resulting in the formation of a cleavable complex (7). Treatment of this complex with a protein denaturant results in DNA strand breaks and the covalent attachment of topoisomerase I to the 3’-end of the DNA break. Studies have suggested that DNA topoisomerase I may be important in resolving topological problems which accompany DNA transcription and replication (8,9). Our lab has been interested in studying the role of topoisom- erase I in regulating the function of cellular chromatin and how this relates to the cytotoxic action of the drug campto- thecin. In this paper, we report on our studies on the effects of camptothecin on the structure and expression of the Asp 70 heat-shock genes in cultured Drosophila cells. MATERIALS AND METHODS Enzymes, nucleic acids, and drugs.—DNA restriction enzymes and E coli DNA polymerase I were obtained from Bethesda Research Laboratories (Gaithersburg, Md.). Drosophila hsp 70 DNA clones pPW232.1 and pPW229.1 (10) were kindly provided by Matthew Meselson (Harvard University, Cambridge, Mass.). Sodium camptothecin was a generous gift from Leroy F. Liu (Johns Hopkins Univer- sity, Baltimore, Md.). Cell culture.—The Drosophila D1 tissue culture cell line was obtained from Mendy Simcox (Johns Hopkins Univer- sity, Baltimore, Md.). Cells were maintained at 25°C in Schneider modified Drosophila medium (GIBCO Labora- tories, Grand Island, N.Y.) supplemented with 10% heat- inactivated fetal calf serum. Cells were heat shocked in a 37°C water bath. DNA purification.—Cellular DNA was purified as de- scribed before (11). Cells (10 ml) grown to a density of 5-10X 109 cells/ml were pelleted in a Beckman RT6000 centrifuge at 2000 rpm for 5 min. After removal of the 49 supernatant, the cells were lysed by the addition of 1 ml of buffer A (20 mM Tris-HCI, pH 8.0, 1% SDS). Proteinase K and EDTA were then added to final concentrations of 200 pg/ml and 20 mM, respectively, and the lysates incubated overnight at 37°C. Lysates were then phenol extracted 3 times and the nucleic acid precipitated with ethanol. The precipitates were then resuspended in 250 ul of buffer B (10 mM Tris-HCI, pH 8.0, | mM EDTA) containing 20 ug/ml RNase A and incubated 2 h at 37°C. The DNA was then phenol extracted and ethanol precipitated before digestion with DNA restriction endonucleases. Alkaline gel electrophoresis.—Restricted DNA samples were ethanol precipitated and resuspended in 25 ul of a solution containing 50 mM NaOH, 1 mM EDTA, 2.5% Ficoll, 0.025% bromocresol green. Samples were then ana- lyzed by electrophoresis in a 1.4% agarose gel containing 30 mM NaOH, | mM EDTA at 4°C for 12 h at 70 V. Southern transfer and hybridization.— After neutralizing to gels in 0.5 M Tris-HCI, pH 8.0, and 1.5 M NaCl, the DNA was transferred to nitrocellulose filters (BASS, Schleicher & Schuell, Keene, N.H.) and hybridized to nick- translated probes as described by Maniatis et al. (12). RNA isolation and dot blotting.—RNA was isolated from cells by CsCl centrifugation as described before (11). The purified RNA solution was then dot-blotted onto nitrocellulose and hybridized to labeled DNA probes as described before (11). Autoradiography.— Autoradiography was done at —70°C using pre-flashed Kodak XAR-5 film and a Dupont Lightning-Plus intensifying screen. Quantitation of the autoradiographs was done by densitometry with a laser densitometer. RESULTS Camptothecin Inhibits the Expression of Drosophila Heat-Shock Genes When eukaryotic or prokaryotic cells are exposed to temperatures above those which are optimum for growth, they respond by synthesizing a family of proteins called heat-shock proteins (hsp’s) (13-16). Expression of heat- shock proteins is primarily regulated at the level of gene transcription. However, the mechanisms underlying the transcriptional control of these genes are just beginning to be understood. The Asp 70 gene codes for the major heat- shock protein (hsp 70) in Drosophila (15,16). The hsp 70 gene consists of a 2.2-kilobase (kb) conserved transcribed region (coding region) with a 0.35-kb conserved nontran- scribed regulatory region (NCR) adjoining the 5’-end of the gene (17,18) (Fig. 1). Earlier studies have shown that transcription of the Drosophila hsp 70 gene could be induced by the topoisomerase II inhibitor VM-26 and it was proposed that this enzyme might negatively regulate Asp 70 expression (/1). Consistent with this idea is the binding of topoisomerase II to a region at the 5-end of the Asp 70 gene which is involved in negative regulation of Asp 70 expres- sion (11,19,20). Camptothecin has been shown to inhibit nucleolar and hn RNA synthesis (4-6). To determine what effect campto- thecin had on Asp 70 transcription, RNA isolated from drug-treated cells was dot-blotted onto nitrocellulose filters and hybridized to labeled pPW232.1 plasmid DNA (con- tains the 5"-half of the Asp 70 gene). The autoradiographs 50 Ie. Transcription BAM SAL [Ne | CODING REGION ————+| pPW 2290.1 PPW 232.1 400 bp — FIGURE 1.—Map of the Drosophila hsp 70 heat-shock gene. DNA clones spanning the 3’ and 5’ region of the gene are shown below the gene. from the dot blots were then scanned by densitometry to measure the relative levels of hsp 70 RNA in each sample. Normally there is little or no detectable transcription of the hsp 70 genes in Drosophila cells at 25°C. However, hsp 70 transcription is stimulated approximately 20-fold upon shifting the cells to 37°C for 15 min (Table 1). Unlike the topoisomerase II inhibitor VM-26, camptothecin does not induce Asp 70 transcription. Treatment of Drosophila cells with 20 uM camptothecin for 30 min at 25°C does not increase Asp 70 RNA levels. However, 20 uM camptothecin does inhibit heat-induced Asp 70 transcription greater than 70%, suggesting that topoisomerase I is possibly involved in hsp 70 transcription. Camptothecin-induced Breaks on Hsp 70 Chromatin Topoisomerase I catalyzes topological changes in DNA structure by breaking and rejoining the DNA phospho- diester backbone (8,9). Camptothecin has been shown to stabilize the transient broken DNA intermediate in the topoisomerase I DNA breakage-rejoining reaction (7). Treatment of this intermediate with protein denaturants (i.e., SDS) reveals the topoisomerase-mediated single- strand break sites in the DNA. To understand mechanistically how topoisomerase I might be involved in Asp 70 transcription, we mapped the camptothecin-induced topoisomerase I single-stranded DNA cleavage sites within the Asp 70 gene region using a modification of the indirect end-labeling procedure (17). A schematic illustrating this procedure is shown in Fig. 2. DNA isolated from cells treated with camptothecin was TABLE 1.—Inhibition of hsp 70 transcription by camptothecin’ Camptothecin HSP 70 RNA (%) (uM) 25°C 37°C None 5 100 20 4 28 @ Logarithmically growing Drosophila D1 tissue culture cells (6 X 10°) were incubated at 25°C for 15 min in the presence or absence of campto- thecin. Incubation was then continued an additional 15 min at either 25°C or 37°C. The cells were then collected by centrifugation and lysed in 1 ml of a solution containing 4 M guanidine isothiocyanate, 20 mM sodium citrate, pH 7.0, 0.1 M 2-mercaptoethanol, 0.5% sarkosyl. RNA was isolated by the CsCl-centrifugation procedure and serial dilutions containing equal amounts of RNA from each sample dot-blotted onto a nitrocellulose filter as described previously (/7). The filter was hybridized to 32P-labeled pPW232.1 DNA (contains the 5’-half of the Asp 70 gene) and then autoradiographed. The levels of hsp 70 RNA were quantitated by weighing the densitometer tracings of the autoradiograph and are cal- culated relative to the non-drug-treated, heat-shock control. NCI MONOGRAPHS, NUMBER 4, 1987 T0P0 BAM HI | HSP 70 GENE | Drug-induced DNA Cleavage by Topoisomerase BAM HI [| 11 HSP 70 GENE | RESTRICT WITH BAM HI [| || usp zo || GENE | 1. GEL ELECTROPHORESIS 2. SOUTHERN TRANSFER 3. HYBRIDIZATION TO PROBE 3 I || usp 70 || GENE | + —] FIGURE 2.—Indirect end-labeling technique (see text for details). restricted and the DNA fragments separated by alkaline denaturing agarose gel electrophoresis. The DNA was then blotted onto a nitrocellulose filter and hybridized to an Asp 70 probe homologous to the region immediately adjacent to the DNA restriction site. In effect, the cellular heat shock DNA is indirectly end-labeled at a specific DNA restriction site. Using such an approach, the distance (d) of any topo- isomerase cleavage sites relative to a chosen DNA restric- tion site can be measured. The results of this experiment are shown in Fig. 3. To map the camptothecin-induced DNA cleavage sites at the 5” and 3’ regions of the gene, the DNA was cut with either Sa/l or BamHI and the blots hybridized to labeled plasmid pPW229.1 DNA which contains the Bam-Sal fragment of the Asp 70 gene (see bottom of Fig. 3). The multiple restriction bands present in the control lanes of Fig. 3 (lanes 1, 3, 5, and 7) represent the 5-6 copies of the Asp 70 gene at the 87 4 and 87 C loci of Drosophila chromosome 3 (10,21). As is evident from lanes 2 and 6, there is little drug-induced cleavage of the hsp 70 chromatin in cells incubated at 25° C. However, following heat shock, the Asp 70 DNA is cleaved at multiple and specific sites in drug-treated cells (lanes 4 and 8). Approximately 40% of the total Asp 70 DNA sequences are cleaved as judged by densitometry scans of the autoradiograph, suggesting that each copy of the Asp 70 gene is probably cleaved less than once. These results suggest that topoisomerase I is asso- ciated with transcriptionally active but not inactive hsp 70 chromatin. This is consistent with previous immunofluo- rescence studies in Drosophila which show that topoisom- erase I is enriched at heat-shock loci following heat induction (22). Photo-crosslinking experiments also sug- gest that topoisomerase I is recruited to the hsp 70 chro- matin during heat shock of Drosophila cells (23). The locations of the camptothecin cleavage sites on the hsp 70 chromatin are summarized in Fig. 4. It is striking that most of these sites fall within the coding region of the DNA TOPOISOMERASES IN CANCER THERAPY Sal Bam 25° 36° 25° 36° E 0 : 8 E ¢ g Oo EE. 2 FE BE: § 2 1 2 3 4 35 ¢ 7 8 KB 2.8 =p | 2.2 mp 1.5 ip 1.1 ——p | 75 ——p 5' (SSB or DSB) and cell killing for 3 3 | chemically different classes of inter- uw ® calators. L1210 cells were treated 2 with m-AMSA, 5-iminodaunorubi- > cin, or 2-methyl-9-hydroxyellipti- 2 cinium for 60 min, at which time 2 SSB or DSB were measured. The - Dm 2-CH,-9-OH-E* +4 2-CH,-9-OH-E* drug was washed away and survi- 3 val of colony-forming ability was - determined. Reproduced from (9). ; =r i ra (These drugs exited rapidly from 5-Iminodaunorubicin 5-iminodaunorubicin the cells and the DNA lesions were reversed within 20 min.) A B | | ] | L. L 1 | 1 | ] 0 1000 2000 0 2000 4000 6000 DNA SINGLE-STRAND BREAK FREQUENCY DNA DOUBLE-STRAND BREAK FREQUENCY (SSB-Rad-equivalents) (DSB-Rad-equivalents) 66 NCI MONOGRAPHS, NUMBER 4, 1987 It is possible that PLSB were the major source of cell killing for m-AMSA, the drug that showed the least cell killing at a given PLSB level. The other two drugs, espe- cially the anthracycline, 5-iminodaunorubicin, showed sub- stantially more cell killing at a given PLSB level and may kill cells largely by other more potent mechanisms. An interesting alternative however is that the cytotoxic potency of PLSB depends on where they are located in the genome. Indeed it has been found that the 3 chemical classes of intercalating agents studied here produce mark- edly different preferential sites of DNA cleavage by purified topoisomerase II [(20-22); Pommier Y, Kerrigan D, and Kohn KW, in preparation]. Comparisons Between Congeners of a Single Class of Intercalators Since m-AMSA was considered possibly to kill cells through the trapping of PLSB, several congeners of m-AMSA were compared. Four congeners having widely different cytotoxic potencies were found to produce quan- titatively coherent results when PLSB (DPC) production was plotted against survival or colony-forming ability (34). Thus PLSB formation may be causally related to cell kill- ing by intercalators of the m-AMSA type. The distribution of DNA sites at which the m-AMSA congeners induced scission by purified topoisomerase II in SV40 DNA were indistinguishable. This contrasted markedly with the large differences in the DNA cutting patterns produced by 5-iminodaunorubicin and ellipticine. These results are com- patible with the possibility that the degree of cytotoxic potency of PLSB varies in relation to their genomic loca- tions. If this is true, then comparisons among congeners of a more toxic class, such as anthracyclines, generating sim- ilar sets of DNA cutting sites, should also give coherent relationships between cell killing and PLSB, whereas this would not necessarily be the case if different killing mecha- nisms were involved. Relation to Cell Proliferation Proliferating cells were compared with quiescent (Gy) cells in regard to PLSB responses to topoisomerase II inhibitors (35). In this study mouse 3T3 fibroblasts were used because of their ability to arrest in a G, state. Dose- dependent PLSB responses were seen in proliferating cells treated with m-AMSA or with etoposide, whereas quies- cent cells exhibited much weaker responses, and the differ- ences were even more striking when responses in isolated nuclei were compared. Thus topoisomerase II is either absent in isolated nuclei of quiescent cells or its PLSB responsiveness to drugs is inhibited. When quiescent 3T3 cells are replaced in fresh medium, they progress synchronously into the cell cycle, with S- phase centered about 24 hr after replating (fig. 9). Drug- stimulated PLSB responses rose sharply during early S- phase, usually reaching a maximum about 2 hr before the S-phase peak. Results using m-AMSA or etoposide were similar. In this experiment the cell DNA was labeled with ['“C]thymidine during the last cell cycle prior to quiescence and continuing after release from quiescence. An early small rise in DPC (peak at about 5 hr) was frequently seen which occurred in the absence as well as in the presence of drug and which remains unexplained. When the labeling protocol was changed, so that [“C]thymidine was present only during the approach to quiescence, and not after release from quiescence, the results were different (fig. 10). Now the drug-induced PLSB response during S-phase was smaller and did not show a clear peak. The results are as would be expected if drug- induced PLSB occur preferentially and to some degree transiently in replicating regions of DNA, affecting equally the template and daughter strands. The question remained whether the suppression of PLSB response requires a normal Gy state or whether it would occur in nutritionally arrested malignant cells. Mouse leukemia L.1210 cells growing in suspension culture were 1200 DNA-PROTEIN CROSS LINKS (rad-equivalents)- FIGURE 9.—PLSB responses to m-AMSA and etoposide (VP16) in nuclei isolated from 3T3 cells during the course of the first cell cycle following release from quiescence. The dashed curve shows the wave of DNA synthesis ([?H]thymidine incorporation in 15 min). Cell DNA was uniformly labelled with [!4C]thymidine which was present in the medium contin- uously from 40 hr prior to the harvesting of quiescent cells until the time of isola- tion of nuclei. Nuclei were treated and assayed as in figure 4, except that the concentration of m-AMSA was 0.5 uM. Symbols: (®) no drug; (4) 0.5 uM m- AMSA; (Hl) 20 pM etoposide. Repro- [®H]-THYMIDINE INCORPORATION (dpm/10® cells) 0 duced from (35). TIME AFTER RELEASE FROM QUIESCENCE (Hours) DNA TOPOISOMERASES IN CANCER THERAPY 67 1200 T T DNA-PROTEIN CROSSLINKS (rad-equivalents) T 10 20 30 TIME AFTER RELEASE FROM QUIESCENCE (Hours) FIGURE 10.—Effect of DNA labeling protocol on observed PLSB responses during the first cell cycle following release of 3T3 cells from quiescence. The experiment was similar to that of figure 9, except that the time period of DNA labelling with [!4CJthymidine was either con- tinuous (@, A) or confined to the period prior to release from quiescence (0, 2). The isolated nuclei were either treated with 0.5 uM m-AMSA (A, A) or were left untreated (O,® ). Reproduced from (35). tested during the growth curve leading to proliferation arrest (fig. 11). As the cells entered stationary phase, the PLSB responses to m-AMSA and to etoposide almost completely disappeared. The loss of the topoisomerase 11 effect in non-proliferating cells hence does not require a normal Gi state. Chromosomal Aberrations and Mutations Since type II topoisomerases produce genetic recombina- tion in prokaryotes (36-38), the question arises whether inhibitors of topoisomerase II can generate chromosomal exchanges. m-AMSA and S-iminodaunorubicin were indeed found to stimulate several types of chromosome aberra- tions and also mutations in Chinese hamster V79 cells (39). 5-Iminodaunorubicin produced more chromosomal aber- rations than did m-AMSA at doses producing given fre- quencies of SSB or DSB. In contrast, sister chromatid exchanges appeared to give coherent relations for the two drugs. Mutation frequency (at the HGPRT locus) was stimulated coherently by the two drugs relative to DSB production. Relative to SSB production, however, 5- iminodaunorubicin generated more mutants than did m-AMSA. In this cell system, survival of colony-forming ability following treatment with drugs gave coherent results relative to DSB, whereas relative to SSB 5-iminodauno- rubicin was more lethal than m-AMSA. Although the current data do not allow firm conclusions, further correlative studies of this type could determine the significance of the observed DNA lesions in the production of chromosome aberrations, mutations and cell death. ARE FREE RADICALS INVOLVED IN THE PRODUCTION OF DNA LESIONS BY INTERCALATORS Some DNA intercalators such as Adriamycin are me- tabolized by cytoplasmic enzymes, and there is evidence of free radical production in the course of this metabolism (40) or by iron-mediated electron transfer (41). Several 68 observations however show that PLSB do not arise by a free radical mechanism. First, DNA strand breaks pro- duced by free radicals in cells treated with ionizing radia- tion, bleomycin (42), or H,0, (43) are generally not protein-associated: they are readily observed in filter elu- tion without the use of proteinase. Secondly, intercalator- induced PLSB are rapidly reversible upon removal of drug even in permeabilized cells and isolated nuclei, whereas x- ray induced strand breaks are not repaired in these systems (44,18,17). Moreover the PLSB produced in permeabilized cells by the intercalator, m-AMSA, were not accompanied by the increased synthesis of poly(ADP-ribose), commonly produced by DNA breaking agents and which was demon- strated to accompany similar frequencies of x-ray-induced SSB (44). Treatment of cells with high enough concentrations of an intercalator or epipodophyllotoxin may result in free SSB which can be observed by filter elution without proteinase. Free SSB generally are seen only above the pharmacologi- cally reasonable dose range. These free SSB could in some cases be due to free radical mechanisms, although they also may result from nuclease action in dying cells. Potmesil et al. (45) obtained evidence for the production of both PLSB and free-radical induced SSB by Adriamycin in L1210 cells and were able to distinguish these as 2 separ- ate types of DNA damage. Adriamycin at low concentra- tion (2.8 uM) produced exclusively PASB (i.e., no free SSB were detectable), and these PASB were unaffected by superoxide dismutase, catalase, or DMSO. At 10- to 100- fold higher Adriamycin concentrations, free SSB appeared, in addition to increasing numbers of PLSB. The produc- tion of free SSB (assayed in the absence of proteinase) was partially inhibited by DMSO, ethanol, superoxide dismu- tase, and catalase, but the production of PLSB (assayed in the presence of proteinase) was unaffected by these free- radical scavengers. Although free-radical production by Adriamycin may contribute to cytotoxicity in some systems, this is probably T T I I 20 — | — 6000 A 10 ha 2 - ~ = J® > E / \ FR 5 m-AMSA \ ~4 4000 is > of / Bre iy VP-16 £35 2 74 as A 50 —_ 23 & A SB a S B x A hl ® a —| 2000 2 Oo \\ ® Control ~ TN ™ ~~ - \, 1 8 | 1 0 0 48 96 Time (Hours) FIGURE 11.—PLSB responses to m-AMSA and etoposide (VP16) in nuclei isolated from L1210 cells at various times during the growth phase. Nuclei were treated with I uM m-AMSA or 20 uM etoposide for 30 min at 37°C and then assayed for DNA-protein crosslinking. The solid sym- bols show the growth curve of the cell culture. Reproduced from (35). NCI MONOGRAPHS, NUMBER 4, 1987 not generally the case, since sensitivity to Adriamycin has been found to be unrelated to the intracellular level of glu- tathione (46,47). WORKING HYPOTHESES Mechanisms of the Drug Effects on Mammalian Topoisomerase Il It is clear that many DNA intercalating agents and epi- podophyllotoxins act on DNA-topoisomerase II complexes so as to stabilize one or more intermediate states (“cleavage complexes”) in which DNA strands are broken and in which the 5’ termini are covalently linked to the enzyme. Figure 12 is a scheme that includes the essential known facts regarding the structure of the cleavage complexes. The diagram is for the case of a complex having a double-strand cut. The enzyme is known to be linked to the 5” ends of staggered strand breaks, as illustrated. Although the shape of the enzyme molecule is unknown, it must extend from one 5’ terminus to the other 5’ terminus. Since the enzyme is a homodimer, the 2 DNA linking sites must be symmetri- cally located on each of the two monomers. In order to be able to carry out the topological strand passing reaction, the enzyme must have an internal cavity that can accom- modate a double helix. Figure 12 is a simple representation of these facts. The enzyme must also have sites that interact with the passing DNA helix. Since the enzyme is a homodimer, there must be one such site symmetrically situated on each of the 2 monomers. It seems likely that the interactions between the passing strand and the enzyme may, in fact, provide part of the driving force that opens the enzyme molecule and produces the DNA strand breaks through which strand passage will occur. Therefore, the stabiliza- tion of cleavage complexes by DNA binding drugs could be due to binding either in the region of DNA cleavage or in the DNA segment that interacts with the strand passing sites of the enzyme. A drug-DNA complex may, in effect, act as a substrate analog for the DNA that normally binds to a site on the enzyme. An interesting alternative however is that the enhanced stability of cleavage complexes is due to confor- FIGURE 12.—Schematic view of a topoisomerase II DNA double-strand cleavage complex. Each of 2 identical enzyme subunits are shown linked to the 5’ terminus of a strand break. A 4-base pair overhang between single-strand cuts is shown. The DNA strand is shown after passage into the cavity in the enzyme, the enzyme having re-closed the double- strand gap in the DNA but not yet having resealed the breaks. The passing strand is presumed to interact with regions of the enzyme pro- tein. Sites are shown for the binding of ATP and epipodophyllotoxins. DNA TOPOISOMERASES IN CANCER THERAPY mational distortion of the DNA produced by drug binding near, but not immediately at, the location of DNA-enzyme binding. Mutual stabilization would arise if drug and enzyme each tends to produce similar conformational changes in a particular DNA region. (The DNA region thus affected need not include the sites of enzyme and drug binding.) The observed non-linear patterns of drug-induced stimu- lation and inhibition are more readily explained on the basis of the latter picture. Moreover, this picture could account for the wide variety of DNA-binding chemical structures that have stimulatory and/or inhibitory effects. The epipodophyllotoxins, since they apparently do not bind to DNA significantly, may act via a different mecha- nism, perhaps by binding to sites on the enzyme. An interesting speculation is that these modified natural products may be analogs of a regulator molecule that normally acts at this site to switch the enzyme from a DNA topoisomerization function to a structural role in linking DNA to the nuclear scaffold [of which topoisomerase 11 is a major component (48)]. Mechanisms of Cytotoxicity The most immediate clue to a possible mechanism of cytotoxicity is the drug-induced stabilization of topoisom- erase II-DNA cleavage complexes. If such a blocked complex is located in the path of another DNA process, such as replication, transcription, or repair, the resulting encounter could have a high probability of being lethal for the cell. The probability of a lethal outcome may depend on (1) the form of the trapped cleavage complex, e.g., whether it is a single- or double-strand cleavage complex, (2) the degree of stabilization of the complex, which may depend on the chemical nature of the drug, (3) the location of the trapped complex in the genome, and (4) the proliferation state of the cell. Although the role of topoisomerase II as a cytotoxic target for DNA intercalators and epipodophyllotoxins is further supported by studies of resistant cell lines, which are discussed in other contributions to this volume, there is no reason to believe that this mechanism is either exclusive or universal. It should be noted that the observations of trapped topoisomerase complexes have been made possible by the easy detectability of covalent linkage of DNA to the topoisomerase protein. The drugs may however also trap or stabilize DNA complexes with other DNA-related enzymes, such as polymerase, and such complexes may not be as easily detected by current methodology. The stabilization of possibly lethal DNA-protein complexes as a result of intercalator-induced alterations in DNA conformation could apply to polymerases or other nuclear proteins, as well as to topoisomerases. 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(25) POMMIER Y, MINFORD JK, SCHWARTZ RE, et al: Effects of the DNA intercalators 4’-(9-acridinylamino)methanesul- fon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage. Biochemistry 24:6410-6416, 1985. (26) POMMIER Y, SCHWARTZ RE, ZWELLING LA, et al: Effects of DNA intercalating agents on topoisomerase II induced DNA strand cleavage in isolated mammalian cell nuclei. Biochemistry 24:6406-6410, 1985. (27) ROWE TC, CHEN GL, HSIANG YH, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (28) ZWELLING LA, KERRIGAN D, MICHAELS S, et al: Coopera- tive sequestration of m-AMSA in L1210 cells. Biochem Pharmacol 31:3269-3277, 1982. (29) POMMIER Y, MATTERN MR, SCHWARTZ RE, et al: Absence of swiveling at sites of intercalator-induced protein- associated deoxyribonucleic acid strand breaks in mam- malian cell nucleoids. Biochemistry 23:2922-2927, 1984. (30) BAUER WR, Crick FH, WHITE JH: Supercoiled DNA. Sci Am 24:100-113, 1980. (31) Cook PR, BRAZELL IA: Conformational constraints in nuclear DNA. J Cell Sci 22:287-302, 1976. (32) POMMIER Y, MATTERN MR, SCHWARTZ RE, et al: Changes in deoxyribonucleic acid linking number due to treatment of mammalian cells with the intercalating agent 4’-(9- acridinylamino)methanesulfon-m-anisidide. Biochemistry 23:2927-2932, 1984. (33) POMMIER Y, ZWELLING LA, SCHWARTZ RE, et al: Absence of a requirement for long-range DNA torsional strain in the production of protein-associated DNA strand breaks in isolated mammalian cell nuclei by the DNA intercalat- ing agent 4’-(9-acridinylamino)methanesulfon-m-anisidide (M-AMSA). Biochem Pharmacol 33:3909-3912, 1984. (34) Covey JM, KoHN KW, TILCHEN EJ, et al: Topoisomerase II-mediated DNA damage produced by 4’-(9-acridinyl- amino)methanesulfon-m-anisidide (m-AMSA) and related acridines in L1210 cells and isolated nuclei; relation to cytotoxicity. Submitted for publication. (35) MARKOVITS J, POMMIER Y, KERRIGAN D, et al: Topoisom- erase II-mediated DNA breaks and cytotoxicity in relation to cell cycle. Cancer Res 47:2050-2055, 1987. (36) IKEDA H: Illegitimate recombination: Role of type II DNA topoisomerase. Adv Biophys 21:149-160, 1986. (37) IKEDA H: Illegitimate recombination mediated by T4 DNA topoisomerase in vitro, recombinants between phage and plasmid DNA molecules. MGG 202:518-520, 1986. (38) IKEDA H: Bacteriophage T4 DNA topoisomerase mediates illegitimate recombination in vitro. Proc Natl Acad Sci USA 83:922-926, 1986. (39) POMMIER Y, ZWELLING LA, KAO-SHAN CS, et al: Correla- tions between intercalator-induced DNA strand breaks NCI MONOGRAPHS, NUMBER 4, 1987 and sister chromatid exchanges, mutations, and cytotoxic- ity in Chinese hamster cells. Cancer Res 45:3143-3149, 1985. (40) GUTIERREZ PL, GEE MV, BACHUR NR: Kinetics of anthra- cycline antibiotic free radical formation and reductive glycosidase activity. Arch Biochem Biophys 223:68-75, 1983. (41) GIANNI L, ZWEIGER JL, LEVY A, et al: Characterization of the cycle of iron-mediated electron transfer from Adria- mycin to molecular oxygen. J Biol Chem 260:6820-6826, 1985. (42) IQBAL ZM, KOHN KW, EWIG RAG, et al: Single-strand scis- sion and repair of DNA in mammalian cells by bleomycin. Cancer Res 36:3834-3838, 1976. (43) BRADLEY MO, ERICKSON LC: Comparison of the effects of hydrogen peroxide and X-ray on toxicity, mutation and DNA damage/ repair in mammalian cells. Biochim Bio- phys Acta 654:135-141, 1981. DNA TOPOISOMERASES IN CANCER THERAPY (44) ZWELLING LA, KERRIGAN D, POMMIER Y, et al: Formation and resealing of intercalator-induced DNA strand breaks in permeabilized L1210 cells without the stimulated syn- thesis of poly (ADP-ribose). J Biol Chem 257:8957-8963, 1982. (45) POTMESIL M, ISRAEL M, SILBER R: Two mechanisms of Adriamycin-DNA interaction in L1210 cells. Biochem Pharmacol 33:3137-3142, 1984. (46) CAPRANICO G, BABUDRI N, CASCIARRI G, et al: Lack of effect of glutathione depletion on cytotoxicity, mutagenic- ity and DNA damage produced by doxorubicin in cul- tured cells. Chem Biol Interact 57:189-201, 1986. (47) ROMINE MT, KESSEL D: Relationship between glutathione (GSH) levels and resistance to daunorubicin (DNR) in the P388 cell lines. Proc Am Assoc Cancer Res 27:242, 1986. (48) EARNSHAW WC, HALLIGAN B, COOKE CA, et al: Topo- isomerase II is a structural component of mitotic chromo- some scaffolds. J Cell Biol 100:1706-1715, 1985. 71 i ht oes” os =n fis ie % of and zk i, SE uh hl erg al . ! 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Ts LR pile Hs Uy naa me IEEE, all : Bap * hil CL at ir i: ¥ | i « Re JA 4 » Ts =i all Se a Role of Proliferation in Determining Sensitivity to Topoisomerase ll-active Chemotherapy Agents Daniel M. Sullivan,’ Kuan-Chih Chow, ' Bonnie S. Glisson,? and Warren E. Ross %* ABSTRACT —We have examined the relationship between topoisomerase II content and the DNA cleavage activity and cyto- toxicity of etoposide during proliferative and quiescent culture conditions. In proliferating cultures of Chinese hamster ovary (CHO) cells, human lymphoblastic CCRF cells, and mouse leukemia L1210 cells, there was easily detectable topoisomerase II by immunoblotting. In contrast, quiescent CHO cells contained virtually no detectable topoisomerase II, while the content of L1210 cells was unchanged. Enzyme content of quiescent CCRF cells was diminished but detectable. DNA cleavage activity induced by etoposide correlated well with enzyme content in pro- liferating and quiescent cells. Quiescent CHO and CCRF cultures were highly resistant to the cytotoxic effects of etoposide as expected. However, despite unchanged enzyme content and DNA cleavage activity, there was also significant resistance observed in plateau L1210 cells. We have also investigated topoisomerase con- tent and drug activity as a function of cell cycle progression. Fol- lowing serum stimulation of confluent BalbC/3T3 cells, maximal etoposide-induced DNA cleavage activity is observed in G/M and is associated with an increase in topoisomerase II content. Maximum cytotoxicity, however, occurs during mid to late S phase. Our data suggest that topoisomerase II content may be an important determinant of chemotherapeutic sensitivity during alterations in the proliferative status of the cell. However, it is clear that other factors must be involved in cell sensitivity, and elucidation of these may contribute to our understanding of the mechanism of action of these drugs.— NCI Monogr 4:73-78, 1987. INTRODUCTION Cellular proliferation plays an important role in deter- mining the sensitivity of mammalian cells to a variety of cancer chemotherapy agents. In some instances, the basis for this is obvious. Examples of these include cell cycle phase-dependent drugs such as the vinca alkaloids and cy- tosine arabinoside. Even before the role of topoisomerase II in mediating the anti-tumor effect of some anti-cancer agents became clear, there was evidence that these drugs also exhibited proliferation-dependent cytotoxicity (7,2). Now, with a better understanding of the mechanism by which these agents kill mammalian cells, it is worthwhile to ! Departments of Pharmacology and Medicine, College of Medicine, The J. Hillis Miller Health Center, University of Florida, Gainesville. 2 Department of Thoracic Oncology, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston. 3 We acknowledge the secretarial assistance of Angelique Fair, the tech- nical assistance of Mike Latham and Charles King, and helpful discussions with Dr. Tom Rowe. * Reprint requests: Warren E. Ross, M.D., Department of Pharmacol- ogy, College of Medicine, Box J-267, The J. Hillis Miller Health Center, University of Florida, Gainesville, FL 32610. re-examine the basis for proliferation-dependent cytotoxic- ity and, in particular, to better understand the behavior of topoisomerase II under conditions of proliferation and quiescence. Several lines of evidence indicate that topoisomerase 11 enzyme activity is stimulated during periods of cell cycling. An increase in enzyme activity has been demonstrated in regenerating rat liver following partial hepatectomy (3), in mitogen-stimulated lymphocytes (4), and in cultured cells stimulated by serum or epidermal growth factor (5,6). In each of these studies, topoisomerase II catalytic activity was assayed in nuclear extracts. In addition to alterations in enzyme activity, however, proliferation also imposes another set of conditions which might be expected to alter the sensitivity of the cell to chemotherapy agents. These conditions arise from changes in the cell as it progresses through the cell cycle. Previous studies have indicated that late S and G, were the phases of the cell cycle in which the greatest sensitivity to etoposide cytotoxicity was exhibited. Interestingly, while there has been some work on topo- isomerase II activity as a function of cell cycle traverse (7,8), the data are conflicting. In order to more fully delineate the alterations in topo- isomerase II activity and content during changes in prolifer- ative status and throughout the cell cycle and to, therefore, provide a rational basis for predicting the activity of topoisomerase-active chemotherapy agents, this laboratory has undertaken a series of studies in which we have corre- lated drug sensitivity, both in terms of cleavable complex formation and cytotoxicity, with alterations in topoisom- erase II. Our studies indicate that during periods of quies- cence, topoisomerase II activity is diminished as a result of loss of enzyme content and that this is an important com- ponent of resistance to intercalating agents and epipodo- phyllotoxins. In addition, however, our results indicate that the extent of cleavable complex formation following drug treatment does not fully account for cell sensitivity to the cytotoxic effects of these drugs and that other factors, some of which appear to be linked to cell cycle traverse, must be sought to more fully understand the mechanism of cell death by these drugs. MATERIALS AND METHODS Mouse leukemia L1210 cells and human lymphoblastic CCREF cells were grown in suspension cultures in RPMI 1640 medium with 209% fetal calf serum and alpha-MEM medium with 10% fetal calf serum, respectively. Chinese hamster ovary cells and BalbC/3T3 cells were grown in monolayer in alpha-MEM supplemented with 5% and 10% 73 fetal calf serum, respectively. In order to achieve quiescent cultures, the monolayer cells were allowed to grow to con- fluence, while the suspension cultures were harvested at a point where they no longer exhibited a net increase in cell number. In all cases, DNA synthesis, as measured by radioactive thymidine incorporation, was reduced to less than 5% of exponentially dividing cells. Viability by trypan blue exclusion was greater than 909%. Etoposide, a gift of Bristol Laboratories, was dissolved in dimethyl sulfoxide. Control cells received an equal volume of DMSO. All cells were resuspended in fresh medium at 37°C for 1 hr prior to drug treatment. Cleavable complex formation in whole cells was assayed as DNA single-strand break frequency using the alkaline elution technique as previously described (9). Strand break frequency is expressed as rad-equivalents. Cytotoxicity was assayed in the CHO and BalbC/3T3 cells by colony formation on plastic dishes. Cytotoxicity was quantitated in CCRF and L1210 cells by colony forma- tion in 0.19% agar. Flow cytometry was employed to assay cell cycle distri- bution according to a method previously described (5). Topoisomerase II content was assayed in nuclear extracts using the Western immunoblotting technique. The method for obtaining the 0.35 M NaCl extract from isolated nuclei has been previously published. Protein concentrations were determined using the Bio-Rad protein assay. Immunoblot- ting was performed following electrophoresis on an SDS- polyacrylamide gel (4% stacking and 10% running) at 8 watts for 5 hr. Protein was transferred to nitrocellulose at 1 amp for 4 hr, and topoisomerase II was detected using a polyclonal rabbit anti-calf thymus topoisomerase II anti- serum (kindly provided by Dr. Leroy Liu, Johns Hopkins University) as well as a polyclonal mouse anti-HeLa cell topoisomerase II anti-serum generated by this laboratory. The Bio-Rad immunoblot assay, which uses goat anti- rabbit and goat anti-mouse alkaline phosphatase conju- gates, was used to detect topoisomerase II. Peripheral blood lymphocytes from normal human volunteers were obtained using Mono-Poly Resolving Medium (Flow Laboratories, McLean, Virginia). The re- covered mononuclear cells were washed with phosphate buf- fered saline and resuspended in RPMI 1640 with 10% fetal calf serum and 1% phytohemagglutinin (Wellcome Diag- nostics, Greenville, North Carolina). After a 48-hr incuba- tion, cells were suspended into medium containing inter- leukin-2 (human T-cell Polyclone, Collaborative Research, Inc., Lexington, Massachusetts) at 5 half-maximal units/ml and ["*C]thymidine at 0.1 mCi/ml. Cell density was main- tained at 2-5X 10° cells/ml. These conditions supported exponential growth with a doubling time of approximately 24 hr. After 36 hr, cells were resuspended in label-free medium with interleukin-2 and incubated for an additional 12 hr. They were then resuspended in fresh medium without the lymphokine. RESULTS Sensitivity to the cytotoxic effects of the epipodophyllo- toxin etoposide was assayed in three cell lines exhibiting varying degrees of growth regulation. This drug was chosen because based on available evidence topoisomerase II appears to be its only important intracellular target and 74 because it rapidly effluxes from the cell following removal of drug from the culture medium. An indication of the degree of growth regulation in cultured cells is provided by their ability to arrest with a G, content of DNA during quiescence. The data in Figure 1 indicate that CHO cells do, indeed, accumulate in quiescence with a G; DNA con- tent. Mouse leukemia L1210 cells, on the other hand, show no perceptible alteration in their DNA content distribution following growth arrest, while the human lymphoblast CCREF line is intermediate. When these lines were exam- ined for sensitivity to the cytotoxic effects of etoposide under conditions of growth and quiescence (Figure 2), all three demonstrated a degree of resistance under the latter condition. Both CHO cells and CCRF cells were highly resistant under conditions which do not allow proliferation, while the L1210 plateau cultures were somewhat resistant compared to the growing L1210 culture but to a lesser degree than the other two cell lines. Since cleavable complex formation, assayed as DNA strand breaks by the alkaline elution technique, appear to play an important role in the cytotoxic action of etoposide, it was of interest to examine the CHO, CCRF, and L1210 lines for sensitivity to etoposide-induced DNA breaks under proliferating and quiescent conditions. As shown in Figure 3, plateau phase CHO cells are highly resistant to etoposide-induced DNA cleavage. Plateau phase CCRF cells exhibit a somewhat reduced sensitivity to etoposide compared to the proliferating cultures, while in the case of L1210 cells, there is no difference in sensitivity to the DNA cleaving effect when log and plateau phase cultures are compared. The participation of DNA topoisomerase II in the for- mation of cleavable complexes following etoposide treat- ment would suggest that alterations in intracellular enzyme content might be one basis for the changes in drug sensitiv- ity observed in Figure 3. In order to elucidate this point further, we prepared nuclear extracts from the three cell lines under each of the growth conditions and assayed topoisomerase II content using a rabbit polyclonal anti- topoisomerase II antiserum. The results, seen in Figure 4, demonstrate that in log phase cells topoisomerase 11 is eas- ily detectable in all three cell lines using this method. Dur- ing quiescence, however, striking differences in enzyme content exist between the cell lines. The L1210 cells show no decrease in enzyme content during plateau phase, while the CHO cells appear to have lost all enzyme content, and 100 80 | . % G, aot . 20 E CHO CCRF LI210 FIGURE 1.—Flow cytometry analysis of log phase and confluent CHO, CCREF, and L1210 cells. The percentage of cells in G; phase is expressed as a function of proliferation (open, log phase; hatched, plateau phase). NCI MONOGRAPHS, NUMBER 4, 1987 100 FIGURE 2.—Etoposide-induced cytotoxicity as a function of proliferation in CHO (@), CCRF (A), and L1210 (0) cells. Log (Panel A) and plateau (Panel B) phase cells were treated with 2-50 uM etopo- side, and colony-forming assays were done as described under Methods. A B 10 | : g £ > a 3 wn — 1.0 | 1 Zz uJ O oc J a olf : 0.01 1 1 1 1 1 1 1 1 . 0 10 20 30 40 50 0 10 20 30 40 50 VP-16 (uM) the CCRF culture is intermediate. Because these assays were performed in nuclear extracts, thus raising the possi- bility of differences in enzyme recovery during the proce- dure, we have also examined whole cell lysates and found similar results (data not shown). We have also assayed the isolated nuclei following salt extraction and find that all topoisomerase II appears to be extracted under the condi- tions of our experiments. Thus, there appears to be a gen- eral correlation between enzyme content and drug sensitiv- Rad- Equivalents 1 3 1 a 30 40 50 O I0 20 30 40 50 VP-16 (uM) FIGURE 3.—DNA single-strand break frequency induced by etoposide in CHO (®), CCRF (A), and L1210 cells (0) as a function of prolifera- tion. DNA damage was quantified by alkaline elution and is expressed as the equivalent radiation dose which would result in that degree of strand scission. (Panel A, log phase; Panel B, plateau phase). DNA TOPOISOMERASES IN CANCER THERAPY ity with respect to DNA cleavage, although the three cell lines differ somewhat in their response to different growth conditions. Previously published evidence has suggested that topo- isomerase II activity in normal peripheral blood lympho- cytes is much lower than that exhibited by those which are mitogen stimulated (4). Based on this, we sought to deter- mine if the increase in enzyme activity was accounted for by increased enzyme content and if this would influence sensi- tivity to the DNA cleaving effects of a topoisomerase 11 active agent. When assayed by the alkaline elution tech- nique, nonstimulated peripheral blood lymphocytes exhibit markedly diminished responsiveness to etoposide (Figure 5), while those which have been stimulated with the lymphokine interleukin-2 are substantially more sensitive to the drug. Indeed, the dose-response curve for stimulated lymphocytes appears to be roughly co-linear with that of proliferating CCRF cells in culture. This increase in drug sensitivity is associated with an increase in enzyme content as well (data not shown). In addition to the many metabolic differences between proliferating and quiescent cells, there are differences within the cell cycle which might be expected to influence sensitivity to drugs which act via topoisomerase II. We have explored this point using BalbC/3T3 cells synchronized by serum stimulation of confluent cultures. At various times following serum stimulation, we have treated the cells with etoposide and assayed them for DNA cleavage and cytotoxicity (Figure 6). As expected, sensitivity to cleavable complex formation is diminished in the quiescent cultures (800 rad-equivalents) compared to those in asynchronous log phase (2,400 rad-equivalents; data not shown). An increase in sensitivity is not exhibited until well into S 75 CHO FIGURE 4.—DNA topoisomerase II content determined by immunoblotting of nuclear extracts of CHO, CCRF, and LI1210 cells as a function of proliferation. Equivalent amounts of nuclear protein (50 ug) were electrophoresed (P= plateau; L =log). phase. From this point, DNA cleavage response to etopo- side increases to a maximum which appears to occur between the end of S and mitosis. These results are similar to those of Estey et al (8) using m-AMSA in a HeLa cell system. Of interest is the fact that the increase in sensitivity to etoposide-induced DNA cleavage is accompanied by an increase in intracellular enzyme content as assayed by immunoblotting (data not shown). Sensitivity to the cyto- toxic effects of etoposide, however, exhibits a somewhat different pattern. Maximal sensitivity is seen in late S phase. Indeed, at the time of maximal cleavable complex formation in late G,, little cytotoxicity is seen. It should be noted that under no circumstance have we observed a significant difference in drug uptake under var- ious growth conditions. DISCUSSION The role of DNA topoisomerase II in mediating the action of chemotherapy agents such as epipodophyllotox- ins and intercalating agents is somewhat unique, and con- sideration of this is essential to appreciating the basis for proliferation-dependent cytotoxicity (/0). In contrast to most chemotherapy targets, topoisomerase II is an active participant in the formation of the critical lesion, the cleav- 4000 O CCRF A LYMPHS-IL2 3000 OJ LYMPHS +IL2 2000 RAD-EQUIVALENTS 1 1 1 Til 1 1 Kah 10.0 60.0 4M ETOPOSIDE FIGURE 5.—DNA cleavage activity of etoposide in resting and interleukin- 2 stimulated peripheral lymphocytes and cultured CCRF cells. The cells were exposed to various concentrations of etoposide for 1 hr at 37°C. Alkaline elution of the resting lymphocytes was performed using a fluo- rometric assay for DNA (27). 76 able complex. Therefore, any significant reduction in intra- cellular content of the enzyme is likely to diminish cell sensitivity to the DNA cleaving activity of the drugs. Our data would suggest that, indeed, cellular sensitivity to cleavable complex formation following exposure to etopo- side correlates well with enzyme content. This correlation is seen in the three cell lines directly compared (Figure 3), in mitogen stimulated peripheral blood lymphocytes (Figure 5), and when drug sensitivity is assayed during cell cycle traverse (Figure 6). In fact, the general correlation between drug-induced cleavable complex formation and enzyme content would suggest that under certain well-defined con- ditions, this drug sensitivity may be used as an in situ assay for enzyme content. While there are certainly a number of factors which could alter this relationship, the use of this assay has the advantage of reflecting the activity of the enzyme in its natural intracellular milieu. There is substantial evidence that drug-induced cleavable complex formation is the major, initial lesion accounting for the cytotoxicity of drugs which act through topoisom- erase II (/0). It would appear reasonable to predict, therefore, that the alterations in cleavable complex forma- tion observed during quiescence would be associated with a —100 9 0 - m 2 xo 2 or z u o =] w ge 12 © =) — -l < ]l = > 0 1 0 6 12 1618 2123 27 36 Time (hr, Post-Serum-Stimulation) FIGURE 6.—Topoisomerase II specific drug sensitivity and cytotoxicity during cell cycle progression. Sensitivity (closed circles) to DNA cleav- age by etoposide was assayed in vivo by the alkaline elution technique. The drug sensitivity curve was assessed by treating cells with 40 uM etoposide at 37°C for 1 hr. The values shown are expressed as the mean + SD, n=4. Open circles represent cytotoxicity assays. These results are from cells treated with 10 uM etoposide at 37°C for I hr, and the values shown are expressed as the mean® SD, n=6. NCI MONOGRAPHS, NUMBER 4, 1987 high degree of resistance to cytotoxicity. To some extent, this is true. The striking decrease in cleavable complex for- mation observed in quiescent CHO cells undoubtedly plays an important role in their resistance to cytotoxicity. The data with L1210 cells, however, would suggest that resis- tance is multifactorial. These cells exhibit no loss of enzyme content or resistance to cleavable complex formation and yet remain somewhat resistant to drug-induced cytotoxicity. Whether this is due to the absence of active cell cycle tra- verse or to other metabolic changes which attend plateau phase in these cultures is not clear, but a more complete delineation of the basis for this phenomenon would proba- bly contribute substantially to our understanding of the mechanism by which cleavable complex formation results in cell death. In this regard, it is also interesting to note the sensitivity of late S phase cells (Figure 6), despite the fact that greater cleavable complex formation is observed in G,/M. Again, this finding indicates that the circumstances in which cleavable complex formation occurs may have great bearing on the consequences thereof. Stated some- what differently, it is apparent that cleavable complex for- mation is necessary, but not sufficient, for cytotoxicity. As noted in the Introduction, a number of studies have suggested that topoisomerase II activity is higher in prolif- erating than quiescent cells (3-6). As assayed, this could result from either alterations in enzyme content or specific activity. Topoisomerase II can be phosphorylated in vitro by casein kinase (11), several oncogene directed tyrosine kinases (12), and protein kinase C (13). Enzyme activity is apparently stimulated upon phosphorylation. Darby et al (/4) have shown that the enzyme can also be poly- (ADP)ribosylated in vitro which reduces enzyme activity. Furthermore, it is conceivable that changes in intracellular location could affect the enzyme’s ability to participate in the drug-induced DNA cleavage event. Our data would suggest that in spite of these possibilities, the principal mechanism for the observed decrease in enzyme activity in quiescence is a result of loss of enzyme content. This is consistent with what is seen with other enzymes involved in DNA metabolism (15). It is of interest that enzyme content is not lost in all cell lines during quiescence. We suspect this is largely a function of their ability to enter a Gy-like state and that topoisomerase II content may be regulated as part of that process. Further data on this point are needed. It is interesting to speculate on the basis for the sensitiv- ity of the late S phase cells to the cytotoxic actions of etoposide. To date, the only clearly documented function for DNA topoisomerase II is the desegregation of daughter DNA at the conclusion of replication (16,17). Therefore, one possibility is that the presence of the drug interferes with this function, prohibiting the cells from entering mito- sis. Etoposide does, indeed, inhibit topoisomerase II- mediated strand passage events, but this generally occurs at a concentration considerably above that needed for cleava- ble complex formation (78,19). Furthermore, the intercal- ating agents ethidium bromide and 0-AMSA are weakly potent with respect to cytotoxicity and cleavable complex formation, and yet both can inhibit strand passage (20,21). Another possibility is that the highly decondensed state of DNA during S phase makes it a vulnerable target for other events initiated by cleavable complex formation. Pommier et al (22) found that induction of sister chromatid exchanges correlated with the cytotoxicity of intercalating DNA TOPOISOMERASES IN CANCER THERAPY agents, suggesting the possibility of illegitimate recombina- tion events which might disable the cell. Inhibition of DNA synthesis per se does not appear to be a reasonable explana- tion for S phase sensitivity since published evidence would indicate that epipodophyllotoxins have little direct effect on DNA synthesis (23). The potential clinical relevance of our work is readily apparent. Several studies indicate that human leukemia cells are strikingly more resistant to the DNA cleaving effects of epipodophyllotoxins and intercalating agents than cultured cells (24-26). In some instances, this has been correlated with diminished topoisomerase II content (25,26). The demonstration that the lymphokine inter- leukin-2 can increase the sensitivity of normal lymphocytes would suggest that efforts to identify means of accomplish- ing this in malignant cells may improve therapeutic efficacy. REFERENCES (I) ToBEY RA, DEAVEN LL, OKA MS: Kinetic response of cul- tured Chinese hamster cells with 4’-[(9-acridinyl)-amino]- methanesulphon-m-anisidide-HCl. J Natl Cancer Inst 60:1147-1153, 1978. (2) Liu LF, Davis JL, CALENDAR R: Novel topologically knotted DNA from bacteriophage P4 capsids: Studies with DNA topoisomerases. Nucleic Acids Res 9:3979- 3989, 1981. (3) DUGUET M, LAVENOT C, HARPER F, et al: DNA topoisom- erases from rat liver: Physiological variations. Nucleic Acids Res 11:1059-1075, 1983. (4) TAuDOU G, MIRAMBEAU G, LAVENOT C, et al: DNA topoi- somerase activities in concanavalin A-stimulated lympho- cytes. FEBS Lett 176:431-435, 1984. (5) SuLLIVAN DM, GLISSON BS, HODGES PK, et al: Prolifera- tion dependence of topoisomerase II mediated drug action. Biochemistry 25:2248-2256, 1986. (6) MISKIMINS R, MISKIMINS WK, BERNSTEIN H, et al: Epi- dermal growth factor-induced topoisomerases. Exp Cell Res 146:53-62, 1983. (7) TricoL1JV, SAHAI BM, MCCORMICK PJ, et al: DNA topo- isomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells. Exp Cell Res 158:1-14, 1985. (8) ESTEY E, ADLAKHA R, ZWELLING L, et al: Hypersensitivity of mitotic cells to topoisomerase II (topo II)-mediated DNA cleavage. Proc Am Assoc Cancer Res 27:240, 1986. (9) KoHN KW, EwiG RAG, ERICKSON LC, et al: Measurement of strand breaks and cross-links by alkaline elution. In DNA Repair: A Laboratory Manual of Research Tech- niques (Friedberg EC, Hanawalt PC, eds). New York: Marcel Dekker, 1981, pp 379-401. (10) Ross WE: DNA topoisomerases as targets for cancer ther- apy. Biochem Pharmacol 34:4191-4195, 1985. (11) ACKERMAN P, GLOVER CV, OSHEROFF N: Phosphorylation of DNA topoisomerase II by casein kinase II: Modulation of eukaryotic topoisomerase II activity in vitro. Proc Natl Acad Sci USA 82:3164-3168, 1985. (12) TSe-DINH Y-C, WONG TW, GOLDBERG AR: Virus- and cell- encoded tyrosine protein kinases inactivate DNA topo- isomerases in vitro. Nature 312:785-786, 1984. (13) SAHYOUN N, WOLF M, BESTERMAN J, et al: Protein kinase C phosphorylates topoisomerase II: Topoisomerase acti- vation and its possible role in phorbol ester-induced dif- ferentiation of HL-60 cells. Proc Natl Acad Sci USA 83:1603-1607, 1986. 77 (14) DARBY MK, SCHMITT B, JONGSTRA-BILEN J, et al: Inhibi- tion of calf thymus type II DNA topoisomerase by poly(ADP-ribosylation). EMBO J 4:2129-2134, 1985. (15) Liu HT, GIBSON CW, HIRSCHHORN RR, et al: Expression of thymidine kinase and dihydrofolate reductase genes in mammalian ts mutants of the cell cycle. J Biol Chem 260:3269-3274, 1985. (16) DINARDO S, VOELKEL K, STERNGLANZ R: DNA topoisom- erase II mutant of Saccharomyces cerevisiae: Topoisom- erase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 81:2616-2620, 1984. (17) HoLM C, GoTo T, WANG JC, et al: DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553-563, 1985. (18) GLISSON BS, SMALLWOOD SE, Ross WE: Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells. Biochim Biophys Acta 783:74-79, 1984. (19) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative anti- tumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1984. (20) NeLsON EM, TEWEY KM, Liu LF: Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisom- erase II on DNA by 4’-(9-acridinylamino)-methanesulfon- m-anisidide. Proc Natl Acad Sci USA 81:1361-1365, 1984. (21) ROWE T, KUPFER G, Ross W: Inhibition of epipodophyllo- 78 toxin cytotoxicity by interference with topoisomerase- mediated DNA cleavage. Biochem Pharmacol 34:2483- 2487, 1985. (22) POMMIER Y, ZWELLING LA, KAO-SHAN C-S, et al: Correla- tions between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxic- ity in Chinese hamster cells. Cancer Res 45:3143-3149, 1985. (23) MisRA NC, ROBERTS D: Inhibition by 4’-demethyl-epi- podophyllotoxin 9-(4,6-O-2-thenylidene-B-D-glucopyrano- side) of human lymphoblast cultures in G, phase of the cell cycle. Cancer Res 35:99-105, 1975. (24) BrROX LW, BELCH A, NG A, et al: Loss of viability and induction of DNA damage in human leukemic myelo- blasts and lymphocytes by m-AMSA. Cancer Chemother Pharmacol 17:127-132, 1986. (25) EDWARDS C, KENTRO S, Ross W: Heterogeneity in cellular topoisomerase II content and epipodophyllotoxin sensitiv- ity. Clin Res 34:562A, 1986. (26) SILBER R, LIU LF, HSIANG YH, et al: DNA topoisomerase II was not detected in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL). Proc Am Assoc Cancer Res 27:248, 1986. (27) Stout DL, BECKER FF: Fluorometric quantitation of single-stranded DNA: A method applicable to the tech- nique of alkaline elution. Anal Biochem 127:302-307, 1982. Topoisomerase Il as a Target of Antileukemic Drugs Leonard A. Zwelling, * Elihu Estey, Milorad Bakic, Lynn Silberman, and Diana Chan? ? ABSTRACT —The identification of topoisomerase II as a target of antineoplastic drug therapy is traced from the original observations by Ross ef al. (1,2) in murine leukemia cells through studies with m-AMSA -resistant human leukemia cells. Recently developed quantitative biochemical assays of topoisomerase II activity and the susceptibility of topoisomerase II to the effects of m-AMSA have allowed the principles identified in murine and human leukemia cell culture systems to be applied to clinical material; a prospective trial is testing the utility of such assays for individualizing antineoplastic drug therapy.—NCI Monogr 4:79-82, 1987. One of the greatest successes of chemotherapy against human disease is in the treatment of bacterial infections. Antibiotics can exert specific cytocidal actions on invading organisms because the biochemistry of the organisms and the biochemistry of the cells of the host are significantly different. The drugs exploit this difference. Thus, they can be viewed as probes of the phenotypic differences in the cellular biochemistry of bacteria and human cells. Differences between malignant and normal cells are not as readily exploitable yet. However, some forms of dissem- ABBREVIATIONS: m-AMSA = 4’~(9-acridinylamino)methanesulfon- m-anisidide; SDS = sodium dodecyl sulfate. 'Supported by Public Health Service grants CA-40090 (L. A. Zwelling) and CA-39809 (E. J Freireich) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; grant CH-324 (L. A. Zwelling) from the American Cancer Society; a grant from the Leukemia Research Foundation, Inc. (E. Estey); and a gift to the M. D. Anderson Annual Fund for the Chemotherapy Research Program from Mr. Henry C. Beck, Jr., of Dallas, TX. E. Estey is a special fellow of the Leukemia Society of America, Inc. M. Bakic is a visiting scientist from the Internal Clinic of Medical Faculty, University of Nis, Yugoslavia. 2 Departments of Medical Oncology (L. A. Zwelling, M. Bakic, L. Silberman, and D. Chan) and Hematology (E. Estey), Division of Medicine, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston. 3 We thank Mrs. Lola Small for typing the manuscript and Ms. Anne Sullivan for editing it, and we thank our collaborators on this work, including Drs. Miloslav Beran, Borje S. Andersson, Ramesh Adlakah, Emil J Freireich, Bonnie S. Glisson, Yves Pommier, Leonard C. Erickson, Richard Ungerleider, Jon Minford, Michael Mattern, and Warren Ross. We also thank Stephen Michaels, Telly Khademi, Laurie Ricketts, Donna Kerrigan, Ronald Schwartz, Margaret Nichols, and Deborah Miears for their assistance. We give special acknowledgment to Dr. Kurt W. Kohn, Chief of the Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, in whose laboratory all of the murine cell work was performed. * Reprint requests: Leonard A. Zwelling, M.D., Department of Medical Oncology, Division of Medicine, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, P.O. Box 52, 6723 Bertner Ave., Houston TX 77030. inated human neoplasia are treatable and, at times, curable with the use of chemicals. Among the forms of human malignancy occasionally yielding to chemotherapeutic intervention is adult acute myelogenous leukemia. Some antileukemia agents, for example, daunorubicin and 4’-(9- acridinylamino)methanesulfon-m-anisidide (m-AMSA), have in common an interaction with a specific nuclear enzyme, topoisomerase II (/,3). If a drug-topoisomerase II interaction is the critical one that results in cell death, then the selective eradication of malignant leukemia cells from human blood and bone marrow may result from a drug- enzyme interaction that differs in malignant and normal cells. Thus, a cytotoxic drug-topoisomerase II interaction may be an example of a therapeutically exploitable, tumor- specific biochemical reaction analogous to penicillin’s inter- ference with Gram-positive cocci cell wall biosynthesis. If a test could be devised to rapidly quantify the drug-topo- isomerase II interaction within target tumor cells prior to therapeutic intervention, it could supplant the empiricism currently employed in drug selection for individual patients. Further, if a complete understanding of topoisomerase 11 biochemistry was at hand, newer target-specific agents could be designed and tested in a fashion similar to the way in which novel antimicrobials are currently being developed. In this paper, we will review our work that led to the identification of topoisomerase II as a target of some of the drugs that effectively treat human leukemia. Our initial work was in the murine L1210 leukemia system. Subse- quently, we have performed experiments in drug-sensitive and drug-resistant human leukemia lines. Our most recent efforts have examined topoisomerase II activity in ex- planted human blood cells from patients with acute myelog- enous leukemia. PROTEIN-ASSOCIATED DNA CLEAVAGE IN MURINE LEUKEMIA CELLS The initial observations of Ross et al. (1-3) were critical in establishing that a novel form of DNA cleavage was produced by DNA intercalating agents. Intercalating agents are drugs that interdigitate between adjacent DNA base pairs and untwist the DNA helix. Some intercalators are among the most potent clinically active antineoplastic agents such as Adriamycin, daunomycin, and m-AMSA. The DNA cleavage produced by these drugs had been thought to be mediated by free radicals as drugs like Adri- amycin were known to produce free radicals (4-6). How- ever, the Adriamycin-induced DNA cleavage differed from that produced by chemical (e.g., bleomycin) (7) or physical (e.g., x-radiation) agents known to cleave DNA via free radical mechanisms. Although free radical-mediated DNA cleavage was readily quantified using the standard alkaline 79 elution method of Kohn (8), DNA cleavage produced by Adriamycin could only be detected if, prior to DNA filter elution, cell lysates were exposed to detergent protein denaturants and enzymatic proteolysis. Additionally, the frequency of the DNA cleavage produced by intercalating agents was accompanied by an equal frequency of DNA- protein crosslinking. Thus, the cleavage was both “protein- concealed” and “protein-associated.” Further, intercalating agents that were not prolific producers of free radicals were potent generators of protein-associated DNA cleavage (2). It seemed unlikely that intercalator-induced, protein-asso- ciated DNA cleavage in murine leukemia cells was pro- duced by free radicals. Our own work with m-AMSA further characterized this DNA cleavage (9). DNA cleavage production was satura- ble with increasing drug dose; it was also rapidly reversible and temperature-dependent. Further, all of these cellular effects could be reproduced in isolated cell nuclei (10). This suggested that the production of DNA cleavage by drugs such as m-AMSA was mediated by an enzymatic activity localized to the cell nucleus. m-AMSA-INDUCED, PROTEIN-CONCEALED DNA CLEAVAGE ACTIVITY COPURIFIES WITH TOPOISOMERASE II The nuclei from L1210 murine leukemia cells were used as the starting material. The nuclei were extracted with 0.35 M NaCl. The extracted m-AMSA-dependent DNA-protein crosslinking (i.e., DNA cleaving) activity was purified using gel filtration chromatography, DNA cellulose chromatog- raphy, and glycerol gradient centrifugation (/1,12). Aga- rose gels were used to monitor the effects of these isolated proteins on the conformation of covalently closed circular SV-40 DNA. We found that two activities from the glycerol gradients could alter the DNA structure (Figure 1). Both fractions 12-13 and 22-24 contained topoisomerase. Only activity from the fraction 12-13 cleaved DNA in the pres- ence of m-AMSA (second lane from right; generation of linear or form III DNA which is cleaved in both strands). Catenanes — Form ll— Form lll — Form |— Fraction Number —m-AMSA 80 7 12 13156182 22412 2, Other biochemical tests revealed that those fractions con- taining m-AMSA-dependent DNA double-strand cleaving activity also contain m-AMSA-dependent DNA-protein crosslinking activity and DNA catenating activity (ring interlocking, a topoisomerase II-specific reaction [see (12) and also fourth and fifth lanes from left in Fig. 1], whereas fractions 22-24 did not. This activity is topoisomerase II. The activity in 22-24 is putatively topoisomerase I. Drugs such as m-AMSA are believed to stablilize an intermediate in the normal topoisomerase II cycle of strand cleavage-strand passage-break resealing. Once stabilized, this enzyme-DNA cleavable complex forms a cleaved complex with protein covalently bound to the 5’ terminus of the cleavage site when exposed to protein denaturants such as sodium dodecyl sulfate (SDS). SDS is used to lyse cells prior to alkaline elution or to stop the biochemical reaction between proteins and SV-40 DNA prior to agarose gel electrophoresis. Breaks do not exist within the living cell as free swivels (1/3); rather, they are a laboratory phenom- enon representing a site of enzyme action rendered more detectable by drug stabilization followed by protein dena- turation. As such, it is not immediately obvious how this drug effect might lead to cell death. HUMAN LEUKEMIA CELLS SENSITIVE AND RESISTANT TO m-AMSA Three lines of evidence support a mechanistic connection between the topoisomerase 11-mediated, protein-associated DNA cleavage produced by antineoplastic agents and the cytotoxic action of these agents. First, inactive analogs of topoisomerase Il-reactive agents produce much lower quantities of DNA cleavage in cells or isolated biochemical systems than do their more active sister compounds (9,14-17). Second, some antineoplastic antimetabolites such as cytosine arabinoside, hydroxyurea, or 5-azacytidine can concordantly and cotemporally enhance m-AMSA- induced DNA cleavage and cytotoxicity (78,19). Third, some cells resistant to certain topoisomerase II-reactive drugs possess a topoisomerase II activity with a reduced FIGURE 1.—The more rapidly sedimenting of two topoisomerase activities from the nuclei of murine leukemia L1210 cells has m-AMSA-dependent DNA double-strand cleaving activity. Fraction numbers refer to the fraction from a 15 to 40% glycerol gradient [see (1/2) for details]. All pro- tein fractions were reacted with SV-40 covalently closed supercoiled DNA in the presence of 0.5 mM ATP, and reactions were stopped with 0.4% SDS and proteinase K. Form I is the starting material. Form II is DNA with a single stranded nick. Fully relaxed but intact DNA (form I°) migrates to a similar position as form II. Form III is linearized DNA (double-strand cleavage). The material in the well of the fourth and fifth lanes is putatively topoisomerase II-produced catenated (multiply interlocked rings) DNA. Reproduced from (12) with permission. +m-AMSA NCI MONOGRAPHS, NUMBER 4, 1987 FIGURE 2.—The production of m-AMSA-induced DNA cleavage in three lines of leukemia cells. DNA single-strand cleavage was quantified using the alkaline elution method of Kohn (8) with proteinase. Results are expressed as rad-equiva- lents (8,9). All treatments were for 60 min at 37° C. Insets are the results of experiments per- formed at low concentrations of m-AMSA using a high-sensitivity alkaline elution assay (9). The remaining points represent results obtained at higher m-AMSA concentrations that produced break frequencies requiring the use of a low- sensitivity assay for accurate quantification. HL- 60/ AMSA is a daughter line of HL-60 made re- i sistant to m-AMSA by repetitive intermittent m-AMSA Concentration (uM) capacity for stabilization of the DNA complex by the drugs to which the cells are resistant (20-23). Using m-AMSA-sensitive (HL-60) and m-AMSA resis- tant (HL-60/ AMSA) (about 20-fold to 50-fold more resis- tant) human myeloid leukemia HL-60 cells developed by 1 2 3 4 5 8 7 Bl _ (3HIKDNA (catenated) Decatenated [3HIKDNA 1.0 T : T < Closed symbols = Centrifugation z _— 0 amy gS 0.8 (Catenated DNA in pellets) . x : Open symbols = Agarose gel ®, {Catenated DNA in well-numbers oo A \ correspond to lanes in gel above) 2c 061 - Lc SE © o = £ © - — SE 0.4 - © c S 0.2 N Q ®,O=HL-60 NX> & A, H=HL-60/AMSA 2 0 1 1 0 1.0 2.0 Amount of Nuclear Protein Extract (ug) FIGURE 3.—Quantitative measurement of topoisomerase II activity in 0.35 M salt extracts from the nuclei of human leukemia cells. Salt extracts were made as previously described (/2). Target DNA is the highly intertwined [*H]dT-labeled kinetoplast (k)DNA isolated from Crithi- dium fasciculata. Reaction products (fraction remaining catenated) are analyzed in one of two ways. First, the products may be electrophoresed in agarose, stained with ethidium bromide, and the DNA in the well (catenated starting material) or in the lane (decatenated by ATP- dependent topoisomerase II activity) may be excised and the radioactiv- ity determined in each using liquid scintillation spectrometry. Secondly, the reaction products may be centrifuged at 12,000 X g for 5 min. This separates the catenated pellet from the decatenated supernatant, which can then be counted in a liquid scintillation spectrometer. Results with either assay are comparable for the two cell lines HL-60 (lanes 2-4) and HL-60/ AMSA (lanes 5-7). DNA TOPOISOMERASES IN CANCER THERAPY > 2 EPR T T T T Jo HL-60/AMSA HL-60 Eo Lx + S m c T T T° 8 S 5 : 0.4} b n> 0.2} 2? oe 28 % or oz R= ° ry —— g 5 10 5 10 a exposure to m-AMSA. HL-60/ AMSA are stably resistant and do not require the continued pres- ence of the drug to remain resistant. KBM/3 is another m-AMSA-sensitive human leukemia cell line. Reproduced from (20) with permission. Drs. M. Beran and B. Andersson at M. D. Anderson Hos- pital, we demonstrated a marked reduction in the m- AMSA-induced DNA cleavage production in these cells (Figure 2). Cellular uptake of ['“C]m-AMSA was identical in the three lines (20). Topoisomerase II activity from these two cell lines was comparable (Figure 3), yet only that from the HL-60 line would produce enhanced 5-end DNA-protein linking in response to m-AMSA (Figure 4) (24). Thus, the resistance of the topoisomerase II in HL-60/AMSA to m-AMSA action as measured in an isolated biochemical system mir- rored both the resistance to m-AMSA-induced cleavage production in intact cells (Figure 2) and the resistance of the cells to the cytotoxic action of m-AMSA. CLINICAL STUDIES Topoisomerase II activity from human leukemia cells in culture and its susceptibility to m-AMSA’s action are readily monitored and quantified by a combination of aga- rose gel electrophoresis and newer quantitative radioactive DNA assays (see legends to Figs. 3 and 4). The effects of drugs on topoisomerase II parallel the cytotoxic effects of the drugs in the cells from which the topoisomerase II activ- < 15} a = > 3 HL-60 53 oo 10 | - ™ x v= 2a 58 5 ] Q © HL-60/AMSA o a 0 1 1 0 5 10 m-AMSA Concentration (uM) FIGURE 4.—m-AMSA-induced DNA-protein crosslinking using 3’-end labeled SV-40 DNA. The same topoisomerase II-containing extracts used in the experiments depicted in Figure 3 were used in this experi- ment. The target DNA was 3’-end labeled [Z2P]DNA (24). Only DNA covalently bound to protein via a 5" linkage will be precipitated follow- ing SDS and KCI treatment; thus, this reaction is relatively topoisom- erase II specific. m-AMSA could stimulate DNA-protein crosslinking in the extract from HL-60, but not in that from HL-60/ AMSA. 81 ity was derived. An obvious clinical question presents itself. Will examination of drug effects on topoisomerase 11 activities from explanted human leukemia cells allow the establishment of a reproducibly predictive assay that could supplant empiric drug selection for the individual patient? We are currently examining this question prospectively in patients with acute myelogenous leukemia. CONCLUSION We have briefly reviewed how studies in murine leukemia cells have led to our present clinical studies. In addition to these studies, more basic questions still must be answered regarding the mechanistic connection between drug effects on topoisomerase II and drug-induced cytotoxicity. How- ever, the evidence suggests that some connection exists. If some clinical utility can be made of these observations, other biochemical approaches with other tumors and other drugs may also be usefully employed. A melding of clinical and basic approaches may still allow greater individualiza- tion of anticancer therapy much as culture and sensitivity assays of bacteria infecting patients have allowed individu- alization of antimicrobial therapy. REFERENCES (I) Ross WE: DNA topoisomerases as targets for cancer therapy. Biochem Pharmacol 34:4191-4195, 1985. (2) Ross WE, GLAUBIGER DL, KOHN KW: Protein-associated DNA breaks in cells treated with Adriamycin or ellipti- cine. Biochim Biophys Acta 519:23-30, 1978. (3) ZWELLING LA: DNA topoisomerase II as a target of anti- neoplastic drug therapy. Cancer Metastasis Rev 4:263-276, 1985. (4) BACHUR NR, GEE MV, FRIEDMAN RD: Nuclear catalyzed antibiotic free radical formation. Cancer Res 42:1078- 1081, 1982. (5) BACHUR NR, GORDON SL, GEE MV: Anthracycline antibi- otic augmentation of microsomal electron transport and free radical formation. Mol Pharmacol 13:901-910, 1977. (6) POMMIER Y, ZWELLING LA, MATTERN MR, et al: Effects of dimethyl sulfoxide and thiourea upon intercalator-induced DNA single-strand breaks in mouse leukemia (L1210) cells. Cancer Res 43:5718-5724, 1983. (7) CHABNER BA: Bleomycin. In Pharmacologic Principles of Cancer Treatment (Chabner BA, ed). Philadelphia: Saunders, 1982, pp 377-401. (8) KoHN KW, EWIG RAG, ERICKSON LC, et al: Measurements of DNA strand breaks and crosslinks by alkaline elution. In DNA Repair. A Laboratory Manual of Research Tech- niques (Friedberg EC, Hanawalt PC, eds). New York: Marcel Dekker, 1981, pp 379-401. (9) ZWELLING LA, MICHAELS S, ERICKSON LC, et al: Protein- associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4’-(9-acridinylamino)methanesulfon-m-anisidide and Adriamycin. Biochemistry 20:6553-6563, 1981. (10) POMMIER Y, SCHWARTZ RE, KOHN KW, et al: Formation 82 and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents. Biochemistry 33:3194-3201, 1984. (11) SHELTON ER, OSHEROFF N, BRUTLAG DL: DNA topo- isomerase II from Drosophila melanogaster. J Biol Chem 258:9530-9535, 1983. (12) MINFORD J, POMMIER Y, FILIPSKI J, et al: Isolation of intercalator-dependent protein-linked DNA strand cleav- age activity from cell nuclei and identification as topo- isomerase II. Biochemistry 25:9-16, 1986. (13) POMMIER Y, MATTERN MR, SCHWARTZ R, et al: Absence of swivelling at sites of intercalator-induced protein-asso- ciated deoxyribonucleic acid strand breaks in mammalian cell nucleoids. Biochemistry 23:2922-2927, 1984. (14) LoNG BH, MUSIAL ST, BRATTAIN MG: Comparison of cyto- toxicity and DNA breakage activity of congeners of podo- phyllotoxin including VP16-213 and VM26: A quantita- tive structure-activity relationship. Biochemistry 23:1183— 1188, 1984. (15) NELSON EM, TEWEY KM, Liu LF: Mechanism of antitumor drug action: Poisoning of mammalian DNA topoisomer- ase II on DNA by 4’-(9-acridinylamino)methanesulfon- m-anisidide. Proc Natl Acad Sci USA 81:1361-1365, 1984. (16) Ross W, ROWE T, GLISSON B, et al: Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleav- age. Cancer Res 44:5857-5860, 1984. (17) TEWEY KM, CHEN GL, NELSON EM, et al: Intercalative antitumor drugs interfere with the breakage-reunion reac- tion of mammalian DNA topoisomerase II. J Biol Chem 259:9183-9187, 1984. (18) MINFORD J, KERRIGAN D, NICHOLS M, et al: Enhancement of the DNA breakage and cytotoxic effects of intercalating agents by treatment with sublethal doses of 1-8-D- arabinofuranosylcytosine or hydroxyurea in L1210 cells. Cancer Res 44:5583-5593, 1984. (19) ZWELLING LA, MINFORD J, NICHOLS M, et al: Enhancement of intercalator-induced DNA scission and cytotoxicity in murine leukemia cells treated with 5-azacytidine. Biochem Pharmacol 33:3903-3906, 1984. (20) BAKIC M, BERAN M, ANDERSSON BS, et al: The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4’-(9-acri- dinyl-amino)methanesulfon-m-anisidide (m-AMSA). Bio- chem Biophys Res Commun 134:638-645, 1986. (21) GLISSON B, GUPTA R, SMALLWOOD-KENTRO §, et al: Char- acterization of acquired epipodophyllotoxin resistance: Loss of drug-stimulated cleavage activity. Cancer Res 46:1934-1938, 1986. (22) GLISSON B, GUPTA R, HODGES P, et al: Cross resistance to intercalating agents in an epipodophyllotoxin-resistant cell line: Evidence for a common intracellular target. Cancer Res 46:1939-1942, 1986. (23) POMMIER Y, SCHWARTZ R, ZWELLING LA, et al: Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibi- tors. Cancer Res 46:611-616, 1986. (24) Liu LF, Rowe TC, YANG L, et al: Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258: 15365-15370, 1983. Topoisomerase Alterations Associated With Drug Resistance in a Line of Chinese Hamster Cells Yves Pommier, * Donna Kerrigan, and Kurt W. Kohn DNA topoisomerases 11 seem indispensable to cell survi- val. They are found both in prokaryotic and eukaryotic cells (7). Their inhibition is thought to be a key mechanism in the therapeutic effects of various drugs used in infectious diseases and human cancers. It has also been established that temperature-sensitive mutants of DNA topoisomerase II in yeast cannot multiply at nonpermissive temperatures (2). DNA topoisomerases 1I play a crucial role in DNA replication, chromosome segregation (1,3) and the scaffold- ing of DNA in eukaryotic cells (4). Both antitumor DNA intercalators and 4’-demethylepipodophyllotoxin deriva- tives are potent inhibitors of mammalian DNA topoisom- erase II. These compounds trap topoisomerase II-DNA complexes that can be detected as protein-linked DNA strand breaks upon sodium dodecyl sulfate (NaDoSOy) addition (5-12). Topoisomerase II trapping by the drugs inhibits enzyme activity (5-7). A common characteristic of topoisomerase II inhibition by either DNA intercalators or 4’-demethylepipodophyllotoxins is the rapid reversibility (within minutes) of this effect after drug removal (9,10,12). Nevertheless, short drug exposures result in a very potent cytotoxic effect. The causal relationship between drug- induced topoisomerase II inhibition and cytotoxicity re- mains to be proven. There have been several experimental approaches to the question of the relationship between drug-induced topoisomerase II inhibition and cytotoxicity. In all cases topoisomerase II inhibition is determined by measuring protein-linked DNA strand breaks by alkaline elution and cytotoxicity by colony formation assays. The first approach is to correlate topoisomerase II inhibition and cytotoxicity in a given cell line for a large number of compounds. The results of such studies indicate that the number of drug-induced topoisomerase II-mediated DNA strand breaks does not fully account for the cytotoxicity of all drugs (13), although DNA double-strand breaks seem a better indicator of cytotoxicity than DNA single-strand breaks (14). A second approach is to study the effects of one drug in one cell line under conditions that affect the drug- induced topoisomerase II-DNA complexes and/or the cy- totoxicity. Chemicals such as DMSO, thiourea (1/5), and ethidium bromide (/6) have been used. More recently stud- ! Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. * Reprint requests: Yves Pommier, M.D., Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 5A-19, Bethesda, MD 20892. ies with cells at various stages of proliferation have indi- cated that more drug-induced topoisomerase II-DNA com- plexes were formed in proliferative and in S-phase cells than in quiescent cells and that cytotoxicity followed a simi- lar pattern (17,18). The third approach is to study resistant cell lines. In an attempt to elucidate the relationship between drug- induced topoisomerase II inhibition and cytotoxicity cell lines resistant to topoisomerase II inhibitors can be used. Jacquemin-Sablon and co-workers have developed a sub- line of Chinese hamster lung cells (DC3F) resistant to 9-hydroxyellipticine (DC3F/9-OHE). These cells were ob- tained by continuous exposure to stepwise increasing concentrations of 9-hydroxyellipticine (19). They have been shown to be cross-resistant to other ellipticines and to dac- tinomycin, vincristine, and methotrexate. Their resistance to ellipticines and particularly to the hydrophilic antitumor analog, 2-methyl-9-hydroxyellipticinium, is not related to any detectable modification of cellular drug uptake (20). It is clear, however that the resistant cells (DC3F/9-OHE) have a reduced global uptake for dactinomycin and gross morphological changes that suggest that they differ mark- edly from the parent cell line (DC3F). The first step of our study was to investigate the cross- resistance of the DC3F/9-OHE cells to a variety of chemi- cally different topoisomerase 11 inhibitors and to determine whether the eventual cross resistance was due to drug uptake or drug-induced protein-linked DNA strand break modifications (27). The three topoisomerase II inhibitors selected for this study were 2-methyl-9-hydroxyellipti- cinium (2-Me-9-OH-E+), 4’-(9-acridinylamino)methanesul- fon-m-anisidide (m-AMSA), and etoposide (VP-16). 2-Me- 9-OH-E+ is an ellipticine derivative, m-AMSA is one of the most potent topoisomerase II inhibitors whose interac- tion with the enzyme have been well characterized in puri- fied systems and in whole cells, and VP-16 is also a well- characterized topoisomerase II inhibitor which does not intercalate into DNA and has shown promising anticancer activity. DC3F/9-OHE cells appeared highly resistant to the three drugs (Figure 1). This resistance was not due to reduced drug cellular uptake. This was known for 2-Me-9- OH-E+ (20) and was shown for m-AMSA (217). Drug- induced protein-linked DNA strand breaks were then stud- ied by alkaline elution. The production of both DNA strand breaks and DNA-protein crosslinks was markedly lower in DC3F/9-OHE than in DC3F cells exposed to either of the 3 drugs (Table 1). The mechanism(s) involved in the lower sensitivity of DC3F/9-OHE cells to drug- induced topoisomerase II inhibition was further investi- gated by using the isolated nuclei methodology. It was, 83 : 39 DC3F/9-OHE™ 2 LOG SURVIVAL FRACTION bs 1 a o DC3F/9-OHE FIGURE 1.—Cytotoxicity of m-AMSA, 2-Me-9-OH-E+, and VP-16 in Chinese hamster DC3F cells and drug resistant 4 DC3F/9-OHE cells. DC3F (filled sym- E bols) and DC3F/9-OHE cells (open sym- bols) were drug-treated for 30 min at 37°C. Drugs were removed by rinsing i % i i each 25-sq cm flask twice with 10 ml DC3F Hank’s balanced salt solution and by a adding 5 ml fresh medium. 8+ 1 days -4 + : a : He 5 20 5 Ne = : 5 5 5 Le # % A 2 later, colonies were stained and counted. m-AMSA 2-Me-9-OH-E VP-16 CONCENTRATION (uM) CONCENTRATION (uM) indeed known that drug-induced topoisomerase II inhibi- tion could be detected by measuring the formation of protein-linked DNA strand breaks in isolated nuclei (10,22). The isolated nuclei system has the advantage of minimizing drug uptake and drug metabolism variations between cell types. Comparable exposures of isolated nuclei from DC3F/9-OHE and DC3F cells to either m-AMSA, 2-Me-9-OH-E+, or VP-16 showed that DC3F/9-OHE nuclei formed less protein-linked DNA breaks than DC3F nuclei (Table 1). An additional observa- tion was that DC3F/9-OHE nuclei exhibited a higher level of DNA strand breaks than DC3F nuclei in the absence of drug treatment. This endogenous level of DNA breakage was not associated with equal frequencies of DNA-protein crosslinks, which would have been expected in the case of topoisomerase II-mediated DNA breaks. These endoge- nous breaks were, however, reversed by 2-Me-9-OH-E+ (Figure 2). Although the topoisomerase II-mediated DNA breaks are known to be reversed by relatively high concen- tration of 2-Me-9-OH-E+ and other intercalators (5,23), which can be seen in Figure 2 in the case of DC3F nuclei, the nature of the endogenous DNA breakage produced by nuclei isolation of DC3F/9-OHE cells remains unknown. Aside from this, the fact that resistant DC3F/9-OHE cells produced less drug-induced DNA strand breaks was con- sistent with the involvement of topoisomerase II in the cytotoxic effect of m-AMSA, 2-Me-9-OH-E+, and VP-16. A second step in the analysis of the resistance of DC3F/9-OHE cells was then to characterize the nuclear modifications involved in the reduced formation of drug- induced protein-linked DNA breaks. Salt extracts of DC3F and DC3F/9-OHE nuclei were first compared. They had CONCENTRATION (uM) similar topoisomerase I and topoisomerase II activities, as measured by DNA relaxation in the absence of ATP [data not shown, see (25)] and kinetoplast DNA decatenation in the presence of ATP, respectively (Figure 3). DC3F nu- clear extracts contained 2- to 3-fold more topoisomerase 11 molecules than did DC3F/9-OHE extracts, as measured by immunoblotting with L1210 topoisomerase II antibodies (24). Despite the lower amount of topo II, DC3F/9-OHE nuclear extracts had more DNA-protein linking activity in the absence of drug than did DC3F extracts. The DNA- DNA DOUBLE-STRAND BREAKS (DBS-rad-equivalents) 0 1 1 1 1 L 1 1 0 20 40 60 80 2-Me-9-OH-E+ CONCENTRATION (uM) FIGURE 2.—DNA double-strand breaks induced by 2-Me-9-OH-E+ in DC3F (®, solid lines) and DC3F/9-OHE nuclei (O; dashed lines). Nuclei were isolated from DC3F and DC3F/9-OHE cells and treated with the indicated 2-Me-9-OH-E+ concentrations for 30 min at 37°C. Nuclei were then diluted 20 fold in drug-free nucleus buffer at 0°C and assayed for DNA double-strand breaks by DNA nondenaturing alka- line elution. Error bars are the standard deviations of at least three independent determinations. TABLE 1.—DNA-Protein Linking in Sensitive and Resistant Cells? m-AMSA 2-Me-9-OH-E+ VPI16 No drug 2uM SuM 10 uM 40 uM ile odl DC3F 0 1090 N.D.? 511 5936 Die cells DC3F/9-OHE 0 517 N.D. 64 1215 Viozsied masta] DC3F 164 1285 2377 205 5242 Joied pio DC3F/9-OHE 137 539 479 0 1195 “ DNA-protein crosslinks were measured by alkaline elution after 30 min exposures of cells or isolated nuclei to the drugs and are expressed in rad-equivalents. 5 N.D.: not determined. 84 NCI MONOGRAPHS, NUMBER 4, 1987 e ® FRACTION OF MINICIRCLES ° > 0 5 10 15 20 NUCLEAR EXTRACT (ng) FIGURE 3.—Topoisomerase II activity of 0.35 M NaCl extracts from DC3F and DC3F/9-OHE nuclei. Nuclei were isolated from DC3F and DC3F/9-OHE cells. These nuclei were incubated for 30 min at 4°C in 0.35 M NaCl nucleus buffer and then spun down. The supernatants were then collected (salt extracts) and assayed for their decatenation activity. 0.2 ug kDNA was reacted for 30 min at 37°C in the absence (open symbols, dashed lines) or presence of 1 mM ATP (filled symbols, solid lines) and various amounts of DC3F (@®,0) or DC3F/9-OHE (A, A) nuclear extract. Reactions were stopped by adding NaDoSO4 and proteinase K (1% and 0.5 mg/ml final concentrations, respectively). Reaction products were run in 1% agarose gels. Gels were stained with ethidium bromide and the minicircle and kDNA bands cut and counted for [*H] radioactivity. protein activity in DC3F/9-OHE extracts, however, was insensitive to m-AMSA (Figure 4, left columns of panels A and C). DNA topoisomerases and DNA-protein linking activities were then purified from DC3F and DC3F/9-OHE salt nuclear extracts by gel filtration, DNA cellulose affinity chromatography and glycerol gradient centrifugation (24,25). The fractionation of DC3F and DC3F/9-OHE salt nuclear extracts by S-400 gel filtration showed that DC3F nuclear extracts had a single DNA- protein linking activity peak and that this peak was m-AMSA sensitive (Figure 4), which was similar to the case of mouse leukemia L1210 nuclear extracts (25). By con- 1. 2 34 5 6 7 8 FIGURE 5.—ATP-dependence and m-AMSA-induced DNA cleavage activity of DC3F/9-OHE topoisomerases. 0.4 ug SV40 DNA was reacted in 30 ul nucleus buffer at 37°C with 5 ul of purified topoisom- erase II (lanes 3, 6), topoisomerase I (lanes 4, 7), or pool III (lanes 5, 8) in the absence (lanes 3-5) or presence of 10 uM m-AMSA (lanes 6-8). No ATP was added. Reactions were stopped after 30 min with NaDoSO4 and proteinase K (1% and 0.5 mg/ml, respectively). Lanes 1 and 2: native and linear SV40 DNA, respectively. I, IT and III: migration posi- tions of supercoiled, relaxed, and linear SV40 DNA, respectively. trast, DC3F/9-OHE nuclear extracts had two peaks of DNA-protein linking activity. The early peak was m-AMSA sensitive and eluted at a position similar to that of DC3F nuclear extract and the late peak had an unusually high DNA linking activity which was not m-AMSA-sensitive (Figure 4). Each of these two peaks was further purified as described previously (25). The early, m-AMSA-sensitive peak contained both DNA topoisomerases I and II, and the two enzymes could be separated by glycerol gradient cen- trifugation. The DC3F/9-OHE topoisomerase II appeared similar to the DC3F enzyme [same S values, molecular weights, ATP and m-AMSA sensitivities (Figures 5 and 6) and reactivity to L1210 topoisomerase II antibodies]. The fractions of the late peak were also purified by glycerol gradient centrifugation. A topoisomerase activity peak was found and named Pool III. Its sedimentation was similar or T TT T T T t T T lL A t 4 B - \ 7 0.40 A — - —40 } o » FIGURE 4.—Fractionation of salt extracts from 2 - ! \ - - ol T DC3F and DC3F/9-OHE nuclei by gel filtra- £2 ! > tion. 0.35 M NaCl nuclear extracts were obtained x w 0.20 ; — L — 20 a2 as described in the legend of Figure 3. These uw 5 I 3 extracts (1.7 mg DC3F salt extract, panels A, B; < w - + - = 1 mg DC3F/9-OHE salt extract, panels C, D) 2 2 = were loaded onto Sephacryl S-400 columns and 89 1 1 1 1 1 1 0 = eluted with nucleus buffer (24) containing 0.35 oh o M NaCl at 4°C. 3 ml fractions were collected and = © J D | I I T T 5 assayed for protein concentration (panels B, D) =z 1 L © and DNA-protein linking activity (panels A, C). 5 s 10 Z DNA-protein linking was assayed by filter bind- E 3 i | i 5 ing with 0.2 pg protein in 0.1 ml nucleus buffer Ea Es containing either no drug (@) or 10 uM Q m-AMSA (A). Columns on the left of panels A a 2 720 and C are DNA-protein linking activities of 0.2 ug protein of salt extracts before Sephacryl S- 7] B i 400 fractionation in the absence (left columns) or | | \ iq 0 presence of m-AMSA (right hatched columns). 10 20 30 40 50 10 20 30 40 50 SALT EXTRACT FRACTION NUMBER DNA TOPOISOMERASES IN CANCER THERAPY 85 ON POLYVINYL CHLORIDE FILTER ° 3 FRACTION OF [*H]-SV40 DNA RETAINED | 0.30 T T T T A Pool | @ Pool II QO Pool Ill 0.20 — — — No Drug FIGURE 6.—DNA-protein linking activity of DC3F/9-OHE topoisomerases. 0.05 ug [*H]-SV40 DNA was reacted in 0.2 ml nucleus buffer for 30 min at 37°C in the absence of drug (left) or in the presence of 20 uM m-AMSA (right). The DNA- protein linking activities were determined by filter binding assay. 0 0.2 0.4 0 slightly faster than that of topoisomerase I. This topo- isomerase activity was neither ATP-dependent nor m-AMSA-sensitive (Figure 5), and copurified with the unusually high DNA-protein linking activity (Figure 6). It contained two major bands in SDS-PAGE gels at 100 and 55 KD, neither of which binds L1210 topoisomerase II antibodies (24). In summary, it seems that resistance of DC3F/9-OHE cells to topoisomerase II inhibitors is due to complex nuclear modifications, which involve both an approximately 2-fold reduction in drug-sensitive topo- isomerase II and the appearance of an unusual topoisom- erase activity with high DNA-protein linking activity. Whether the new topoisomerase could compensate for the reduced drug-sensitive topoisomerase II and accomplish some of the topoisomerase II functions at the nuclear scaf- fold level is yet unknown. The mechanism(s) by which drug-induced topoisomerase II trapping along the DNA leads to cell death is not known. The drug-induced DNA breaks and chromatin modifica- tions, as measured by nucleoid sedimentation (26), are indeed reversible after drug removal under conditions where more than 99.9% of the cells die. Assuming that the protein-linked DNA strand breaks are involved in the lethal effect of the drugs, these breaks would have to be converted into irreversible cellular damages that are not accessible to alkaline elution measurements. Sister chroma- tid exchanges (SCEs) and mutations are possible can- didates for such damages because they involve DNA breaking-rejoining processes and are produced by topoisom- erase II inhibitors. The relationship between SCEs or mutations and topoisomerase II inhibition is correlated quantitatively (/4), but causality has not been demon- strated. Resistant topoisomerase II modified cells may be an interesting system in which to test such a relationship. The clastogenic effects of topoisomerase II inhibitors were thus compared in DC3F and DC3F/9-OHE cells. DC3F cells produced SCEs after 30-min exposures to either m-AMSA, 2-Me-9-OH-E+, or VP-16 (Figure 7). By con- trast, DC3F/9-OHE cells did not (Figure 7). Such a finding is thus consistent with the possibility that topoisomerase 11 trapping by drugs could induce SCEs. A model involving 86 0.2 0.4 PROTEIN CONCENTRATION (ug/200A reaction volume) enzyme subunit exchanges has been proposed to explain such an effect (/4). Other possible activities of topoisom- erase II can be tested by comparing sensitive and resistant cell lines. Topoisomerase II has been implicated in DNA replication for initiation, elongation, and daughter mole- cule segregation. Cell killing by topoisomerase II inhibitors is preceded by an accumulation of the cells in S and then in the G2 of their last cell cycle (27). DNA synthesis altera- tions can also be detected as a reduction of thymidine incorporation during short pulses in the presence of topo- isomerase II inhibitors. VP-16 decreased DNA synthesis in DC3F cells but had only weak inhibitory effect in DC3F/9-OHE cells (Figure 8). This result is consistent with the involvement of topoisomerase II in DNA synthesis and with a possible role of DNA synthesis alterations in the cytotoxic effect of topoisomerase II inhibitors. In conclusion, comparative studies of cell lines sensitive and resistant to topoisomerase II inhibitors should help clarify the causal relationship between drug-induced topo- isomerase II-mediated DNA strand breaks and the anti- 100 > N TT ry TT rd Z DC3F DC3F/9-OHE G80 - = H | gd eo - - oO 2 7 ® ai FH wl O nw 20 = = gimmie Ll ld bedi ll 0 12 204080 © 1 2 20 40 80 DRUG CONCENTRATION (1M) FIGURE 7.—Sister chromatid exchanges observed in DC3F and DC3F/9- OHE cells exposed to topoisomerase II inhibitors. Cells were treated with m-AMSA (2), 2-Me-9-OH-E+ (O), or VP-16 (J) for 30 min at 37°C. Drug treatments were stopped by rinsing each culture flask twice with Hanks’ balanced salt solution. Fresh medium containing 5-bromo-2- deoxyuridine (10 ug/ml) was then added for an additional 24- to 36-hr period. Chromosomes were then prepared after cell treatment with col- cemid (0.1 ug/ml). Left panel, sensitive DC3F cells; right panel, resis- tant DC3F/9-OHE cells. NCI MONOGRAPHS, NUMBER 4, 1987 100 T T T >, N, \ _ 80 oo | 09 T~o—___ DC3F/9-OHE ac T= £8 Of © £5 0 = E 40 . 8 °8 =n DC3F 0 1. L 1 0 10 25 50 VP-16 Concentration (nM) FIGURE 8.—Inhibition of DNA synthesis by VP-16 in sensitive DC3F and resistant DC3F/9-OHE cells. Cells whose DNA had been prelabeled with ['4C]thymidine, were treated with the indicated VP-16 concentra- tions for 45 min at 37°C. [*H] Thymidine (I uCi/ml) was then added for an additional 15 min at 37°C. Drug treatments and thymidine labeling were stopped by rinsing each culture flask in an excess volume of Hanks’ balanced solution at 4°C twice. Cells were then scraped in 5 ml Hanks’ balanced salt solution at 4°C. DNA was precipitated in 10% TCA and counted for [?H] and [!4C] radioactivity. tumor effect of drugs. Resistance could arise from drug uptake or metabolism modifications, mutant enzymes (see Sullivan et al. in this volume) or chromatin modifications with the appearance of unusual DNA topoisomerases (such seems to be the case of DC3F/9-OHE cells). Comparison of sensitive and resistant cell lines may also be important in determining the molecular structure of DNA topoisomer- ases and their various functions. REFERENCES (I) WANG JC: DNA topoisomerases. Annu Rev Biochem 54:665-697, 1985. (2) UEMURA T, YANAGIDA M: Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: Uncoordinated mitosis. EMBO J 5:1003-1010, 1986. (3) GELLERT M: DNA topoisomerases. Annu Rev Biochem 50:879-910, 1981. (4) EARNSHAW WC, HECK MMS: Localization of topoisom- erase II in mitotic chromosomes. J Cell Biol 100:1716- 1725, 1985. (5) TEWEY KM, ROWE TC, YANG L, et al: Adriamycin-induced DNA damage mediated by mammalian DNA topoisom- erase II. Science 226:466-468, 1984. (6) CHEN GL, YANG L, ROWE TC, et al: Non-intercalative anti- tumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1985. (7) POMMIER Y, MINFORD JK, SCHWARTZ RE, et al: Effects of the DNA intercalators 4’-(9-acridinylamino)methanesul- fon-m-anisidide 2-methyl-9-hydroxyellipticinium on topo- isomerase II-mediated DNA strand cleavage and strand passage. Biochemistry 24:6410-6416, 1985. (8) Ross WE: DNA topoisomerases as targets for cancer therapy. Biochem Pharmacol 34:4191-4195, 1985. (9) ZWELLING LA, MICHAELS S, ERICKSON LC, et al: Protein- associated DNA strand breaks in L1210 cells treated with the DNA intercalating agent, 4’-(9-acridinyl-amino)- methanesulfon-m-anisidide and Adriamycin. Biochemistry 20:6553-6563, 1981. (10) POMMIER Y, SCHWARTZ RE, KOHN KW, et al: Formation and rejoining of deoxyribonucleic acid double-strand breaks produced in isolated cell nuclei by antineoplastic antitumor intercalating agents. Biochemistry 23:3194- 3201, 1984. DNA TOPOISOMERASES IN CANCER THERAPY (11) Ross WE, ROWE T, GLISSON B, et al: Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleav- age. Cancer Res 44:5857-5860, 1984. (12) LoNnG BH, MusiAL ST, RATTAIN MG: Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP16-213 and VM26: A quan- titative structure-activity relationship. Biochemistry 23: 1183-1188, 1984. (13) ZWELLING LA, MICHAELS S, KERRIGAN D, et al: Protein- associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl- 9-hydroxyellipticinium. Biochem Pharmacol 31:3261-3267, 1982. (14) POMMIER Y, ZWELLING LA, SHAN CS, et al: Correlation between intercalator-induced DNA strand breaks, sister chromatid exchanges, mutations and cytotoxicity in Chi- nese hamster cells. Cancer Res 45:3143-3149, 1984. (15) POMMIER V, ZWELLING LA, MATTERN MR, et al: Effects of dimethylsulfoxide and thiourea upon intercalator-induced DNA strand breaks in mouse leukemia (L1210) cells. Cancer Res 43:5718-5724, 1983. (16) ROWE T, KUPFER G, ROSS W: Inhibition of epipodophyllo- toxin cytotoxicity by interference with topoisomerase- mediated DNA cleavage. Biochem Pharmacol 34:2483- 2487, 1985. (17) SuLLIVAN DM, GLISSON BS, HODGES PK, et al: Prolifera- tion dependence of topoisomerase II mediated drug action. Biochemistry 25:2248-2256, 1986. (18) MARKOVITS J, POMMIER Y, KERRIGAN D, et al: Topoisom- erase [I-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle. Cancer Res. In press. (19) SALLES B, CHARCOSSET JY, JACQUEMIN-SABLON A: Isola- tion and properties of Chinese hamster lung cells resistant to ellipticine derivatives. Cancer Treat Rep 66:327-338, 1982. (20) CHARCOSSET JY, SALLES B, JACQUEMIN-SABLON A: Uptake and cytofluorescence localization of ellipticine derivatives in sensitive and resistant Chinese hamster lung cells. Bio- chem Pharmacol 32:1037-1044, 1983. (21) POMMIER Y, SCHWARTZ R, ZWELLING LA, et al: Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibi- tors. Cancer Res 46:611-616, 1986. (22) POMMIER Y, KERRIGAN D, SCHWARTZ R, et al: The forma- tion and resealing of intercalator-induced DNA strand breaks in isolated L1210 cell nuclei. Biochem Biophys Res Commun 107:576-583, 1982. (23) POMMIER Y, SCHWARTZ RE, ZWELLING LA, et al: Effects of DNA intercalating agents on topoisomerase II-induced DNA strand cleavage in isolated mammalian cell nuclei. Biochemistry 24:6406-6410, 1985. (24) POMMIER Y, KERRIGAN D, SCHWARTZ R, et al: Altered topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res 46:3075-3081, 1986. (25) MINFORD J, POMMIER Y, FILIPSKI J, et al: Isolation of intercalator-dependent protein-linked DNA strand cleav- age activity from cell nuclei and identification as DNA topoisomerase II. Biochemistry 25:9-16, 1986. (26) POMMIER Y, MATTERN MR, SCHWARTZ RE, et al: Changes in deoxyribonucleic acid linking number due to treatment of mammalian cells with the intercalating agent, 4’-(9- acridinylamino)methanesulfon-m-anisidide. Biochemistry 23:2927-2932, 1984. (27) SMITH PJ, ANDERSON CO, WATSON JV: Predominant role for DNA damage in etoposide-induced cytotoxicity and cell cycle perturbation in human SV40-transformed fibro- blasts. 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EL pti pT 1; ES le ade ai +, wo gal < TO An ue . y is i ii : “a nH, 4 PAT ar wo hE ay fai i LPN =! i aly “he an PL En pT =H os Bh Si a IY = HL Ax SE fa BA] mah FARA ei i Prats pr=h A "Apna “oo hi ema + i eh dpe lye oP Lis Sapte = Spy ak i Na ink id iA BS atlas si w oe resi be lk i : ie 1 x a Yea Ca a Ay LO Ress rth 28 ; BRA ni $62 LF el =e, eng E a. 1k = : om phd uitia Lat Feadit, bes A te AB os oe qi! hie afb ade tik 4 Rapa rn BfY, gal da J i Ey si : it gr fe } gf de B Fe qa | : lil i ELAN TaleR RS RODE gi nas, 1 len SL Jek wif 13, et RA Sry i Eatin wy 5 Fr: 3 0 fi ¥ dl oe dais Fibs ome ; i fh FRR etd Patel Enragitic. sk hi Tite vu ith dan lr a foi Shed ia EF at | gi 48 hs Hk oF i wef 4 et = 2 E SNe = in Les ate = bk Shu - Mediation of Multi-drug Resistance in a Chinese Hamster Ovary Cell Line by a Mutant Type Il Topoisomerase Bonnie S. Glisson," * Daniel M. Sullivan,’ Radhey Gupta,? and Warren E. Ross? ABSTRACT —Identification of DNA topoisomerase II as the intracellular target for the DNA cleavage activity of the epipodo- phyllotoxins and several intercalating agents is well established. In contrast, definite correlation of cleavable complex formation with eventual cell death has been more difficult to document. Our stud- ies with an epipodophyllotoxin resistant cell line not only pro- vide additional evidence that the enzyme is a multi-drug target, but also implicate drug-stimulated cleavage activity as a critical component of cytotoxic effect. When compared to WT (wild type) cells, the mutant Chinese hamster ovary cell line, Vpm~-5, exhib- its marked resistance to both the cytotoxic and DNA cleavage activity of etoposide as well as various intercalating agents. Steady state concentrations of these drugs in both cell lines is identical. Enzyme content as measured by immunoblot and by total decate- nation activity in crude nuclear extracts is equal. Catalytic activity is also equally sensitive to inhibition by etoposide. In contrast, cleavage activity in purified enzyme from VpmR-5 cells is pro- foundly resistant to stimulation by drug. These data indicate that a multi-drug resistant phenotype may be acquired by a qualitative change in the enzyme that alters its interaction with drug. Further, the data strongly support a direct role for cleavable complex for- mation in cell death.—NCI Monogr 4:89-93, 1987. INTRODUCTION Evidence accumulated in the past eight years has given rise to the concept that the nuclear enzyme, DNA topoisom- erase II, acts as the mediator of DNA cleavage produced by a diverse group of drugs, including the epipodophyllotox- ins, which do not bind to DNA, and several intercalating agents (/-3). The most persuasive evidence of this is the demonstration that m-AMSA-induced, protein-associated DNA breaks created intracellularly can be immunoprecipi- tated with polyclonal antibody specific for type II topo- isomerase (4). Mammalian type II topoisomerases are homodimeric proteins (Mr 170 kDa) whose critical role in regulating ABBREVIATIONS: m-AMSA =4’-(9-acridinylamino)methanesul- fon-m-anisidide; CHO= Chinese hamster ovary; WT=wild type; HBSS =Hank’s balanced salt solution; KkDNA=kinetoplast DNA; 0-AMSA =4'-(9-acridinylamino)methanesulfon-o-anisidide. ! Departments of Pharmacology and Medicine, College of Medicine, University of Florida, Gainesville. 2 Department of Biochemistry, McMaster University, Hamilton, Onta- rio, Canada. 3 We thank Patricia Coldiron for help in preparation of the manuscript. * Reprint requests: Bonnie S. Glisson, M.D., Department of Medical Oncology, Box 39, Division of Medicine, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 1515 Hol- combe, Houston, TX 77030. structure and function of DNA is just now becoming understood [for reviews, see (5,6)]. In vitro, the enzyme catalyzes the topological passing of two double-stranded DNA segments by transiently introducing a reversible dou- ble strand break in one of the crossing segments. This dou- ble strand break, through which strand-passing occurs, actually represents a reaction intermediate topologically constrained by enzyme subunits which are covalently bound to the 5” end of each broken strand and also tightly associated with the 3’ ends. Enzyme dissociation and nick resealing typically follow the strand-passing event to complete the reaction (6). Certain cancer chemotherapeutic agents, including the epipodophyllotoxins and several DNA intercalators, appear to block the reversibility of this reaction by inhibiting enzyme dissociation and nick closure, thereby stabilizing the so-called cleavable complex (2,3). Exposure of this stabilized reaction intermediate to a pro- tein denaturant allows it to be detected as protein-linked breaks in DNA. These drugs also appear to have a second effect on the enzyme by inhibiting the strand-passing func- tion, i.e., catalytic activity (3,7). While it is clear that the drugs do behave in this manner in cellular and reconsti- tuted systems, the precise relationship of these two separate effects to eventual cell death remains unclear. The data presented herein represent detailed study of an epipodophyllotoxin-resistant CHO cell line, VpmR-5, in an effort to further define drug-enzyme interactions that are critical to antitumor effect. The Vpm®-5 line is markedly resistant to the DNA cleavage and cytotoxic activity of etoposide and is cross-resistant to three chemically unre- lated intercalating agents, all of which stimulate type II topoisomerase mediated cleavage of DNA. Studies with a somatic cell hybrid line demonstrate that etoposide sensitiv- ity is dominant and accompanied by reconstitution of drug- mediated DNA cleavage activity. Enzyme content as mea- sured by immunoblotting and decatenation activity in crude nuclear extracts is equal in both WT and Vpm?R-5 cell lines. Decatenation activity is also equally sensitive to inhi- bition by drug. In contrast, DNA cleavage activity in puri- fied enzyme from VpmR®-5 cells is quite resistant to stimula- tion by both etoposide and m-AMSA. Our findings serve to underscore the critical role of DNA topoisomerase II as a multi-drug target and further implicate formation of the cleavable complex as a necessary component of cytotoxic activity. MATERIALS AND METHODS Chemicals.—Cell culture medium, fetal calf serum, trypsin, and HBSS were purchased from GIBCO (Grand Island, NY), ["*C]thymidine (53 mCi/mmole) and 89 [-*P]dATP (3200 Ci/mmole) were obtained from ICN (Irvine, CA). Etoposide was a gift from Bristol Laborato- ries (Syracuse, NY). m-AMSA was obtained from the National Cancer Institute, Adriamycin from Adria Labs (Columbus, OH), and mitoxantrone from Lederle (Pearle River, NY). All drugs were dissolved in dimethyl sulfoxide except Adriamycin which was dissolved in water. Tetra- propylammonium hydroxide was purchased from RSA Corp. (Ardsdale, NY). HindIll and Klenow fragment were obtained from BRL (Gaithersburg, MD). All other chemi- cals were purchased from Sigma Chemical Co. (St. Louis, MO). Cell lines and culture techniques.—CHO cells were grown in monolayer and in suspension in glass spinner flasks in e-minimal essential medium (without ribonucleo- sides) with 5% fetal calf serum, penicillin, and strepto- mycin. The VpmR®-5 line was formed by mutagenization of parental (WT) CHO cells with ethylmethane sulfonate and selected for teniposide resistance as described by Gupta (8). The somatic cell hybrid line M,J,-7 is the product of fusion of VpmR-5 cells with an epipodophyllotoxin-sensitive CHO line EOT-3 using the method of Norwood (9). Colony forming assays were performed as previously described (10). Alkaline elution.—High and low frequency single strand breaks in DNA were assayed as described in (/0) using a modification of a previous technique (/7,12). Assay of intracellular drug concentration.—Steady state concentrations of [*H]etoposide, [*Clm-AMSA, and ['“C]daunomycin were measured as previously described (13); 1 X 10” monolayer cells in mid-log phase were treated with drug for 60 min. at 37°C, harvested, washed with phosphate-buffered saline 2 times and assayed. Preparation of DNA substrates.—Kinetoplast DNA was purified from sarkosyl extracts of Crithidia fasciculata cul- tures by cesium chloride-ethidium bromide density centrif- ugation as described in (/4). Supercoiled dimer of plasmid pBR322 was prepared from FE. coli strain HB101 as in (15). The procedure for end-labeling of DNA restriction frag- ments is fully described in (10). Crude nuclear extracts and enzyme purification.—Nuclei were isolated from 1X 108 cells in suspension and extracted with 0.35 M NaCl as described in (10). Aliquots of crude extracts were stored at —20°C in 50% glycerol. Enzyme from 2X10" WT and VpmR-5 cells was purified from a 1 M NaCl extract of the nuclear pellet. An FPLC system (Pharmacia, Piscataway, NJ) and a three column purifica- tion protocol involving sequential use of hydroxyl apatite (LKB, Gaithersburg, MD) MONO-Q, and Superose-12 (Pharmacia, Piscataway, NJ) routinely resulted in a prepa- ration 98% pure on silver stain. Topoisomerase assays.— The standard reaction mixture for all assays contained 50 mM Tris-HCI (pH 7.5), 85 mM KCl, 10 mM MgCl,, 5 mM dithiothreitol, 0.5 mM EDTA, bovine serum albumin (0.03 mg/ml), and 1 mM ATP. WT and VpmR-5 extracts were adjusted to equalize total protein concentration. Decatenation of kDNA was carried out as previously described (1/0). Quantitative analysis of DNA cleavage activity was assayed using 3'[*’P]-end-labeled DNA restriction fragments and the SDS-K Cl precipitation of topoisomerase II-DNA complexes as originally de- scribed by Liu (16). Western blotting.—Nuclear extracts and whole cell 90 lysates were electrophoresed on PAGE-SDS gels as de- scribed by Laemmli (17). Protein was transferred to nitrocel- lulose and blots were incubated with mouse anti-topoisom- erase II antiserum obtained by immunization with partially purified Hela topoisomerase II. The Bio-Rad Immuno- Blot assay kit (Bio-Rad Laboratories, Richmond, CA) was used to develop the blot as described previously (18). RESULTS For the purposes of this study we have quantified the degree of resistance by defining it as the factor by which drug dose must be multiplied to produce equitoxic effects in the Vpm®-5 line when compared to WT cells. The 30-fold level of resistance of the VpmR-5 cells to etoposide-induced DNA cleavage activity (Fig. 1) is well correlated with the 20-fold level of resistance to etoposide-mediated cytotoxic- ity (Fig. 2). Based on studies with a somatic cell hybrid line, the product of fusion of VpmR-5 cells with drug-sensitive CHO cells, drug sensitivity appears to be a dominant trait. M,J,7 cells retain only a 2-fold level of resistance to the cytotoxic effects of etoposide (8) and show a parallel dim- inution in resistance to etoposide-mediated strand-breaking activity (10). The VpmR®-5 cell line is also cross-resistant to the inter- calators, m-AMSA and mitoxantrone, though the degree of resistance is less profound, on the order of 5- to 10-fold. The results with Adriamycin, however, were qualitatively quite different than those obtained with the other drugs and bear closer scrutiny. Specifically, VpmR-5 cells, while exhib- iting a 5-fold level of resistance to the cytotoxic effects of Adriamycin at concentrations less than 2 uM, undergo a striking increase in the degree of resistance at higher con- centrations of drug, associated with a sharp plateau in the dose-response curve (10). This effect is persistent at an Adriamycin concentration as high as 100 uM and is mir- rored in the dose-response curve for Adriamycin-induced single strand breaks. High concentrations of other strong intercalators, namely, ethidium bromide and 2-methyl-9- hydroxyellipticine, have previously been reported to inhibit drug-stimulated type II topoisomerase DNA cleavage activ- wT 150 vpmR-5 SSB Rad Equivalents 50 1 1 1 J 51 5 10 15 25 VP-16 (uM) FIGURE 1.—VP-16-induced single strand break frequency (SSB) in WT and VpmR-5 cells as measured by rad equivalents, i.e., that amount of radiation required to produce equivalent DNA damage. Cells in mono- layer were treated with drug for 1 hr at 37°C, rinsed with cold PBS and harvested by scraping in 1 ml of HBSS/.02% EDTA. DNA was eluted at pH 12.1 with a speed of .03-.04 ml/min for 15 hrs. Proteinase K was used in the lysis step. NCI MONOGRAPHS, NUMBER 4, 1987 O.l o c 2 E Vpm'=5 0 c 2 = o 2 w 0.0! wT 0.001 I — L 1 J 51015 50 100 150 pM VP-16 FIGURE 2.—Effect of VP-16 on colony-forming ability of WT and VpmR-5 cells. Points, mean of at least four separate experiments; bars, SE. Reproduced from (10) with permission. ity and cytotoxicity (19,3). Although the mechanism for this is not well defined, it may be related to progressive intercalation of drug interfering with accessibility of enzyme to interactive sites on DNA. In an effort to deter- mine if this effect might explain the peculiar behavior of the Vpm?R-5 line in response to Adriamycin, we examined the intercalator’s effect on etoposide-induced strand breaks in VpmR-5 cells. We found a progressive decrease in etoposide-induced strand breaks as Adriamycin concentra- tion is increased from 2 to 80 uM (13), an effect which was not explained by altered drug transport. Confirmation that this effect is dependent upon Adriamycin concentrations comes from the observation that when WT and Vpm?®-5 cells are exposed to very low doses of Adriamycin con- tinuously, the plateau in the dose-response curve for the VpmR-5 line is completely eliminated (13). The level of re- sistance in these assays is 5-fold reproducing the results with pulse doses of 0.2-2 uM. Identical steady-state concentrations of radiolabeled etoposide, mn-AMSA, and daunomycin in WT and Vpm®-5 cells effectively rule out altered transport as a contributing factor in VpmR-5 resistance (10,13). Type II topoisomerase catalytic activity in crude 0.35 M NaCl extracts of WT and VpmR-5 cells was assayed by decatenation of kinetoplast DNA, a highly catenated net- work DNA that is resolved into minicircles upon exposure to type II topoisomerase. Fig. 3 illustrates that catalytic activity in nuclear extracts from both cells is very nearly equipotent, a finding which was confirmed repeatedly with different extracts. In addition, immunoblots with polyclo- nal antibody to type II topoisomerase from HeLa cells have shown that total enzyme content and molecular weight in whole cell lysates and nuclear extracts from WT and VpmR-5 cells are approximately equal (manuscript in prep- aration). Interestingly, we also found that decatenation activity from both cell lines is equally sensitive to inhibition by etoposide (Fig. 4), an effect which, notably, required rather high doses of drug. DNA cleavage activity in extracts of VpmR®-5 cells was first found to be resistant to stimulation by etoposide using DNA agarose electrophoresis to monitor formation of lin- earized DNA (Form III) from supercoiled pBR322 DNA (10). This finding was reproduced in a more specific and quantitative manner using the SDS-KCI precipitation assay of Liu, which measures 5’ protein-linked DNA cleav- age products (10). Of most significance, we have more recently been able to confirm the data from crude nuclear extracts with enzymes purified to homogeneity from WT and VpmR-5 cells. Decatenating activity in purified enzymes from both cell lines is equally sensitive to inhibition by etoposide (data not shown). In contrast, the striking resistance of the purified VpmR-5 enzyme to stimulation of cleavage by etoposide and m-AMSA is demonstrated in Fig. 5. DISCUSSION Historically, the study of drug-resistant cell lines has con- tributed significantly to our comprehension of the nature of drug action at the molecular level. Certainly, this applies to the observations that we have made in the VpmR-5 cell line. We have clearly demonstrated that a multi-drug-resistant phenotype to structurally diverse agents which stimulate type 11 topoisomerase-mediated cleavage of DNA can be acquired by a qualitative change in the enzyme. Drug inter- action with this mutant enzyme or enzyme-DNA complex is altered in such a way that formation of the cleavable complex is inhibited while the enzyme retains sensitivity to drug-mediated inhibition of strand passing activity. While this observation offers compelling evidence that the enzyme WT Vpm*-5 1:10 1:100 1:10 1:100 FIGURE 3.—Decatenation of kinetoplast DNA by CTLI1 2 1 2255/11 2 1 2255 serial dilutions of crude nuclear extracts from MINICIRCLES —— DNA TOPOISOMERASES IN CANCER THERAPY WT and VpmR-5 cells. The bottom row of numbers refer to extract dose in microliters in a final volume of 20 ul. Extract protein concentra- tions from the two cell lines were adjusted to equivalence. Reproduced from (/0) with per- mission. 91 WT Vpm"-5 CTLIO 50 100 2001] 0 50 100 200 MINICIRCLES — IB is indeed a multi-drug target in the cell, these data in toto also represent, perhaps, the strongest evidence to date that stabilization of the cleavable complex intermediate by drug is a critical and necessary component of antitumor effect. Previous investigations studying the relationship of enzyme-mediated DNA cleavage and cytotoxicity have util- ized two major approaches, the study of drug congeners and the use of inhibitory agents. Epipodophyllotoxin con- geners as well as several acridine dye derivatives have been compared in both whole cell and reconstituted systems, and consistently, potency in inducing DNA cleavage effects has been directly related to cytotoxic activity (20,21). Disulfi- ram, a sulfhydryl blocking agent, and ethidium bromide, a weakly cytotoxic intercalating agent, have both been used to demonstrate that inhibition of drug-stimulated topo- isomerase II-mediated cleavage of DNA is well correlated with protection from etoposide-induced cytotoxicity (22,19). More recent data obtained in ellipticine-resistant Chinese hamster lung cells supply additional corroborative evidence in that resistance of these cells to several topo- isomerase-active agents was accompanied by loss of strand- breaking activity in whole cells and nuclei (23). Our data indicate that inhibition of normal topoisom- erase function (i.e., catalytic or strand-passing activity) is unlikely to play a significant part in cytotoxicity. This was 50 A B 40 | a & g of 4 J 20} = © ° Pel ; be Jor mie" | -10 1 1 1 1 1 or) 5 1 1 1 1 0 20 40 60 8 100 O 10 20 30 40 50 VP-16 (uM) m-AMSA (uM) FIGURE 5.—Effect of VP-16 (A) and m-AMSA (B) in stimulating type II topoisomerase-mediated cleavage activity using purified WT (@) and VpmR-5 (0) enzymes. 92 FIGURE 4.—Effect of VP-16 on decatenation of kinetoplast DNA. Numbers refer to [VP-16] in micromolar units. Extract dose was 2 ul in a final volume of 20 wl. Reproduced from (10) with permission. first suggested by work with congeners of intercalating agents, the ortho- and meta- isomers of AMSA, and ellipti- cine and 2-methyl-9-hydroxyellipticine (2,3). o-AMSA and ellipticine are weakly cytotoxic compounds with limited ability to stimulate DNA cleavage, yet they each possess nearly equipotent activity in inhibiting catalytic activity of purified enzyme when compared to their respective partners. Similarly, ethidium bromide, a strong intercalator and potent inhibitor of type II topoisomerase strand-passing activity, is not a potent cytotoxic agent and blocks rather than stimulates type II topoisomerase-mediated DNA cleavage (19). Sharp plateaus in the dose-response of Vpm®-5 cells to the cytotoxic and strand-breaking activities of Adriamycin were of note and prompted further examination. This led to documentation that the same doses of Adriamycin which lead to loss of dose-response in the VpmR®-5 line also inhibit etoposide-induced strand-breaking activity. This suggests that high concentrations of this agent can interfere with type II topoisomerase-mediated cleavage as has previously been shown with ethidium bromide and 2-methyl-9-hy- droxyellipticine (19,3). This finding, in combination with elimination of the plateau in the dose-response curve by continuous low dose exposure to Adriamycin, clearly implicates type II topoisomerase as the major intracellular target of Adriamycin in this cell line. While our findings represent the first direct evidence that a mutant topoisomerase II can mediate multi-drug resis- tance in mammalian cells, given the complexities of drug- topoisomerase interactions, it is quite likely that other mechanisms of resistance involving the enzyme will be defined in the future. Indeed, Pommier et al (24) have reported quite different findings in their studies of a Chi- nese hamster lung cell line, DC3F, and an ellipticine resis- tant subline DC3F/9-OHE. Although cleavage activity in DC3F/9-OHE nuclear extracts is resistant to stimulation by drug, purified enzymes from DC3F and DC3F/9-OHE cells are equally responsive to drug. These investigators have hypothesized that a nuclear factor associated with unusual type I topoisomerase activity in the resistant cell line modifies type II topoisomerase-drug interactions. It is of note that at least one other mechanism of drug resistance appears to be operative in this cell line. Unlike VpmR®-5 cells, which were selected for resistance in a single step and are not cross resistant to agents which do not interact with type II topoisomerase, the DC3F/9-OHE cells were derived by exposure of parental cells to increasing concentrations of 9-hydroxyellipticine (25) and are cross-resistant to a number of agents which are not topoisomerase active. This NCI MONOGRAPHS, NUMBER 4, 1987 broad cross-resistance is explained at least in part on the basis of altered drug transport (23,24). Obviously, resis- tance in the DC3F/9-OHE cell line is complex. Many of the differences apparent between this cell line and Vpm®-5 cells may partially be explained by the differing conditions under which resistance was selected. The possible clinical implications of multi-drug resis- tance mediated by altered drug-topoisomerase II interac- tions remain speculative. Preliminary results from the study of an m-AMSA resistant human promyelocytic leukemia cell line suggest that a mechanism involving the enzyme may be operative (26,27). Clearly, investigation of other human tumor cell lines is a necessary first step in defining the significance of this phenomenon in the clinical realm. In summary, we have defined a new mechanism of multi- drug resistance mediated by a mutant type II topoisom- erase; and, in so doing, have provided strong evidence of the enzyme’s critical role as a multi-drug target in cancer chemotherapy. The data also clearly indicate that forma- tion of the cleavable complex, rather than loss of normal enzyme activity, is intrinsic to cytotoxic effect. Provocative questions remain concerning the precise nature of drug- topoisomerase interactions at the molecular level. Future work with the mutant enzyme from VpmR-5 cells should prove quite useful in addressing these issues. REFERENCES (I) Ross W, ROWE T, GLISSON B, et al: Role of topoisomerase IT in mediating epipodophyllotoxin-induced DNA cleav- age. Cancer Res 44:5857-5860, 1984. (2) TEWEY KM, ROWE TC, YANG L, et al: Intercalative anti- tumor drugs interfere with the breakage-reunion of mam- malian DNA topoisomerase II. J Biol Chem 259:9182- 9187, 1984. (3) TEWEY KM, ROWE TC, YANG L, et al: Adriamycin-induced DNA damage mediated by mammalian DNA topoisom- erase II. Science 226:466-468, 1984. (4) YANG L, ROWE TC, NELSON EM, et al: In vivo mapping of DNA topoisomerase II specific cleavage sites on SV40 chromatin. Cell 41:127-132, 1985. (5) WANG JC: DNA topoisomerases. Annu Rev Biochem 54:665-697, 1985. (6) Liu LF: DNA topoisomerases—Enzymes that can catalyze the breaking and rejoining of DNA. CRC Crit Rev Bio- chem 15:1-24, 1983. (7) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative anti- tumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:13560-13566, 1984. (8) GupTA RS: Genetic, biochemical, and cross-resistance stud- ies with mutants of Chinese hamster ovary cells resistant to the anticancer drugs, VM-26 and VP-16-213. Cancer Res 43:1568-1574, 1983. (9) NorwooD TH, ZEIGLER CJ, MARTIN GM: Dimethyl sulfox- ide enhances polyethylene glycol-mediated somatic cell fusion. Somatic Cell Genet 2:263-270, 1976. (10) GLISSON B, GUPTA R, SMALLWOOD-KENTRO S, et al: Char- acterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: Loss of drug-stimulated DNA cleavage activity. Cancer Res 46:1934-1938, 1986. DNA TOPOISOMERASES IN CANCER THERAPY (11) KoHN KW, ERICKSON LC, EwIG RAG, et al: Fractionation of DNA from mammalian cells by alkaline elution. Bio- chemistry 14:4629-4637, 1976. (12) ZWELLING LA, MICHAELS S, ERICKSON LC, et al: Protein- associated deoxyribonucleic acid strand breaks in L1210 cells treated with the deoxyribonucleic acid intercalating agents 4’-(9-acridinyl-amino)methanesulfon-m-anisidide and Adriamycin. Biochemistry 20:6553-6563, 1981. (13) GLISSON B, GUPTA R, HODGES P, et al: Cross-resistance to intercalating agents in an epipodophyllotoxin-resistant Chinese hamster ovary cell line: Evidence for a common intracellular target. Cancer Res 46:1939-1942, 1986. (14) ENGLUND PT: The replication of kinetoplast DNA networks in Crithidia fasciculata. Cell 14:157-168, 1978. (15) CLEWELL DB: Nature of Col E; plasmid replication in Escherichia coli in the presence of chloramphenicol. J Bac- teriol 10:667-676, 1972. (16) Liu LF, Rowe TC, YANG L, et al: Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258: 15365-15370, 1983. (17) LAEMMLI UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685, 1970. (18) EDWARDS CM, GLISSON BS, KING CK, et al: Etoposide- induced DNA cleavage in human leukemia cells: Correla- tion with type II topoisomerase content. Cancer Che- mother Pharmacol. Submitted for publication. (19) Rowe T, KUPFER G, Ross W: Inhibition of epipodophyllo- toxin cytotoxicity by interference with topoisomerase- mediated DNA cleavage. Biochem Pharmacol 34:2483- 2487, 1985. (20) LoNG BH, MusIAL ST, BRATTAIN MG: Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP-16-213 and VM-26: A quantitative structure-activity relationship. Biochemistry 23:1183-1188, 1984. (21) ROWE TC, CHEN GL, HSIANG YH, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (22) WozNIAK AJ, GLISSON BS, HANDE KR, et al: Inhibition of etoposide-induced DNA damage and cytotoxicity in L1210 cells by dehydrogenase inhibitors and other agents. Cancer Res 44:626-632, 1984. (23) POMMIER Y, SCHWARTZ RE, ZWELLING LA, et al: Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibi- tors. Cancer Res 46:611-616, 1986. (24) POMMIER Y, KERRIGAN D, SCHWARTZ RE, et al: Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. Cancer Res 46:3075-3081, 1986. (25) SALLES B, CHARCOSSET JY, JACQUEMIN-SABLON A: Isola- tion and properties of Chinese hamster lung cells resistant to ellipticine derivatives. Cancer Treat Rep 66:327-338, 1982. (26) BAKIC M, BERAN M, ANDERSSON BS, et al: The production of topoisomerase II-mediated DNA cleavage in human leukemia cells predicts their susceptibility to 4’~(9-acridi- nylamino)methanesulfon-m-anisidide (m-AMSA). Bio- chem Biophys Res Commun 134:638-645, 1986. (27) ESTEY E, SILBERMAN L, BAKIC M, et al: m-AMSA-induced topoisomerase II-mediated DNA cleavage: An indicator of sensitivity to m-AMSA in human leukemia cells. Proc Am Assoc Cancer Res 27:261, 1986. 93 TT —% sal Ray Elevated Topoisomerase Il Activity and Altered Chromatin in Nitrogen Mustard-resistant Human Cells K. B. Tan,* M. R. Mattern, R. A. Boyce, R. P. Hertzberg, and P. S. Schein’? ABSTRACT —A human Burkitt’s lymphoma cell line (Raji- HN;) made resistant to nitrogen mustard, a bifunctional alkylat- ing agent, was used to study the mechanism of resistance to nitro- gen mustard. A comparative study of Raji-HN, and the parental sensitive Raji cell lines revealed the following: (1) The DNA of Raji-HN, cells was crosslinked by nitrogen mustard to a lower extent than Raji DNA; (2) once interstrand crosslinks were formed, they were repaired at the same rate in both cell lines; (3) DNA crosslink formation in Raji-HN,, but not in Raji cells, was enhanced by novobiocin, a topoisomerase II inhibitor; (4) Raji- HN; cells had elevated topoisomerase II activity and were hyper- sensitive to topoisomerase inhibitors (amsacrine, novobiocin, teniposide); (5) similar amounts of topoisomerase I were found in both cell lines; and (6) the chromatin of Raji-HN, but not of Raji cells, was hypersensitive to DNase I digestion. The relationship between DNA repair, topoisomerase II activity, chromatin struc- ture and drug resistance is discussed.—NCI Monogr 4:95-98, 1987. INTRODUCTION The establishment of cell lines that are resistant to alky- lating agents can be achieved only with great difficulty (1,2). Cells made resistant to one class of alkylating agents are usually not cross-resistant to other classes of alkylating agents (1,2). For example, a human Burkitt’s lymphoma cell line (Raji) made resistant to nitrogen mustard is not cross-resistant to melphalan, mitomycin C, N,N’-bis(2- chloroethyl)- N-nitrosourea, hydroxycyclophosphamide, cis- platin, or other alkylating agents (2). The mechanism of resistance to nitrogen mustard is not known. Since nitrogen mustard induces DNA interstrand crosslinks, prevention of crosslink formation and/or efficient repair of crosslinks would lead to drug resistance (/,3). We investigated the nitrogen mustard-resistant Raji cells to determine whether DNA repair is involved in drug re- sistance and whether topoisomerases might be involved in DNA repair and drug resistance. ABBREVIATIONS: HN, = bis(2-chloroethyl)methylamine, nitrogen mustard; U= units of enzyme activity. Molecular Oncology Group and Departments of Molecular Pharma- cology and Medicinal Chemistry, Smith Kline & French Laboratories, Philadelphia, PA. 2We thank E. Frei III and C. Cucchi for providing the Raji and Raji- HN, cells and Drs. R. K. Johnson, C. K. Mirabelli, and D. Trainer for providing P388 and Colo 201 cells. * Reprint requests: K. B. Tan, Ph.D., Department of Molecular Phar- macology, Smith Kline & French Laboratories, 709 Swedeland Road, Swedeland, PA 19479. MATERIALS AND METHODS Cells.—Cells were maintained in RPMI medium contain- ing 109% fetal bovine serum. Nitrogen mustard-resistant cells were treated weekly with 10 uM HN, to maintain the resistant phenotype. All cells used were in the exponential growth phase. Drug treatment.—Drug treatment was with serum-free RPMI medium. Nitrogen mustard (HN,; 2-chloro-N-[2- chloroethyl]- N-methylethanamine, Sigma Chem. Co.) was dissolved in 0.1 N HCI and stored at —20° C ({). Clonogenic assays.—Cell killing was assayed by colony formation in soft agarose. After drug treatment, cells were diluted 500-fold into 0.15% low melting point agarose (Seakem) prepared in RPMI medium containing 209% fetal bovine serum. Cells were stained with tetrazolium salt 7-10 days after incubation, and colonies were counted in a Bio- tran counter. Alkaline elution.—Labeling of cells with ["“C] or [H]thymidine and alkaline elution analysis of the DNA were performed as described (5). After lysis with sodium dodecyl sulfate and treatment with proteinase K, the DNA was eluted in the presence of 0.1% sodium dodecyl sulfate. DNase I digestion.—Cells labeled with [*H]thymidine as described above were washed with RSB (10 mM NaCl, 5 mM MgCl,, 10 mM Tris-HCI, pH 7.5) and allowed to swell in RSB for 5 min. in ice. Nonidet P40 was added to 0.5%, and nuclei were collected by centrifugation, washed with RSB and resuspended in RSB at 8 absorbance units (260 nm) per ml. DNase I (Worthington) was added at 40 U/ml. After digestion, samples were mixed with equal volumes of cold 10% perchloric acid, chilled in ice for 30 min. and centrifuged at 2000 X g for 20 min. Radioactivity in the supernatant fraction was determined by liquid scintil- lation spectroscopy. Topoisomerase assays.—Topoisomerase 1 was assayed by measurement of the ATP-independent relaxation of pBR322 supercoiled DNA (6). Topoisomerase II was assayed by measurement of the ATP-dependent removal of topological knots from the DNA of bacteriophage P4 (7). Specific activities of topoisomerase I and topoisomerase 11 were estimated by dividing total units of activity by total protein, as determined according to Bradford (8). RESULTS DNA Interstrand Crosslinks Induced by Nitrogen Mustard Nitrogen mustard (HN), a bifunctional alkylating agent, induces DNA interstrand crosslinks rapidly; most of the crosslinks are formed within 30 min. (4). The extent of 95 HN,-induced crosslinking of Raji and nitrogen mustard- resistant (Raji-HN,) DNA was determined by alkaline elu- tion (5). From the elution kinetics, the crosslink index, a measure of extent of DNA interstrand crosslinking, was calculated (5). HN, formed crosslinks in Raji-HN, DNA to a much lower extent than in Raji DNA (Fig. 1). When Raji and Raji-HN, cells were treated with HN, producing equiv- alent numbers of crosslinks, the crosslinks had been repaired at about the same rate 6 hr. after drug treatment (Fig. 2). Topoisomerase Activity Because topoisomerase II has been suggested to play a role in DNA repair (9), and DNA repair is one of the postulated mechanisms of HN, resistance (1,3), we com- pared the topoisomerase activities of Raji and Raji-HN, cells. Raji cells contained extractable topoisomerase II activity similar to that found in other mammalian cells (Table 1). Raji-HN, cells, however, contained about 4 times the amount of enzyme activity found in Raji cells. On the other hand, there was no difference in topoisomerase I activity of Raji and Raji-HN, cells. EFFECTS OF TOPOISOMERASE Il INHIBITORS We found that Raji-HN, cells have elevated topoisom- erase II activity and are more sensitive to the cytotoxic properties of topoisomerase II inhibitors such as novobio- cin, amsacrine, and teniposide (Fig. 3) than are Raji cells. The extreme sensitivity of Raji-HN, cells to novobiocin is unusual because most cultured mammalian cells are not killed by novobiocin at concentrations of less than 1 mg/ml. Of the inhibitors studied, only novobiocin did not induce DNA strand breaks. We, therefore, investigated the effect of novobiocin on DNA crosslink formation by HN. Novobiocin had no effect on either the formation or re- moval of crosslinks from Raji DNA (Fig. 4A). The amount of DNA crosslinking in Raji-HN, cells increased in the presence of novobiocin after 30 min. incubation with HN,, or after 6 hr. incubation following removal of HN,. When the rate of loss of Raji-HN, DNA crosslinks in the presence of novobiocin was compared with that in its absence, no > ® w O08 Q Zz 06 . > ° Soa} 2°1f Loz} J oO 0 1 1 Lk 1 1 1 1 0510 20 30 40 50 60 HN2 (uM) FIGURE 1.—DNA interstrand crosslinks induced by HN. Cells treated 1 hr. with HN, were analyzed by alkaline elution methods. The crosslink index was calculated from the formula: crosslink index= V (I—Ro)/(1—R)—1, where Ro and R are the fractions of DNA re- tained on the filters of control and HN,-treated cells, respectively (5). O=Raji cells; ®=Raji-HN, cells. All samples received 300 rad of y-radiation. 96 FRACTION OF 'C-DNA RETAINED Q ro 1 1 1 1 1 10 08 06 04 0-2 FRACTION OF 3H-DNA RETAINED FIGURE 2.—DNA interstrand crosslink repair. Raji cells were treated with I pM HN; and Raji-HN; cells with 5 uM HN; to produce equivalent crosslinking at 1 hr. after treatment. Repair of crosslinks was deter- mined by alkaline elution methods 6 hr. after HN; treatment. A = Raji, © = Raji-HN, “C-labeled DNA at I hr. after HN; treatment.A = Raji, O =Raji-HN; !4C-labeled DNA at 6 hr. after HN, treatment. ——, 3H-labeled Raji DNA not treated with HN; and included in each sample as a control. All samples received 300 rad of ~y-radiation. difference was found. Thus novobiocin appears to have enhanced crosslink formation, possibly by inhibiting mono- adduct repair, and to have had no effect on their repair. CHROMATIN STRUCTURE OF RAJI AND RAJI-HN, CELLS Topoisomerase 11 is a major structural protein of the nuclear matrix and chromosome scaffold (0,11). Since Raji-HN, cells contain about four times more topoisom- erase II activity than Raji cells, we were interested in knowing whether the increased enzyme activity in Raji-HN, cells is associated with changes in chromatin structure detectable by nuclease digestion. Incubation of nuclei with DNase I revealed that, compared to Raji DNA, Raji-HN, DNA is more susceptible to DNase I digestion (Fig. 5). This observation suggests that the chromatin of the two cell lines is organized differently. DISCUSSION Raji-HN, cells differ from Raji cells in several important aspects: (a) their DNA is crosslinked by HN, to a lower TABLE 1.—Topoisomerase Activity Enzyme activity (U/ug protein) Cell line Topoisomerase 11 Topoisomerase I Raji-HN, 9621 (n=4) 222 Raji 28+6 (n=4) 206 Colo 201° 24 ND¢ P388° 27 ND ? Nuclei were extracted with 0.35 M NaCl and assayed for enzyme activities as described in Materials and Methods. b Colo 201 is a human colon carcinoma cell, and P388 is a mouse leukemia cell. ¢ Not done. NCI MONOGRAPHS, NUMBER 4, 1987 1-00 Z 0-50 o E Q < a w gore > S 0:05 a > a 0-01 Lu Sennh ld Lila 1 Xk A 1 A 1 Ll Ll 1 0 05 1 5 10 50 0 100 200 400 800 1400 O 0-05 04 05 1 5 10 m-AMSA (uM) NOVOBIOCIN (pg/ml) VM-26 (uM) FIGURE 3.—Sensitivity to topoisomerase II inhibitors. Cells were treated with amsacrine or teniposide (VM-26) for 1 hr., or novobiocin for 30 min. at 37°C. Cell survival was determined by colony formation in soft agarose. O= Raji cells; ® = Raji-HN, cells. extent; (b) they contain elevated topoisomerase II activity; (c) they are hypersensitive to topoisomerase II inhibitors; and (d) their chromatin is hypersensitive to DNase I digestion. The reason for the reduced crosslinking of Raji-HN, DNA by HN, when compared with that in Raji cells is not known. HN, crosslinks DNA in a two-step process: forma- tion of a monoadduct on each DNA strand and subsequent formation of interstrand crosslinks, the latter resulting in cell death (3). Our results showed that interstrand cross- links are repaired at the same rate in both cell lines. Resis- 1-0 Q a } FRACTION OF '#C-DNA RETAINED Oo on Li bl ty 1 1 1 10 05 FRACTION OF 3H-DNA RETAINED FIGURE 4.—Effect of novobiocin on DNA interstrand crosslink repair. Both Raji and Raji-HN; cells incubated for 30 min. with novobiocin (200 pg/ml) were then treated with HN; (1 uM) for 30 min. At 30 min. and 6 hr. after HN; treatment, cells were analyzed by alkaline elution. Panel A: Raji cells, Panel B: Raji-HN, cells. C-labeled DNA 30 min. after HN; treatment with (A) or without (®) novobiocin; 6 hr. after HN, treatment with (A) or without (O) novobiocin. (-——-), 3H-labeled Raji DNA not treated with drugs. All samples received 300 rad of y-radiation. DNA TOPOISOMERASES IN CANCER THERAPY tance to HN, could result from factors that affect monoad- duct formation or repair. Efficient repair of monoadducts would result in fewer crosslinks in Raji-HN, DNA. Human Mer" cells are capable of repairing nitrosourea monoad- ducts, whereas Mer™ cells lack this capacity (/2). An analo- gous repair system may be operating in the Raji-HN, and Raji cells. Alternatively, reduced HN, uptake would lead to reduced crosslinking of Raji-HN, DNA. This possibility seems unlikely as HN, uptake was found to be similar for both Raji and Raji-HN, cells (1). A third possibility for reduced crosslinking is enhanced detoxification of HN, in Raji-HN, cells. Elevated glutathione concentration is known to be associated with resistance to melphalan (/3). We found 2-3 times as much glutathione in Raji-HN, cells as in Raji cells (unpublished data). Raji-HN, cells are, how- ever, not cross-resistant to melphalan or nitrosoureas and are hypersensitive to amsacrine, drugs which are detoxified by glutathione (1/4). Therefore, the interpretation of drug resistance in the setting of increased amounts of glutathione must be made with great caution. The possible role of glu- tathione in mediating resistance to HN, remains to be determined. 100 + [2] @ Oo Oo DNA DIGESTED D (@] °le ny Oo 0 1 1 0 5 10 15 20 TIME (minutes) FIGURE 5.—DNase I digestion of Raji and Raji-HN, DNA. Nuclei pre- pared from cells labeled with >H-thymidine were incubated at 37°C with DNase I (40 U/ml). At the indicated times, aliquots were withdrawn for determination of acid soluble radioactivity which was then com- pared to the total radioactivity present in a control aliquot not treated with DNase I. O=Raji DNA; ®=Raji-HN, DNA. 97 Raji-HN, cells have increased topoisomerase 11 (but not topoisomerase I) activity when compared with Raji cells. This increase of topoisomerase II activity is related in- versely to cell growth rate, as the cell doubling time of Raji- HN, cells is twice that of Raji cells (/). Although the func- tion of topoisomerase II in Raji-HN, cells is not known, it could, for example, interact with and modify chromatin structure to promote efficient monoadduct repair in Raji- HN, cells. Compared to that of Raji cells, the DNA of Raji-HN, chromatin is hypersensitive to DNase I diges- tion. O%-Methylguanine monoadducts have been shown to be removed more rapidly from nuclease sensitive (or active) chromatin than from bulk chromatin (15). The efficiency of HN, monoadduct repair may be modulated similarly by differences in the chromatin structures of Raji and Raji- HN, cells. The mechanism of resistance to HN, by Raji-HNj, cells is obviously complex and may involve interactions among DNA repair enzymes, topoisomerase II, chromatin struc- ture, and glutathione. Interstrand crosslink repair and topoisomerase I are not factors in HN, resistance. REFERENCES (1) FrEl E III, CuccHl CA, ROSOWSKY A, et al: Alkylating agent resistance: In vitro studies with human cell lines. Proc Natl Acad Sci USA 82:2158-2162, 1985. (2) TEICHER BA, CuccHI CA, LEE JB, et al: Alkylating agents: In vitro studies of cross-resistance patterns in human cell lines. Cancer Res 4379-4383, 1986. (3) CoNNORS TA: Mechanism of “resistance” towards specific drug groups. /n Handbook of Experimental Pharmacol- ogy (Fox BW, Fox M, eds), vol 72. Berlin: Springer- Verlag, 1984, pp 403-424. (4) Ross WE, EwiG RA, KoHN KW: Differences between mel- phalan and nitrogen mustard in the formation and remo- val of DNA cross-links. Cancer Res 38:1502-1506, 1978. 98 (5) KoHN KW, EWIG RAG, ERICKSON LC, et al: Measurement of strand breaks and cross-links by alkaline elution. In DNA Repair (Friedberg EC, Hanawalt PC, eds), vol 1, part B. New York: Marcel Dekker, 1981, pp 379-401. (6) Liu LF, MILLER KG: Eukaryotic topoisomerases: Two forms of type I DNA topoisomerases from HeLa cell nu- clei. Proc Natl Acad Sci USA 76:3487-3491, 1981. (7) Liu LF, DAVIS JL, CALENDAR R: Novel topologically knot- ted DNA from bacteriophage P4 capsids: Studies with DNA topoisomerases. Nucleic Acids Res 9:3979-3989, 1981. (8) BRADFORD MM: A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248- 254, 1976. (9) MATTERN MR, PAONE RF, DAY RS III: Eukaryotic DNA repair is blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases a and B. Bio- chim Biophys Acta 697:6-13, 1982. (10) BERRIOS M, OSHEROFF N, FISHER PA: In situ localization of DNA topoisomerase II, a major polypeptide compo- nent of the Drosophila nuclear matrix fraction. Proc Natl Acad Sci USA 82:4142-4146, 1985. (11) EARNSHAW WC, HALLIGAN B, COOKE CA: Topoisomerase II is a structural component of mitotic chromosome scaf- folds. J Cell Biol 100:1706-1715, 1985. (12) ERICKSON LC, LAURENT G, SHARKEY NA, et al: DNA cross-linking and monoadduct repair in nitrosourea- treated human tumor cells. Nature 288:727-729, 1980. (13) GREEN JA, VISTICA DT, YOUNG RC, et al: Potentiation of melphalan cytotoxicity in human ovarian cancer cell lines by glutathione depletion. Cancer Res 44:5427-5431, 1984. (14) ARRICK BA, NATHAN CF: Glutathione metabolism as a determinant of therapeutic efficacy: A review. Cancer Res 44:4224-4232, 1984. (15) RYAN AJ, BILLETT MA, O’CONNOR PJ: Selective repair of methylated purines in regions of chromatin DNA. Car- cinogenesis 7:1497-1503, 1986. Heat Shock Proteins: Role in Thermotolerance, Drug Resistance, and Relationship to DNA Topoisomerases Gloria C. Li%%* ABSTRACT —The events following a mild heat exposure to cells that appear to be closely linked are: enhanced synthesis of hsp’s and thermotolerance. In some cases, thermotolerance is also associated with drug resistance. We have recently examined the role that DNA topoisomerase II may play in the induction of these phenomena. VM-26 was found to both initiate hsp synthesis and to cause thermotolerance. Furthermore, the permanent heat resistant or transient thermotolerant cells were more resistant to VM-26 than controls. These results suggest that topoisomerase II, or more likely topoisomerase II-DNA complexes, are affected by heat or by VM-26 in a phenomenologically overlapping manner. Elevated levels of hsp’s apparently protect cells against the cytotoxic action of both heat and VM-26.—NCI Monogr 4: 99-103, 1987. THERMOTOLERANCE AND HEAT SHOCK PROTEINS One of the most intriguing aspects of thermal biology is the response of heated mammalian cells to a subsequent heat treatment. Gerner and Schneider (/) and Henle and Leeper (2) demonstrated that mammalian cells that survive an exposure at elevated temperature have the ability to acquire a transient resistance to a subsequent otherwise lethal heat challenge. This phenomenon has been termed “thermotolerance” (3). The effect of thermotolerance when measured in terms of cell survival can be dramatic, leading to increase in survival by several orders of magnitude. The kinetics of induction, development, and decay of thermo- tolerance constitute a well-studied subject (3-5). In parallel, heat and other environmental stresses (chemical or me- chanical) induce or enhance the synthesis of a family of proteins, usually referred to as heat shock proteins (hsp’s) (6). These proteins have been observed in cells from a vari- ety of organisms ranging from bacteria to yeast, to Droso- phila, to mammalian systems (6-8). In recent years, the heat shock response has been the subject of intensive stud- ies, specifically in the area of gene organization and trans- I' Supported in part by Public Health Service grant CA-31397 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. 2 Radiation Oncology Research Laboratory, Department of Radiation Oncology, University of California, San Francisco, and Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan, Republic of China. 31 thank Johnson Y. Mak for technical assistance and Carol Shea for preparing the manuscript. * Reprint requests: Gloria C. Li, Ph.D., Radiation Oncology Research Laboratory, Department of Radiation Oncology, CED-200, University of California, San Francisco, San Francisco, CA 94143. cription, regulation of heat shock response, identification and characterization of hsp’s, and the physiological role of hsp’s. There are excellent reviews that describe this phe- nomenon in detail (9-11). Qualitatively, there is good temporal correlation between thermotolerance and hsp’s (7). Heat shock induces both hsp’s and thermotolerance under conditions in which the initial heat treatment either does not suppress total protein synthesis (e.g., 41°C) or drastically inhibits it (e.g., 46°C). The kinetics of synthesis of several hsp’s correlate well with the development of thermotolerance. The persistence of some of the hsp’s synthesized after the priming heat treatment correlates well with the persistence of thermotol- erance. Furthermore, a number of agents known to induce thermotolerance induce hsp’s; conversely, agents known to induce hsp’ induce thermotolerance. Table 1 lists the chemical and physical agents that have been examined for both phenomena in the same system. It can be seen that except for the arginine analog canavanine, and proline ana- log L-azetidine-2-carboxylic acid, all hsp-inducers induce thermotolerance. The amino acid analog data suggest that hsp’s as well as other cellular proteins, synthesized in the presence of canavanine or azetidine are probably not func- tional (12). The analog-substituted hsp’s may not protect cells from thermal damage. In further experiments (Laszlo and Li, manuscript in preparation), it was found that (a) the incorporation of amino acid analogs into cellular proteins inhibits the development of thermotolerance, and (b) cells which are already transiently thermotolerant or perma- nently heat resistant are resistant to the thermal sensitizing action of amino acid analogs. Overall, the results generate a strong argument for the functional role of hsp’s in the development of thermotolerance and in protecting cells against thermal damage. In spite of the numerous data that indicate the positive correlation, there are occasional reports in the literature suggesting that thermotolerance can be induced even in the absence of increased synthesis of hsp’s. Recently, we have examined the quantitative relationship between the thermal sensitivity and levels of hsp’s in tran- sient thermotolerant Chinese hamster fibroblasts HA-1 cells and in their heat resistant variants (4). Of the many hsp’s preferentially synthesized after heat shock, the con- centration of 70 kD hsp (hsp 70) appears to correlate best with either permanent or transient heat resistance. As shown in Figure 1, when the concentration of hsp 70 de- creases, the cell survival after a 45°C, 45 min treatment de- creases. These data suggest that the level of hsp 70 appears to be a good predictor of thermal response, at least in tissue culture cell lines. 99 TABLE 1.—Thermotolerance and Heat Shock Protein: Drugs That Induce Thermotolerance and/or Synthesis of Heat Shock Proteins? Induction of Induction of heat Agent tolerance shock proteins Reference Ethanol of + 6,7,21) N-Pentanol Ce NP 30) Sodium arsenite +++ +4 6,7) Cadmium + 4 + (22) ccr +++ + 29) 2,4-Dinitrophenol + + fe (6,29) Disulfiram + + + (29) Dimethyl sulfoxide + — NP (30) Dimethyl fluoride + NP (30) Lidocaine + + = 30) Procaine fe == + (30) Tetracaine — ? 30) Canavanine (arginine analog) _ + 12) L-Azetidine-2-carboxylic acid (proline analog) —- + (12) Epipodophyllotoxin (VM-26) = + 27) 4 Note: +, ++, +++, positive effect with increasing potency; ?, questionable effect; —, no effect; NP, not performed. There is considerable experimental evidence showing that thermotolerance can be induced in tumors and thus modifies their response to fractionated hyperthermia (13-15). We have examined the induction kinetics of ther- motolerance and the synthesis of hsp’s in murine tumor models, a squamous cell carcinoma, and a radiation- induced fibrosarcoma (16). Our data show that mild heat shock induces thermotolerance in murine tumors, a result consistent with those of others (15). The kinetics of induc- tion and decay of thermotolerance are dependent upon the temperature and duration of the priming treatment. Mild heat shock also enhances the rate of synthesis and accumulation of some hsp’s, specifically the hsp 70, during —- J T 6 HSP 70 24 2242 of Aton ® g 23 Fg 4 2 eM QHA-1 Eo 3 2c we QL 0 1 1 1 1 23 gz Oc 18} ACTIN 2k ws C2 oc a HA-1 i 208° 201168 4g 72 80 2242 96 —- a 15 0 Dit 10° 5 | 107" SURVIVING FRACTION FIGURE 1.—Relationship between cell survival and levels of hsp’s in ther- motolerant HA-1 cells and their stable heat resistant variants. Survivals of exponentially growing, thermotolerant HA-1 cells and of heat- resistant cells (strains 3011 and 2242) exposed to 45°C for 45 min are plotted against levels of hsp’s, both induced and constitutive. The levels of hsp’s are expressed as percentage of total [>>S]methionine-labeled proteins. To measure the total accumulated levels of hsp’s, cells were labeled continuously throughout the experiments. Open symbols: unheated HA-1, 3011, and 2242 cells. Solid symbols: exponentially growing thermotolerant HA-1 cells. Cells were made thermotolerant by exposure to 45°C for 15 min, followed by 37°C incubation for 8, 23, 24, 48, 72, or 96 hr. 100 the development of thermotolerance in vivo. The kinetics of induction of hsp synthesis appear to be similar to those found in cells in cultures. Furthermore, the data indicate that the levels of hsp 70 correlate well with thermotoler- ance. Similar experiments were performed using murine normal bone marrow cells, and it is found that heat shock also enhances the synthesis of hsp 70 in total bone marrow cell population during the development of thermotolerance in CFU-GM and CFU-E’. When the protein synthesis profiles of tumor cells from an involved lymph node of a patient with breast carcinoma were examined, it was clearly demonstrated that after an initial heat shock, the synthesis of hsp’s with molecular weight 68-70 kd and 87 kd are greatly enhanced when compared to the unheated control tumor cells (/7). Thus, it appears feasible that the mea- surement of the levels of hsp 70 can be used as an assay to predict the degree of thermotolerance of tissues during frac- tionated hyperthermia as applied in the clinics. THERMOTOLERANCE AND DRUG RESPONSE One of the promising approaches to clinical application of hyperthermia is to combine it with chemotherapy (as termed “thermochemotherapy”). In vivo and in vitro sys- tems (here, the terms refer to whole animals and cultured cell lines, respectively) have been used to demonstrate the synergistic effect of hyperthermia in combination with drugs such as Adriamycin, bleomycin, nitrosourea, cis- platinum, or local anesthetics, etc. Hahn has reviewed this topic in considerable detail (/8-20). The cytotoxicity of some drugs is enhanced at elevated temperatures. Phe- nomenologically, this synergistic effect may be due to an increased rate of reaction of one or more critical processes (for example, the alkylating agents BCNU, CCNU), or due to a threshold phenomenon taking place between 41° and 43°C (for example, Adriamycin or bleomycin), or caused by some drug actions that mimic the action of heat (for example, aliphatic alcohols). It is found that an increase of 0.15 M of ethanol is equivalent to a 1°C increase in temper- ature (27). It is also known that thermotolerance has a significant effect on some drug response and heat-drug interaction (20-22). In the thermotolerant state, not only NCI MONOGRAPHS, NUMBER 4, 1987 do cells become resistant to heat, but their responses to some drugs are also modified. Several examples are given as follows: When cells were made thermotolerant by a one hour exposure to heat (43°C), arsenite (100 uM, 37°C), cadmium (100 uM, 30°C), or ethanol (6%, 37° C), these cells containing elevated levels of hsp 70 also developed toler- ance to Adriamycin (9, 22). This induced tolerance to Adria- mycin is transient; in principle, one can avoid this drug tolerance by properly scheduling multiple treatments. On the other hand, permanent heat-resistant CHO cells that contain elevated levels of hsp 70 at normal growth tempera- ture also demonstrate appreciable resistance to Adriamycin (23). In the case of bleomycin, the observed threshold temperature is shifted 1°C higher for thermotolerant cells, e.g., 43.5°C for tolerant cells and 42.5°C for control non- tolerant cells, respectively. However, if the exposure temperature is at 37°C, thermotolerant and control nontol- erant cells show little difference in their response to bleo- mycin. Table 2 summarizes the effect of thermotolerance on drug cytotoxicity. Lowering pH can greatly enhance the cytotoxicity at ele- vated temperatures of some chemotherapeutic agents, such as BCNU or amphotericin B (24). This pH dependence of drug cytotoxicity at elevated temperatures was much less for transient thermotolerant cells or their stable heat resis- tant variants [Li, unpublished data; (24)]. A significant dif- ference between thermotolerant (or heat resistant) cells and the nontolerant control is that the former contain high lev- els of hsp 70. The concentration of hsp 70 appears to corre- late best with heat resistance, either permanent or transient. Therefore, it is of both biological and clinical interest to elucidate the molecular basis related to the roles of hsp 70 in modulating cellular response to heat and to drugs. THERMOTOLERANCE, HEAT RESISTANCE, AND TOPOISOMERASE INHIBITORS Anti-tumor drugs, VP-16 and VM-26, which are epipo- dophyllotoxins, interfere with the breakage-reunion reac- tion of mammalian DNA topoisomerase II (25). Rowe TABLE 2.—Thermotolerance and Drug Response: Effect of Thermotolerance on Drug Cytotoxicity? Hours after heat exposure 0 6 24 Reference Adriamycin 1 | | (19,31) Actinomycin D t | | (19,32) Amphotericin B 372C t = = (19) Bleomycin 37°C t = - (19,31) 43°C t | | (19,31) BCNU 37°C t t t (19) 41°C 1 t NP (19) 43°C t — — (19) Methyl methanesulfonate 37°C t t NP (19,33) 41°C t t NP (19,33) 43°C t | NP (19,33) 45°C 1 | NP (19,33) VM-26 37°C | t NP (27) ¢ Note: t, increased cytotoxicity; |, decreased cytotoxicity; —, cytotox- icity indistinguishable from that seen against unheated controls; NP, not performed. DNA TOPOISOMERASES IN CANCER THERAPY 10° 6 uM 10 'F = © - oO < oc -2] nw 10° a O : No-0 oO E z 5 10° on A 45°C, 45 MIN. —4 1 1 1 1 L 1 0 4 8 12 16 20 24 28 TIME AT 37°C ( HR) FIGURE 2.—Induction of thermotolerance by VM-26. Exponentially grow- ing HA-1 cells were first exposed to 6 uM VM-26 for | hr at 37°C, and then incubated at 37°C in drug-free medium for various times before a subsequent heat challenge (45°C, 45 min). Survival after the combined treatments (—O —), all normalized to VM-26 toxicity, were plotted as a function of 37°C incubation time between the two treatments. Survival value after heat treatment (45°C, 45 min) was 4 X 1074. Thermotoler- ance was evidenced by the increased survivals (—O —) over the heated controls (A). et al. have shown that VM-26 induced hsp 70 transcription in Drosophila cells (26). Recently, we have examined the effect of VP-16 or VM-26 on the induction of thermotoler- ance, on the stimulation of hsp 70 transcription, and on the synthesis of hsp 70 in Chinese hamster fibroblasts (27). Our 10° 10° A A \ B 1 © A L Sih Na W\ 10 | ENS TAN S L[ = Na 10 \ 5 10 \ 5 E \ x I s Se + v & Ne A \ 0 10°F ~ Se 10°F £ | }, : g 10° i \ [ \. w 10°F N 10°F 7 : } -6 i 1 Knead 1 A Lede ~4 i 1 4 1 1 1 04 8 12 16 20100 4 8 12 VM-26 (uM) VM-26 (uM) FIGURE 3.—The effect of transient thermotolerance or stable heat resis- tance on the cytotoxicity of VM-26. Panel A: Exponentially growing HA-1 cells were made thermotolerant by a 15 min exposure at 45°C followed by 6 hr incubation at 37° C. The thermotolerant cells (®) and control non-tolerant cells (a) were then exposed to graded doses of VM-26 for 1 hr at 37°C, and cell survival determined. Note that ther- motolerant cells were more resistant to VM-26 than the nontolerant controls. Panel B: Exponentially growing HA-1 cells (®) and their stable heat resistant variant 3012 (ww) were exposed to graded doses of VM-26 for 1 hr at 37°C, and cell survival was determined. The heat resistant cells were more resistant to VM-26 cytotoxicity. 101 HSP - D CELL DEATH FIGURE 4.—A working model for the interaction between heat shock pro- teins and VM-26 cytotoxicity: A) Unbound DNA topoisomerase 11; B) Reversible formation of a noncleavable complex between DNA topo- isomerase II and DNA; C) Reversible formation of the cleavable com- plex between DNA topoisomerase II and DNA. DNA at the site of the cleavable complex is presumably broken. The two broken double- strand DNA fragments are held together only by noncovalent protein— protein interaction; D) Processing of the cleavable complex results in irreversible DNA breakage which eventually leads to cell death. ®, covalent linkage site between the 5’-ends of the transient DNA break and each subunit of DNA topoisomerase II (28). data show that VP-16 or VM-26 induce thermotolerance as evidenced by up to 10- to 50-fold increase in cell survival (Figure 2). Tolerance is close to its maximum immediately after the one hour drug treatment. VP-16 or VM-26 also increases the level of hsp 70 transcription approximately 10- to 30-fold over controls. Furthermore, epipodophyllo- toxins enhance the synthesis of the constitutive form of hsp 70. The mechanism underlying the observation that VP-16 or VM-26 induces thermotolerance, stimulates hsp 70 transcription, and enhances synthesis of hsp 70 is unclear at present. One speculates that DNA topoisomerase II may play a role in the heat shock response. We also examined the effect of thermotolerance on the cytotoxicity of VM-26 (Figure 3a). Cells were made ther- motolerant by a fifteen minute exposure at 45°C and fol- lowed by six hours incubation at 37°C. The thermotolerant cells and control nontolerant cells were then exposed to graded doses of VM-26 for one hour at 37°C, and cell survivals were determined. Thermotolerant cells are more resistant to VM-26 than the nontolerant controls. This heat-induced tolerance to VM-26 is not due to alteration of the level of DNA topoisomerase II. The VM-26-induced DNA cleavages as well as the reversal kinetics of this VM- 26-induced DNA cleavage are identical between thermotol- erant and control nontolerant cells. Furthermore, heat re- sistant variants that contain high levels of hsp 70 at normal growth temperature are also more resistant to VM-26 than their parent lines (Figure 3b). It is most likely significant that the transiently tolerant CHO cells and their stable heat resistant variants share the common characteristics in their ability to withstand heat challenge and in their elevated levels of hsp 70. Presently, we do not have enough data on the relationship between hsp 70 synthesis and resistance to 102 topoisomerase inhibitors, e.g., VM-26. The possible mecha- nisms by which heat shock proteins might act to confer increased resistance to VM-26 are not clear. Figure 4 shows schematically a working model for the interaction between heat shock proteins and VM-26 cytotoxicity. As proposed previously (28), a partial reaction of mammalian DNA topoisomerase II may be affected by VM-26. Topoisom- erase II forms at least two types of complexes with DNA, the noncleavable complex and the cleavable complex. Nor- mally, the two complexes are at rapid equilibrium and the equilibrium concentration of the cleavable complex is low. VM-26 may increase the concentration of cleavable complex by binding to the enzyme or enzyme-DNA complex. This cleavable complex may be processed subsequently, which eventually leads to cell death. If hsp’s play a role in protect- ing cells from VM-26 damage, it is plausible that hsp’s may interfere with steps involved in the processing of the drug- altered cleavable complexes. REFERENCES (1) GERNER EW, SCHNEIDER MJ: Induced thermal resistance in HeLa cells. Nature 256:500-502, 1975. (2) HENLE KJ, LEEPER DB: Interaction of hyperthermia and radiation in CHO cells: Recovery kinetics. Radiat Res 66:505-518, 1976. (3) HENLE KJ, DETHLEFSEN LA: Heat fractionation and ther- motolerance: A review. Cancer Res 38:1843-1851, 1978. (4) L1 GC: Elevated levels of 70,000 dalton heat shock protein in transiently thermotolerant Chinese hamster fibroblasts and in their stable heat resistant variants. Int J Radiat Oncol Biol Phys 11:165-177, 1985. (5) MAJIMA H, GERWECK LE: Kinetics of thermotolerance decay in Chinese hamster ovary cells. Cancer Res 43: 2673-2677, 1983. (6) ASHBURNER M, BONNER JJ: The induction of gene activity in Drosophila by heat shock. Cell 17:241-254, 1979. (7) L1 GC, WERB Z: Correlation between synthesis of heat shock proteins and development of thermotolerance in Chinese hamster fibroblasts. Proc Natl Acad Sci USA 79:3219- 3222, 1982. (8) MCALISTER L, FINKELSTEIN DB: Heat shock protein and thermal resistance in yeast. Biochem Biophys Res Com- mun 93:819-824, 1980. (9) SCHLESINGER MJ, ASHBURNER M, TISSIERES A (eds): Heat Shock: From Bacteria to Man. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. (10) ATKINSON BG, WALDEN DW (eds): Changes in Eukaryotic Gene Expression in Response to Environmental Stress. New York: Academic Press, 1984. (11) CRAIG EA: The heat shock response. CRC Crit Rev Bio- chem 18:239-280, 1985. (12) L1 GC, LaszLo L: Amino acid analogs while inducing heat shock proteins sensitize CHO cells to thermal damage. J Cell Physiol 122:91-97, 1985. (13) KAMMA T, NIELSEN OS, OVERGAARD J, et al: Development of thermotolerance during fractionated hyperthermia in a solid tumor in vivo. Cancer Res 42:1744-1748, 1982. (14) ROFSTAD EK, BRUSTAD T: Development of thermotoler- ance in human melanoma xenograft. Cancer Res 44: 525-530, 1984. (15) MEYER JL, VAN KERSEN I, HAHN GM: Tumor responses following multiple hyperthermia and x-ray treatments: The role of thermotolerance at the cellular level. Cancer Res 46:5691-5695, 1986. (16) L1 GC, MAK JY: Induction of heat shock protein synthesis in NCI MONOGRAPHS, NUMBER 4, 1987 murine tumors during the development of thermotoler- ance. Cancer Res 45:3816-3824, 1985. (17) L1 GC: Unpublished data. (18) HAHN GM: Hyperthermia and Cancer. New York: Plenum Press, 1982. (19) HAHN GM: Hyperthermia to enhance drug delivery. In Rational Basis for Chemotherapy. New York: Alan R Liss, 1983, pp 427-436. (20) HAHN GM, LI GC: Interaction of hyperthermia and drugs: Treatments and probes. Natl Cancer Inst Monogr 61: 317-323, 1982. (21) L1 GC, HAHN GM: Ethanol-induced tolerance to heat and to Adriamycin. Nature 274:699-701, 1978. (22) Li GC: Thermal biology and physiology in clinical hyper- thermia: Current status and future needs. Cancer Res (suppl) 44:4886s-4893s, 1984. (23) WALLNER K, Li GC: Adriamycin resistance, heat resistance and radiation response in Chinese hamster fibroblasts. Int J Radiat Oncol Biol Phys 12:829-833, 1986. (24) HAHN GM: Thermotolerance and Drug. CRC Press. In press. (25) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative anti- tumor drugs interfere with the breakage reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259: 13560-13566, 1984. (26) ROWE TC, WANG JC, Liu LF: In vivo localization of DNA DNA TOPOISOMERASES IN CANCER THERAPY topoisomerase II cleavage sites on Drosophila heat shock chromatin. Mol Cell Biol 6:985-992, 1986. (27) L1 GC, Liu LF: Unpublished data. (28) ROWE TC, CHEN GI, HSIANG YH, et al: DNA damage by antitumor acridines mediated by mammalian DNA topo- isomerase II. Cancer Res 46:2021-2026, 1986. (29) HAVEMAN J, L1 GC, MAK JY, et al: Chemically induced resistance to heat treatment and stress protein synthesis in cultured mammalian cells. Int J Radiat Biol 50:51-64, 1986. (30) HAHN GM, SHIU EC, WEST B, et al: Mechanistic implica- tions of the induction of thermotolerance in Chinese hamster cells by organic solvents. Cancer Res 45:4138- 4143, 1985. (31) HAHN GM, BRAUN J, HAR-KEDAR I: Thermochemotherapy; synergism between hyperthermia (42-43° C) and Adria- mycin (or bleomycin) in mammalian cell inaction. Proc Natl Acad Sci USA 72:937-940, 1975. (32) DONALDSON SC, GORDON C, HAHN GM: Protective effect of hyperthermia against the cytotoxicity of actinomycin D on Chinese hamster cells. Cancer Treat Rep 62:1489-1495, 1978. (33) HAHN GM, Li GC: The interactions of hyperthermia and drugs: Treatments and probes. Natl Cancer Inst Monogr 61:317-323, 1982. 103 27 al ~ 1 a 1 o DNA Topoisomerase Ill as a Potential Factor in Drug Resistance of Human Malignancies Milan Potmesil,? * Yaw-Huei Hsiang,? Leroy F. Liu,® Hai-Young Wu,? Frank Traganos,* Bruce Bank,? and Robert Silber?5 ABSTRACT —The stabilization of the cleavable complex be- tween DNA topoisomerase II and DNA by adriamycin (ADR), as well as by other topoisomerase II-targeted drugs, is an essential step in a process associated with drug cytotoxicity. Unlike many other cell types, ADR does not produce DNA cleavage in the lymphocytes of chronic lymphocytic leukemia (CLL). The CLL lymphocytes have been identified as quiescent cells with an ex- tremely low level of topoisomerase II. The low level of this enzyme could constitute a basis for a new mechanism of drug resistance operating not only in CLL, but perhaps in any slow growing can- cer with a large population of quiescent cells. Other factors con- tributing to drug resistance could include changes in enzyme regu- lation or processing of the cleavable complex, or the presence of a “mutant” enzyme which renders cancer cells unresponsive to topoisomerase II-targeted drugs. Suggested strategies in drug de- velopment, aimed at the topoisomerase II-related drug resistance, could include 1) the selection of topoisomerase I as an alternative target for cancer chemotherapy, 2) the development of ADR ana- logs which, unlike ADR, stabilize the topoisomerase II-DNA complex with high efficiency, and 3) the search for agents enhanc- ing the SOS-like repair response, presumably triggered by DNA topoisomerase-targeted drugs. NCI Monogr 4:105-109, 1987. Often a cancer patient is treated with a combination of drugs and goes into remission. This pattern is observed in the treatment of initially responsive tumors such as advanced breast or ovarian cancer (/,2). The response rate of the two types of cancer approximates 50%, but most ABBREVIATIONS: ADR = adriamycin (doxorubicin); CLL= chronic lymphocytic leukemia; mw =molecular weight; PAB= protein-associated DNA breaks. Supported in part by Public Health Service grants CA-11655, CA- 39662, CA-32055, and CA-23296 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by grant CH-348 from the American Cancer Society; by a grant from Farmitalia Carlo Erba; and by the Marcia Slater Society for Research in Leukemia. 2 Departments of Radiology and Medicine, New York University School of Medicine, New York, NY. 3 Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD. 4 Investigative Cytology Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY. 5 We thank Dr. Daniel Knowles for some of the blood and lymph node samples obtained from hematological patients. * Reprint requests: Milan Potmesil, M.D., Department of Radiology, New York University School of Medicine, 550 First Ave., New York, NY 10016. patients ultimately relapse and the cancer becomes drug resistant. Alternatively, the initial therapy is ineffective, and drug resistance is manifested by continuing tumor growth. This is the case in some patients with several types of cancer, among them adenocarcinoma of the colon or rectum. Partial response is registered only in 20-25% of patients with advanced disease, and long-lasting remissions are rarely achieved (3,4). Even in the initially responsive types of cancer or leukemia, the disease could improve fol- lowing treatments with empirically selected drugs and remain refractory to other compounds. For example, ADR is considered by most oncologists to be of little use in the treatment of CLL, despite the wide application of the drug in the management of lymphoma. Traditionally, resistance to drug therapy has been stud- ied in tissue culture cell lines isolated by multistep selection. During this process, cells are adapted to high levels of a selected drug (5,6). Human, murine, or hamster cell lines, established in the process, may demonstrate a multi-drug- resistant phenotype, associated in many instances with reduced drug accumulation in the cell and the appearance of membrane glycoproteins (7). Increased expression and amplification of the gene for P-glycoprotein may accom- pany these events (8). The establishment of a resistant cell line by a continuous drug exposure could be due to the selection of cells with an advantageous phenotype. Efficient drug efflux is very likely just one of several potential mecha- nisms of drug resistance operating in human cancer and its study may be of limited relevance to clinical situations. As discussed elsewhere in this volume, there is ample evidence identifying topoisomerase II as the target of numerous anticancer drugs as diverse as anthracyclines, epipodophyllotoxins, and acridine derivatives (9-11). These drugs affect the breakage-reunion reaction by stabilizing the cleavable complex between DNA and topoisomerase II. The “frozen” topoisomerase II-DNA complex could be the primary cytotoxic lesion and first step in drug exerted cytoxicity (12). The notion that topoisomerases may play a pivotal role in the mediation of antitumor effects of some drugs raises the question of how either a low intracellular topoisomerase II content, changes in the processing of the cleavable complex, or the presence of a “mutant” enzyme would affect drug action in human cancer cells. RESULTS AND DISCUSSION Low DNA Topoisomerase Il Levels Several lines of evidence show that topoisomerase II activity may fluctuate according to the proliferative state of 105 3 4.0 7 = 15 wr BE TNT ae » Se << 05 1 LY “—e 5 1 < 0 NO DRUG h A o ® ADR 1.0 pg/ml San 5s 02 A ADR 5.0 pg/ml i a = o 5 |CLL L1210 oc ke 0.1 TTT FT T T T T SE SE jE T T T T T 1.0 0.5 02 10 50 20 FRACTION OF ™C-DNA RETAINED FIGURE 1.—One of a series of experiments showing DNA alkaline filter elution assay of CLL lymphocytes compared side-by-side with L1210 cells, control and treated with indicated concentrations of Adriamycin. Cell lysates were incubated before elution with SDS and proteinase K. the cell. The activity, as compared to unstimulated cells, was markedly enhanced in the nuclei of regenerating cells following partial hepatectomy (/3), in the mitogen-stim- ulated guinea pig lymphocytes (/4), in human or mouse fibroblasts exposed to epidermal growth factor (15), and in CLL lymphocytes stimulated by interleukin-2 (16). Several studies have shown m-AMSA and VP-16 to be less cyto- toxic for plateau-phase cells as compared to cells in a log phase (17-19). Finally, low levels of topoisomerase II were detected in some but not all plateau-phase culture lines which were resistant to the cytotoxic effects of VP-16 (16). A report coming from our laboratories has shown that ADR, even at high concentrations of 50.0 ug/ml, did not cause DNA cleavage in CLL lymphocytes (20-22). This rather unusual observation (Figure 1), which was first reported in 1983, is in contrast to the findings in numerous ENZYME LY 106 other cell types. In order to explain this phenomenon, DNA topoisomerase II, an intracellular target of ADR, was assayed in CLL and normal lymphocytes by immuno- blotting (Figure 2) (23,24). The blots show the serial titration of purified human DNA topoisomerase II, a polypeptide with an mw of 170,000. The enzyme was neither detected in unseparated CLL lymphocytes, nor in enriched B- and T-cell populations obtained from 21 untreated patients with B-cell CLL (stage 0-IV). It was also not detected in normal lymphocytes obtained from 6 donors (Table 1). Ex- ponentially growing HeLa and L1210 tissue culture lines had approximately 7-9X 10° enzyme copies per cell, sug- gesting that the enzyme content is at least 100 fold lower in CLL or normal lymphocytes than in growing cancer or leukemia cells. Quite consistently, indirect immunofluores- cence with topoisomerase II antibodies detected significant quantities of the enzyme in L1210 cells, while no fluores- cence was seen in nuclei of CLL lymphocytes (Figure 3). Unlike CLL lymphocytes, cells obtained from patients with prolymphocytic leukemia, acute lymphoblastic leu- kemia, Burkitt’s lymphoma, diffuse histiocytic and nodular mixed lymphoma (Table 1) show high levels of topoisom- erase II. The proliferative status of these cells is demon- strated in Figure 4. Cycling cells were detected in L1210 cells and in cells of the prolymphocytic leukemia. The rest of the samples analyzed were composed of quiescent cells confined to the G,/G, phase of the cell cycle. DNA topo- isomerase I levels were approximately the same in CLL and several other hemopoietic malignancies, as well as in nor- mal lymphocytes (Table 1). There was no detectable DNA cleavage, measured as PAB, in CLL or normal lymphocytes exposed to 0.1-50.0 ug/ml of ADR in vitro (23,24). Consistent with this obser- vation, no PAB were observed in enriched CLL B-cells, and normal B- or T-cells, exposed to 5.0 ug/ml of ADR (Table 2). Dose-dependent frequency of PAB was detected cl C0 ABCDEFGHIJKLMND FIGURE 2.—Topoisomerase II immunoblot: Purified human enzyme (arrow) 750 ng (A), 375 ng (B), 188 ng (C), 94 ng (D), 47 ng (E), and 24 ng (F). High molecular weight marker (G). Normal blood lympho- cytes (H), enriched normal T- (I) and B-lymphocytes (J), normal monocytes (K), lymphocytes of chronic lymphocytic leukemia (CLL) (L), enriched T- (M) and B-CLL lymphocytes (N), and HeLa cells in exponential growth (O). Lanes H-N, 2.5% 106 cells per sample, lane O, 106 cells. NCI MONOGRAPHS, NUMBER 4, 1987 FIGURE 3.— Upper panel: Indirect immunofluorescence with topoisomerase II-antibodies in L1210 cells and phase contrast of an identical field. Lower panel: Immunofluorescence and phase contrast of an identical field of chronic lymphocytic leukemia lymphocytes. in L1210 cells treated with ADR. It should be emphasized that the findings cannot be explained by differences in drug uptake (23,24). A point could be made that low topoisomerase II levels in quiescent cells render the cells refractory to the treatment with topoisomerase II-directed drugs. This may constitute a basis for a new, as yet uncharacterized, mechanism of drug resistance of human malignancies. Further studies are needed to investigate drug unresponsiveness of slow grow- ing tumors, such as colorectal cancer, presumably contain- ing a major compartment of quiescent cells. DNA TOPOISOMERASES IN CANCER THERAPY There is, however, evidence (23) indicating that the re- sistance to topoisomerase II-targeted drugs may not be simply explained by the position of the cell in the cell cycle: (i) T- as well as B-cells obtained from patients with B-cell CLL have undetectable levels of topoisomerase II, and both types are in the G,/ G, cell-cycle phase. Yet, ADR pro- duced PAB in CLL T-cells, while no DNA cleavage was detected in CLL B-cells, or in normal T- and B-cells (Table 2). It has been established that CLL T-cells differ from normal T-cells in several aspects and cannot be considered as representative of a normal T-cell population [reviewed in 107 TABLE 1.—DNA Topoisomerase Detection by Western Blot Analysis Number of enzyme copies cell X103 Type of cells Topoisomerase I Topoisomerase II Chronic lymphocytic leukemia 5.4+2.7 (21)¢ n.d. 21)? Normal blood lymphocytes 2311.6 (6) n.d. (6) Prolymphocytic leukemia 3.0(1) 0.4 (1) Acute lymphoblastic leukemia 1.5+0.7 (4) 0.2£0.06 (4) Burkitt’s lymphoma 4.1 (1) 3.0 (1) Diffuse histiocytic leukemia 2.0 (1) 0.4 (1) Nodular mixed lymphoma 2.0 (1) 0.3 (1) Hela tissue culture line® 36.08.14) 9.2+4.8 (4) L1210 cell line® 19.6 +2.6 (3) 7214.2 (3) ¢ Mean * SD (No. of patients, donors, or tissue-culture passages). All assays of hemopoietic malignancies other than CLL were repeated twice. b n.d., not detectable, i.e., less than 7X 103 copies/ cell. ¢ Cells in exponential growth. (25)]. (ii) Cells of a diffuse histiocytic lymphoma were also confined to Gy/G, (Figure 4). However, their topoisom- erase II level was detectable (Table 1) and ADR did cause PAB. (iii) Conversely, cells of a prolymphocytic leukemia were resistant to ADR (Table 2), in spite of a proliferating cell fraction (Figure 4) and a detectable topoisomerase II level (Table 1). In the latter case, it could be suggested that accelerated dissociation or a lack of association between ADR molecules and topoisomerase [I-DNA adducts favors the formation of a “noncleavable” complex, perhaps due to structural or functional changes of the enzyme. More information has to be provided by a systematic study of selected hemopoietic malignancies. Mutant Topoisomerase Il in Cancer It has been shown by others (26-28) that several tissue culture lines, which are resistant to topoisomerase II inhibi- tors, may retain some but not all functions linked to this enzyme. Also, topoisomerase II of a CHO cell line resistant to epipodophyllotoxins differs in some characteristics from the wild-type enzyme. This includes the lack of stabi- lization of the topoisomerase II-DNA complex by VP-16 (28). Since the characteristic of leukemia and cancer reflects clonal selection of abnormal cell types with the most advantageous phenotype arising via mutation, recom- bination, or deletion, it could be suggested that topoisom- erase II with aberrant properties could be present in some cancer cells. Alternatively, regulatory steps in the process- ing of the cleavable complex could be affected. Undoubt- edly, the research in this direction is in its initial stage and more conclusive work in human tumors is needed. @ L1210 CLL LY-B £ 2 oO +64 ~ 0 S GoM ° Fr Oo Tm -.r-rTrTrT Ti rrrrrroroto © Ly-T HL PL ee € o £ Oo ~ 2 © oO rrr rr TT T= Fryer 500 1000 500 1000 500 100 DNA DNA DNA FIGURE 4.—Flow cytometry data represented by one-parameter histo- grams of exponentially growing L1210 cells, chronic lymphocytic leukemia lymphocytes (CLL), enriched normal T- (LY-T) and B- lymphocytes (LY-B), diffuse histiocytic lymphoma cells (HL), and pro- lymphocytic leukemia cells (PL). Note that cycling cells were detected in the first (L1210) and last panel (PL). CONCLUSIONS There is the question of what does it mean to a cancer patient. Extremely low levels of DNA topoisomerase II may explain the lack of ADR effects in B-cell CLL. Even more importantly, CLL cells provide a model situation: Topoisomerase II-mediated drug cytotoxicity could be seriously diminished in any quiescent cancer cell with extremely low levels of DNA topoisomerase II. This may explain the poor results of therapy by ADR and other topoisomerase II-targeted drugs applied to slow growing tumors with a large compartment of quiescent cells (17). If so, there are several directions in drug developmental strategies which could attempt to bypass such limitation: 1) Very consistently, DNA topoisomerase I levels are largely independent of the proliferative status of tumor cells (Table 1). The recent identification of this enzyme as a possible target in cancer chemotherapy may offer a new approach to the management of slow growing tumors. Camptothecin has been identified as an alkaloid active against experimental leukemia and cancer (29) and targeted at topoisomerase 1 (30). 2) While a reversible trapping of the topoisomerase I1- DNA cleavable complex is very likely responsible for the cytotoxicity exerted by various drugs including ADR (12), the overall efficiency of the ADR-topoisomerase II interac- tion is low (9,23,24). This efficiency, and presumably drug effectiveness against cancer cells with low levels of topo- isomerase II, could be increased by a conversion of the ADR molecule into an N-acyl or N-alkylanthracycline (22). TABLE 2.—Protein-Associated DNA Cleavage Enriched lymphocytes Type Drug (ug/ml) Lymphocytes B-Cells T-Cells CLL ADR (5.0) 0.11£0.15 (3) 0.03+0.09 (4) 0.71£0.36 (4) Normal ADR (5.0) 0.01 (2) 0.18 (2) —0.01£0.06 (4) Prolymphocytic leukemia ADR (5.0) —0.07 (1) Diffuse histiocytic lymphoma ADR (5.0) 0.47 (1) 4 Mean+SD (No. of patients or donors). Cells from each patient or donor were assayed twice. Results are shown as the number of PAB/10° nucleotides. Statistically significant increases above the background level are indicated by underlining. 108 NCI MONOGRAPHS, NUMBER 4, 1987 3) Based on an analogy in bacterial systems, an SOS-like repair response may play an important role in the killing mechanism of topoisomerase II-directed drugs in cancer cells (12). A theoretical possibility exists that the com- pounds stimulating the SOS-like repair could be used as enhancers of the cell kill by topoisomerase-targeted drugs. 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Proc Am Assoc Cancer Res 25:303, 1984. (22) GROSSBERG H, POTMESIL M, ISRAEL M, et al: Induction of DNA lesions in chronic lymphocytic leukemia (CLL) by novel derivatives of Adriamycin (ADR). Proc Am Assoc Cancer Res 26:224, 1985. (23) PoTMESIL M, BANK B, GROSSBERG H, et al: Resistance of human leukemic and normal lymphocytes to DNA cleav- age by drugs correlates with low levels of DNA topo- isomerase II. J Clin Invest. Submitted for publication. (24) SILBER R, Liu LF, HSIANG Y-H, et al: DNA topoisomerase II was not detected in lymphocytes from patients with B-cell chronic lymphocytic leukemia (CLL). Proc Am Assoc Cancer Res 27:248, 1986. (25) STARK S, LIEBES LF, NEVRLA D, et al: Decreased actin content of lymphocytes from patients with chronic lympho- cytic leukemia. Blood 59:536-541, 1982. (26) POMMIER Y, SCHWARTZ RE, ZWELLING LA, et al: Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibi- tors. Cancer Res 46:611-616, 1986. (27) GLISSON BS, SULLIVAN DM, GUPTA R, et al: Mediation of multi-drug resistance in a Chinese hamster ovary cell line by a mutant type II topoisomerase. NCI Monogr 4:89-93, 1987. (28) ROWE T, KUPFER G, Ross W: Inhibition of epipodophyllo- toxin cytotoxicity by interference with topoisomerase- mediated DNA cleavage. Biochem Pharmacol 34:2483- 2487, 1985. (29) WALL ME, WANI MC, COOKE CE, et al: The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from ‘Camptotheca acuminata’. J Am Chem Soc 88:3888-3890, 1966. (30) HSIANG Y-H, HERTZBERG R, HECHT S, et al: Camptothecin induced protein-linked DNA breaks via mammalian topoisomerase I. J Biol Chem 260:14873-14878, 1985. 109 Metabolic Activation of N-Acylanthracyclines Precedes Their Interaction With DNA Topoisomerase Il’ Robert Silber,? Leroy F. Liu,® Mervyn Israel,* Annette L. Bodley,® Yaw-Huei Hsiang,® Stanley Kirschenbaum,? Trevor W. Sweatman,* Ramakrishnan Seshadri,’ and Milan Potmesil #5 * ABSTRACT—The N-acylanthracyclines AD32 (N-trifluoro- acetyladriamycin-14-valerate) and AD143 (/V-trifluoroacetyladria- mycin-14-O-hemiadipate) are analogs of Adriamycin (ADR) undergoing clinical or advanced pre-clinical screening. Their prin- cipal metabolites, following the cleavage of the 14-acyl side-chain, are N-trifluoroacetyladriamycin (AD41) and its reduced form N- trifluoroacetyladriamycinol (AD92). Both these compounds are biologically active and detectable in treated patients, laboratory animals, and in tissue culture cells. Unlike ADR, AD32, as well as AD143 and metabolites, show no detectable binding to double- strand DNA. Their effects on DNA have been previously investi- gated in vivo and in vitro using the alkaline filter-elution assay. It has been shown that all of the compounds cause approximately equivalent amounts of protein-associated DNA breaks (PAB) and DNA-protein crosslinks in a mouse lymphoma and in tissue- culture leukemia cells. In order to establish whether the induction of PAB by the drugs requires DNA topoisomerase II mediation, cleavage mapping analysis was done with tested compounds using purified human topoisomerase II. DNA fragmentation was signif- icantly enhanced in the presence of the enzyme and either AD41 or AD92. In contrast, no fragmentation enhancement was observed in the presence of the parental drugs AD32 or AD143. The results strongly suggest that metabolic activation of N-acyl- anthracyclines by nonspecific esterases is a prerequisite for their interaction with DNA topoisomerase II and for stabilization of the cleavable complex.—NCI Monogr 4:111-115, 1987. ABBREVIATIONS: AD32= N-trifluoroacetyladriamycin-14-valerate; AD143= N-trifluoroacetyladriamycin-14-O-hemiadipate; ADR = Adriamycin (doxorubicin); AD41= N-trifluoroacetyladriamycin; AD92= N-trifluoroacetyladriamycinol; PAB=protein-associated DNA breaks; DMSO = dimethyl sulfoxide; i.a. = intra-arterial; SDS =sodium dodecyl sulfate; AMNOL = adriamycinol (14- dihydroadriamycin). I'Supported in part by Public Health Service grants CA-37082, CA- 37209, CA-11655, and CA-39662 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by grant CH-348 from the American Cancer Society; and by the Marcia Slater Society for Research in Leukemia. 2Departments of Medicine and Radiology, New York University School of Medicine, New York, NY. 3 Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD. 4 Department of Pharmacology, University of Tennessee College of Medicine, Memphis. 5 We thank Dr. Marc B. Garnick, Dana-Farber Cancer Institute (Bos- ton, MA), for obtaining blood samples for analysis and Drs. William J. Pegg and Vinod K. Khetarpal for assistance with the clinical and animal pharmacology studies. * Reprint requests: Milan Potmesil, M.D., Department of Radiology, New York University School of Medicine, 550 First Ave., New York, NY 10016. The metabolic conversion of AD32 and AD143 (struc- tural formulas, Table 1), two N-acylanthracycline analogs of ADR in clinical or advanced preclinical testing, involves the formation of AD41 by the action of nonspecific serum and tissue esterases. Some of the metabolite is further transformed into AD92 by ubiquitous aldo-keto reduc- tases. Both of the metabolites lack the acyl function at the side-chain 14-carbinol position. The metabolic pathway has been documented in previous publications (/-3) and is re- examined in this study. Earlier investigations from our laboratories have shown that AD32, AD143, and their principal metabolites induce approximately equivalent amounts of PAB and DNA-pro- tein crosslinks in L1210 mouse leukemia cells in vitro (3-5) and in a mouse lymphoma in vivo (2). DNA topoisomerase II has been established as a target enzyme of ADR (6). ADR appears to stabilize a topoisomerase II-DNA interac- tion, and the resultant “cleavable complex” is detectable by alkaline-elution technique as PAB (7). The present study was undertaken to establish (i) whether the DNA nonbinding anthracyclines, similarly to the intercalating anthracycline ADR, stabilize the topo- isomerase II-DNA cleavable complex, and (ii) whether the conversion of AD32 and AD143 into their chief metabo- lites could be of importance for the drug-topoisomerase II-DNA interaction. MATERIALS AND METHODS Spectrophotometric determination of drug-DNA bind- ing.—A modification of the procedure of Goldberg et al. (8) and Rusconi (9) was used. One hundred mg of calf thymus DNA (sodium salt, Type I, Sigma Chemical Co., St. Louis, MO) was gently stirred overnight at room temperature in 50 ml of Tris buffer, 0.1 M, pH 7.0. The solution was filtered through Whatman No. 2 cellulose fil- ter paper and stored at 4° C. The concentration of the solu- tion was determined by measuring its optical density at 260 nm, using an ultraviolet/visible spectrophotometer. Stock solutions of tested drugs, 0.1 mM, were prepared in Tris buffer, 0.1 M, pH 7.0, containing 5% DMSO. The absorbances of the anthracycline solutions at 480 nm in the absence (Ay) or presence of DNA (A) were determined. The molar ratio of anthracycline to DNA was adjusted to 1:10, respectively, in the drug-DNA mixture. The ratio A/ A, was taken as a measure of the extent of DNA binding, with values 0.55-0.6 representing strong drug-DNA binding and a value of 0.96-0.98 indicating no DNA binding. Pharmacology studies.— As part of Phase I study (10,11), plasma levels of AD32 and metabolites were studied in ten 111 TABLE 1.—Structural Formulas of Adriamycin and N-Acylanthracyclines y C-CH,0Rs “OH 0, CH, HO NA Ri Name Code R, R, R; Adriamycin ADR H oO H N-Trifluoroacetyladria- AD32 COCF; O CO(CH,);CHj3 mycin-14-valerate N-Trifluoroacetyladria- AD143 COCF; O CO(CH,)4COOH mycin-14-0- hemiadipate N-Trifluoroacetyladria- AD41 COCF; O H mycin? N-Trifluoroacetyladria- AD92 COCF; H,OH H mycinol? ¢ Metabolites of AD32 and AD143. consenting patients at various dose levels. One of the patients, with lung metastases of an adenocarcinoma of unknown origin and normal liver function, had received a 24-hr continuous infusion of 400 mg/m? of AD32. Venous blood was obtained during the infusion at 0.5, 3, 6, and 18 hr, as well as 1 and 12 hr after its discontinuation. Sepa- rated plasma was mixed with 150 ml of methanol to precipi- tate protein. Following centrifugation, the supernatant was extracted three times with 2 ml of chloroform. Extracts from each sample were pooled, evaporated to dryness under N, gas at 25°C, frozen and stored at —20°C for further analysis. In animal studies, a group of 5 male A/JAX mice injected with AD32, 50 mg/kg i.v., and a group of 4 male Sprague-Dawley rats receiving AD143, 50 mg/kg i.a., were used. Blood samples were obtained at intervals from 0 to 24 hr (mice) or from 0 to 8 hr (rats), following the drug administration. Serum from all the mice was pooled at each time interval, mixed with equal volumes of Tris buffer, 0.05 M, pH 8.5, and extracted twice with 4 ml of chloro- form-propanol (3:1 by volume). N-Trifluoroacetyladria- mycin-14-octanoate, 250 ng, was added to each aliquot of serum extracts as an internal standard. Plasma samples from individual rats were processed, without pooling, in a similar way. Finally, the samples were evaporated to dry- ness and treated as described in the preceding paragraph. For in vitro studies, CEM cells were maintained in log- phase growth in Eagle’s MEM (Grand Island Biological Co., Grand Island, NY) supplemented with 10% heat- inactivated fetal bovine serum (Grand Island Biological Co.) at 37°C. AD32, 10 uM, was added to the culture for various time intervals from 15 min to 6 hr. At the end of each incubation period, cell pellets and media supernatants were obtained from duplicate cultures. Pelleted cells were washed twice with 3 ml of cold saline (0.15 M NaCl), resus- 112 pended in 1-2 ml of saline and sonicated. Cell sonicates, aliquots of culture media, and aliquots of cell washes were mixed with equal volumes of Tris buffer, 0.05 M, pH 8.5, and extracted three times with 6 ml of ethyl acetate:pro- panol (9:1 by volume). Extracts were pooled, an internal standard was added, and samples were concentrated as detailed above. HPLC analyses.—Human and animal samples were sub- jected to complementary reverse-phase and normal-phase HPLC analysis (12,13). Reverse-phase separation was con- ducted with a uBondapak/ phenyl column (30 cm long and 3.9 mm diameter) using a dual-pump solvent delivery sys- tem (Waters Associates, Milford, MA). The solvent system consisted of ammonium formate buffer, 0.05 M, pH 4.0 (solvent A) and acetonitrile (solvent B). The flow rate was 3.5 ml/min, using a linear gradient from 68% A:32% B to 35% A: 65% B over 6 min, with the final condition held for additional 4 min. Normal-phase chromatographic separa- tion was achieved with a Partisil-10 PAC column (30 cm X 3.9 mm; Whatman Inc., Clifton, NJ) and an eluting sol- vent system consisting of chloroform (solvent A) and a 4-component mixed solvent (chloroform:methanol:acetic acid:water, 85:15:5:1.5, solvent B) at a flow rate of 2.0 ml/min. The initial conditions of 90% A:10% B were modi- fied (Waters gradient profile curve #4) to 100% B over 2 min, with final conditions maintained for an additional 8 min. Samples obtained in the in vitro study were analyzed only by reverse-phase chromatography in a procedure simi- lar to the one described in the preceding section. A phenyl- RADIALPAK 10 pu column (10 cm X 8 mm), mounted in a Z-Module radial compression unit, was used. In both modes, normal and reverse-phase HPLC, column eluate was monitored using a flow fluorometer (Model SF- 970; Kratos Schoeffel Instruments, Ramsey, NJ) fitted with a deuterium light source (482 nm excitation wavelength, and barrier emission filter with 550 nm cut-off). Elution was monitored using a fluorescence detector, and eluting peaks were identified by their retention time relative to that of authentic standards. Peaks were quantified by reference to standard extraction curves for each drug and metabolite, and corrected for recovery of internal standards. DNA topoisomerase I and Il.—Topoisomerase I and II were purified to homogeneity from calf thymus glands and HeLa cells, respectively (14,15). Assay for topoisomerase-mediated DNA cleavage.—The assay was performed as described previously (6). PM C41 plasmid DNA was linearized with EcoRI and then end- labeled at the 3’ termini with the large fragment of E. coli DNA polymerase I in the presence of (alpha->P)dATP and unlabeled dTTP. The test drugs were dissolved in DMSO, then diluted to the desired treatment concentration with incubation buffer. Cleavage reaction (20 ul each) contain- ing 50 mM Tris, pH 7.5, 100 mM KCl, 10 mM MgCl,, 1 mM ATP, 0.5 mM DTT, 0.5 mM EDTA, 30 pg/ml bovine serum albumin, 50 ng 3-end labeled DNA, 50 ng of puri- fied human topoisomerase II, and 0, 0.02, 0.1, 0.5, 2.5, and 12.5 ug/ml of drug, was incubated for 20 min at 37°C. The reaction was stopped with the addition of SDS, to a final concentration of 1%. Proteinase K (0.2 mg/ml) was added to each reaction mixture, followed by 1 hr incubation at 37°C. Reaction products were analyzed by electrophoresis using a 1% agarose gel either in alkaline (topoisomerase I) NCI MONOGRAPHS, NUMBER 4, 1987 TABLE 2.—Binding Affinity of Anthracyclines to Double-Strand Calf Thymus DNA Drug A/Ay° ADR 0.6 AD32 0.98 AD41 0.98 AD92 0.97 AD143 0.96 “ The A/ A, value of 0.96-0.98 corresponds to the background fluctua- tion and indicates no drug-DNA binding. or neutral buffer (topoisomerase II). The cleavage pattern was visualized by autoradiography. RESULTS The binding affinity of AD32, AD143, and their major metabolites AD41 and AD92, to double-strand DNA was negligible (Table 2). ADR, the reference standard, has an A/A, value of 0.6 indicating strong binding affinity to DNA. Pharmacology studies showed a consistent metabolic conversion of AD32 and ADI43 into AD41 and AD92. This pathway was demonstrated in a patient treated with AD32 (Figure 1), and in mice and rats injected with AD32 and AD143, respectively (Figure 2). Both metabolites were also detected in vitro, following the incubation of CEM cells with AD32 for 15 min to 6 hr (Table 3). Trace amounts of ADR were detectable in rats in vivo and CEM cells in vitro. This conversion was minimal and at early incubation times did not exceed 0.4% of the total fluorescence signal recoverable from cultured cells. As shown previously (6), ADR stimulated the topo- isomerase II-mediated cleavage of plasmid DNA at low concentrations (Figure 3). Higher drug doses inhibited cleavage. At ADR concentration of 2.5 ug/ml (4.2 uM) and greater, the cleavage reaction was virtually abolished. N-Acylanthracyclines, AD32 and ADI143, at concentra- tions of 0.1-12.5 ug/ml (0.04-26.5 or 0.05-29.4 uM, respec- tively) did not stimulate topoisomerase I- or II-mediated DNA cleavage (not shown). However their metabolites, prea: 1X10 S ; = 4] D [&) [sms Oo © 7 5 1X10 H i= = ® AD32 LY oO ADH % A AD92 Y X 1X10°8 wall i 1 . . 0326 18 30 42 Time (hr) FIGURE 1.—Levels of AD32 and metabolites in blood plasma of a patient treated with AD32, 400 mg/m? in a continuous 24-hr iv infusion; n.d., not detectable. DNA TOPOISOMERASES IN CANCER THERAPY 509. (B) 50 3S nmoles /ml nmoles / ml 1 001 ——1 + 2468 12 16 24 1 2 3458678 Hrs Post AD32 Administration Hrs Post AD143 Administration FIGURE 2.—(A) Levels of AD32 and metabolites in serum of A/JAX mice following an intravenous bolus of AD32, 50 mg/kg. (B) Levels of AD143 and metabolites in plasma of Sprague-Dawley rats following an intra-arterial injection of AD143, 50 mg/kg. AD41 and AD92, stimulated topoisomerase II-mediated cleavage in a dose-dependent manner at concentrations of 0.1-12.5 pg/ml (0.04-23.5 uM) (Figure 3), while they remained ineffective in the absence of the enzyme. A com- parison between the cleavage pattern of AD41 or AD92 and ADR shows clearly discernible differences in the inten- sity of individual bands. Unlike ADR, increasing concen- trations of AD41 and AD92 produced increasing amounts of cleaved DNA. DISCUSSION It has been shown in this study that the N-acylanthracy- clines, AD41 and AD92, analogs of ADR which do not bind to DNA, interact with DNA topoisomerase II and stabilize the cleavable complex between the enzyme and DNA. The cleavage pattern of the two analogs of ADR, and that of ADR, are identical, suggesting that all these drugs have the same cutting sites of the plasmid DNA. While the side-chain 14-position substitution by O-valer- ate (AD32) or O-hemiadipate (AD143) functions is impor- tant for various biological properties, such as intracellular partitioning, enhanced tumor-mass penetration, alterations of cell-membrane permeability, and reduced overall toxic- ity (16-18), this substitution also appears to prevent the drug molecules from interacting with DNA topoisomerase II. Thus, the metabolic cleavage of the 14-O-acyl substitu- ent of AD32 or AD143, with the resultant formation of AD41 and AD92, could be a prerequisite for the interaction between the drug and enzyme (Figure 4). Among the metabolites, there were inactive aglycones, unidentified species, and trace levels of ADR (Figure 2B and Table 3). The in vitro conversion of AD32 to ADR at 15- and 45-min treatment intervals represents less than 0.4% of the total fluorescence signal. The calculated ADR concentration in the incubation medium was 0.02 ug/ml (0.04 uM). This concentration of ADR is non-toxic (1/9). The in vivo con- version in a murine lymphoma model represents approxi- mately an equivalent of <1 mg/kg of ADR in a single dose, which is totally ineffective (2). A question remains concerning the precise nature of the interaction between N-acylanthracyclines and topoisom- erase II. While the structure-activity relationship between this enzyme and N-acylanthracyclines has not yet been 113 FIGURE 3.—ADR, AD41, and AD92 stimulated topoisomerase II-mediated DNA cleavage. PMC41 DNA, labeled at one end with (alpha->2P)d ATP, was incubated in a reaction mixture containing 50 ng of DNA, approximately 50 ng of human DNA topoisom- erase II, and no drug (A), ADR 0.02 ug/ml (0.03 uM) (B), 0.1 (0.17) (C), 0.5 (0.85) (D), 2.5 (4.25) (E), and 12.5 (21.25) (F); AD41 0.02-12.5 ug/ml (0.04-23.42 uM) (GK); and AD92 0.02-12.5 ug/ml (0.04-23.5 uM) (L-P). DNA samples were electrophoresed in a neutral buffer. topoisomerase II N-Trifluoroacetyl | JADR] [14-0-| adipate | Secsnn nn EEESTEeeENSEENeRT i — a) RX topoisomerase 11 ] anssesss fssssanccssnesne mm > topoisomerase II 1 senuneaenseed eesscesss 1 Ww aldoketoreductases 1 1 v anscssee Jpessssccsssssns N-Trifluoroacetyl][AMNOL ]------- > topoisomerase II ssseescsssses) sssasssees double-strand DNA smces Kasssseamame topoisomerase II sesssssesadl seses enzyme co-valently bound to 5'-termini FIGURE 4.—Model of the metabolic activation of ADI143 and the drug-DNA topoisomerase II-DNA interaction. Noncleavable complex, right upper corner; cleavable complex, lower left corner. 114 investigated in a systematic way, a working hypothesis for the interaction could be suggested. Although these drugs do not bind to DNA, they may bind to the enzyme-DNA adduct forming a ternary complex. Binding of the anthra- cycline to topoisomerase could result in the inhibition of the enzyme and stabilization of the cleavable complex between the enzyme and DNA. Using the filter-DNA elution tech- nique, the stabilized complexes are detected as PAB. The proposed ternary complex, drug-enzyme-DNA, implies that the enzyme molecule has a domain which represents the binding site for the drug. Cleavage of the bulky side- chain of AD32 or AD143 may reduce steric interference by the drug molecule in the formation of drug-topoisomerase II-DNA complexes. A broader study of the interaction between topoisomerase II and various ADR analogs is under way. It remains unclear whether (i) the intercalative mode of drug-DNA binding or (ii) just an increased concentration of drug molecules around topoisomerase II-DNA adducts is essential for the stabilization of topoisomerase II-DNA interaction (6,20). Our results showing that DNA non- binding AD41 and AD92 stabilize the cleavable complex between the enzyme and DNA favor the second possibility. However, a weak drug-DNA binding, not detectable by the present method, could be important for the formation of NCI MONOGRAPHS, NUMBER 4, 1987 TABLE 3.—AD32 Partitioning and Metabolism in CEM Cells 15 min 45 min 3 hr 6 hr 15 min 45 min 3 hr 6 hr Signal Concentration nmol/ 10° cells Concentration pmol/ml media AD32 0.849 0.957 0.948 0.633 8.08 6.39 4.09 1.80 AD41 0.181 0.131 0.235 0.261 0.40 0.69 3.39 3.85 AD92 0.002 0.005 0.003 0.003 n.d. n.d. n.d. 0.04 ADR 0.003 0.004 0.016 0.038 n.d. t.a.c 0.28 0.04 ADMOL n.d. n.d. n.d. 0.003 n.d. n.d. n.d. n.d. Others 0.035 0.022 0.123 0.052 0.08 ¢ Cells were incubated with 10 uM AD32 for the time intervals indicated. b n.d., not detected. ¢ t.a., trace amount. drug-topoisomerase II-DNA ternary complexes. A more extensive analysis is necessary to establish the mode of the interaction between N-acylanthracyclines and DNA topo- isomerase II. REFERENCES (I) ISRAEL M, WILKINSON PM, PEGG WJ, et al: Hepatobiliary metabolism and excretion of Adriamycin and N-trifluoro- acetyladriamycin-14-valerate in the rat. Cancer Res 38: 365-370, 1978. (2) POTMESIL M, LEVIN M, TRAGANOS F, et al: In vivo effects of Adriamycin or N-trifluoroacetyladriamycin-14-valerate on a mouse lymphoma. Eur J Cancer Clin Oncol 19: 109-122, 1983. (3) LEVIN M, SILBER R, ISRAEL M, et al: Protein-associated DNA breaks and DNA-protein crosslinks caused by DNA non-binding derivatives of Adriamycin in L1210 cells. Cancer Res 41:1006-1010, 1981. (4) POTMESIL M, KIRSCHENBAUM S, ISRAEL M, et al: Dose dependency of DNA lesions induced in hypoxic and euoxic L1210 cells by Adriamycin (ADR) and DNA non- binding derivatives. In Proceedings of the 13th Interna- tional Cancer Congress, Seattle, Sept. 1982, p 145. (5) POTMESIL M, ISRAEL M, LIU LF, et al: Cellular metabolism and DNA interaction of N-acyl- and N-alkylanthracy- clines. In Proceedings of the 14th International Cancer Congress, 1986, Budapest, p 158. (6) TEWEY KM, ROWE TC, YANG L, et al: Adriamycin-induced DNA damage mediated by mammalian DNA topoisom- erase II. Science 226:466-468, 1984. (7) Ross WE, GLAUBIGER DL, KOHN KW: Protein-associated DNA breaks in cells treated with Adriamycin or ellipti- cine. Biochim Biophys Acta 519:23-30, 1978. (8) GOLDBERG IH, RABINOWITZ M, REICH E: Basis of actino- mycin action. I. Deoxyribonucleic acid (DNA) binding and inhibition of ribonucleic acid (RNA)-polymerase syn- thetic reactions by actinomycin. Proc Natl Acad Sci USA 48:2094-2101, 1962. (9) Ruscont A: Different binding sites in DNA for actinomycin and daunomycin. Biochim Biophys Acta 123:627-630, 1966. DNA TOPOISOMERASES IN CANCER THERAPY (10) BLUM RH, GARNICK MB, ISRAEL M, et al: Preclinical ratio- nale and phase I clinical trials of the Adriamycin analog, AD32. Recent Results Cancer Res 76:7-15, 1981. (11) BLuM RH, GARNICK MB, ISRAEL M, et al: Initial clinical evaluation of N-trifluoroacetyladriamycin-14-valerate (AD-32), an Adriamycin analog. Cancer Treat Rep 63: 919-923, 1979. (12) ISRAEL M, PEGG WJ, WILKINSON PM, et al: Liquid chro- matographic analysis of Adriamycin and metabolites in biological fluids. J Liquid Chromatogr 1:795-809, 1978. (13) ISRAEL M, KARKOWSKY AM, KHETARPAL VK: Distribution of radioactivity and anthracycline fluorescence in tissues of mice one hour post-[!“C]-labeled AD 32 administration. Evidence for tissue aglycone formation. Cancer Che- mother Pharmacol 6:25-30, 1981. (14) Liu LF, MILLER KG: Eukaryotic DNA topoisomerases: Two forms of type I DNA topoisomerases from HeLa cell nuclei. Proc Natl Acad Sci USA 78:3487-3491, 1981. (15) HALLIGAN BD, EDWARDS KA, Liu LF: Purification and characterization of calf thymus DNA topoisomerase II. J Biol Chem 260:2475-2482, 1985. (16) ISRAEL M, POTTI PG, SESHADRI R: Adriamycin analogs: Rationale, synthesis, and preliminary antitumor evalua- tion of highly-active DNA-nonbinding N-(trifluoroacetyl)- adriamycin-14-O-hemiester derivatives. J Med Chem 28:1223-1228, 1985. (17) ISRAEL M, IDRISs JM, KOSEK1 Y, et al: Comparative effects of Adriamycin and DNA non-binding analogues on DNA, RNA and protein synthesis in vitro. Cancer Chemother Pharmacol. Submitted for publication. (18) ISRAEL M, IDRISS JM: A possible mechanistic basis for the growth-inhibitory properties of N-trifluoroacetyladriamy- cin-14-valerate (AD 32), a non-DNA binding Adriamycin (ADR) analog. In Proceedings of the 13th International Cancer Congress, Seattle, Sept 1982, p 245. (19) POTMESIL M, ISRAEL M, KIRSCHENBAUM 8S, et al: Effects of N-trifluoroacetyladriamycin-14-O-hemiadipate and radia- tion on L1210 cells. Radiat Res 105:147-157, 1986. (20) TEWEY KM, CHEN GL, NELSON EM, et al: Intercalative antitumor drugs interfere with the breakage-reunion reac- tion of mammalian DNA topoisomerase II. J Biol Chem 258:9182-9187, 1984. 115 "Toa } Cte tw, aloe aa a Sra F =e CLE Ts nD yo ah Aa 6 THE S33 =m. = i = ih I n Se I =F | ne | B oo oo a iL : - oo how: AE Sut LE EE = ' a? Bik ane = Protein-linked DNA Strand Breaks Produced by Etoposide and Teniposide in Mouse L1210 and Human VA-13 and HT-29 Cell Lines: Relationship to Cytotoxicity Donna Kerrigan, Yves Pommier, and Kurt W. Kohn * ABSTRACT —The two demethylepipodophyllotoxin glycosides, teniposide (VM-26) and etoposide (VP-16), have previously been reported to interact with DNA topoisomerase II by stabilizing a topoisomerase II-DNA covalent intermediate. This study exam- ined the protein-association aspect of the topoisomerase II-DNA - epipodophyllotoxin lesion. We found that in mouse (L1210) and human (VA-13 and HT-29) log-phase cell cultures, all DNA strand breaks produced by VP-16 or VM-26 were protein-associated. We found also that these protein-associated breaks occurred with a frequency which correlated with cytotoxicity in all three cell lines. For all three cell lines and for both compounds the regression lines were similar. Therefore, for a given class of topoisomerase II inhibitors, it may be possible to generate a characteristic line from which DNA-protein crosslink frequency predicts cytotoxicity.— NCI Monogr 4:117-121, 1987. INTRODUCTION The demethylepipodophyllotoxin derivatives, etoposide (VP-16) and teniposide (VM-26), have been shown to pro- duce DNA single- and double-strand breaks and DNA- protein crosslinks in cultured mammalian cells (/-4). These DNA strand breaks have been postulated as mediated by DNA topoisomerase II because both VP-16 and VM-26 can produce similar DNA effects in isolated DNA with purified enzyme (5). Alternatively, it has been suggested that VP-16 could also induce some protein-free DNA strand breaks in cultured L1210 cells by free radical pro- duction (3). A follow-up study using free radical scavengers disclaimed the free radical mechanism for VP-16 (6). In order to clarify this possibility, VP-16- and VM-26- induced protein-associated and protein-free DNA strand breaks were measured by alkaline elution in L1210 cells and in two other cell lines (HT-29 and VA-13) chosen for their differences in free radical production potential (7,8). If free radical-mediated DNA strand breaks were generated the expected result would be: i) the production of protein-free DNA strand breaks, and ii) a greater frequency of DNA single-strand breaks than DNA-protein crosslinks. If only topoisomerase II-mediated DNA strand breaks were gen- erated, the expected result would be (Figure 1): i) the I Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. * Reprint requests: Kurt W. Kohn, M.D., Ph.D., Laboratory of Molec- ular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bldg. 37, Rm. 5A-19, Bethesda, MD 20892. absence of protein-free DNA strand breaks, and ii) equal frequencies of DNA single-strand breaks and DNA-pro- tein crosslinks (9). MATERIALS AND METHODS The mouse leukemia 1.1210 cell line was grown in RPMI 1630 medium supplemented with 10% horse serum, 2 mM glutamine, and 50 units/ml of both penicillin and strepto- mycin. The human colon carcinoma, HT-29 and the viral- transformed human embryonic fibroblast, VA-13, cell lines were grown in Eagle’s minimum essential medium supple- mented with 10% fetal calf serum, 2 mM glutamine, | mM sodium pyruvate, and 0.1 mM non-essential amino acids. Cultures utilized in these experiments were all in exponen- tial growth phase. Cellular DNA was labeled by incubation with [2-"C]thymidine (0.02 wCi/ml) or with [methyl- 3H]thymidine (0.1 »Ci/ml, 107° M cold thymidine added) for 24 h. Radioactive label was removed for 24 hours prior to the experiments. Etoposide (VP-16, NSC 141540) was a generous gift from Bristol-Myers Co. (Syracuse, NY). Teniposide (VM- 26, NSC 122819) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, NCI. VP-16 and VM-26 were dissolved at 10 mM in di- methyl sulfoxide immediately before each experiment. Drug exposures were for 45 min at 37°C in culture medium or nucleus buffer. Drugs were removed by centrifugation (L1210) or washing the cultures (HT-29, VA-13) with phosphate-buffered saline at 0° C. Nuclei were diluted 1:20 into cold nucleus buffer prior to elution. Nuclei were prepared as described previously (10). Cells were centrifuged and resuspended in nucleus buffer (NB; 150 mM NaCl, 1 mM KH,PO,, 5S mM MgCl,, 1 mM EGTA, 0.1 mM DTT, pH 6.4) at 4° C. Cells were washed in NB and centrifuged again at 4°C, pellets were then resus- pended in 1/10 volume NB at 49°C. A 9/10 volume of NB containing 0.3% Triton X-100 was added. The mixture was incubated at 4°C for 10 min. Nuclei pellets were then obtained by centrifugation and resuspension in NB at 37°C. Drug-induced cytotoxicity was determined by colony formation assay following drug removal. For the L1210 cells, 102-10° cells were seeded in triplicate into sterile tubes containing 3 ml culture medium with soft agar at 0.1% final concentration. Colonies were counted after 12 days incuba- tion at 37°C. The cloning efficiency of untreated cells was approximately 90% and was used to normalize the drug- induced cytotoxicities. For the HT-29 and VA-13 cells, log 117 Topoisomerase II Double-stranded DNA a 1 1 3 No’ ) C SDS Alkali SSB Ng § mr on FIGURE 1.—Schematic distinction between a free radical-mediated SSB and a protein-associated DPC as detectable by alkaline elution. cultures were dispersed with trypsin-EDTA, diluted in warm culture medium and counted. 10-10 cells were seeded in triplicate into 25 cm? flasks in 5 ml medium. Cells were allowed to attach for four hours and were then drug- treated by adding 5 ml of medium containing either VP-16 or VM-26. Drug treatments were ended by removing drug- containing medium and washing the cultures twice with 10 ml fresh medium at 37° C. 5 ml fresh culture medium was then added and the flasks were incubated for 10-12 days in humidified 5% CO, incubators at 37°C. Colonies were counted after methylene blue staining. The average plating efficiencies of untreated cells were 67% for HT-29 and 64% for VA-13. Drug-induced cytotoxicities were normalized to these values. The various alkaline elution procedures were performed as previously described (11) and are summarized in Table 1. DNA single-strand break (SSB) frequencies were deter- mined from vy-irradiation doses that would produce DNA elution curves similar to those observed in drug-treated cells and were expressed in rad-equivalents (11). DNA-pro- tein crosslinks were calculated from the protein bound to one terminus model and expressed in rad-equivalents (11). RESULTS As indicated in Figures 2 and 3, both VP-16 and VM-26 produced DNA-protein crosslinks (DPC) and DNA single- strand breaks (SSB) in a 1:1 ratio for all three cell lines. It is important to note also that neither VP-16 nor VM-26 pro- duced DNA frank breaks (protein-free) in any cell line at any dose examined. Since demonstration of these charac- teristics by the intercalator classes of topoisomerase (12) II inhibitors is held as indicative of DNA damage mediated via the topoisomerase, the demethylepipodophyllotoxins constitute another class of these inhibitors and, at least under the conditions of the present study, do not damage DNA via free-radical production. The potency of VP-16 and VM-26 to induce SSB and DPC and the drug sensitivity of the three cell lines, L1210, VA-13, and HT-29, are compared in Table 2. The drug concentrations producing 1000 rad-equivalents SSB or DPC were obtained from Figures 2 and 3. VM-26 was more potent than VP-16 (9-fold in L1210, 26-fold in VA-13 or HT-29 cells). L1210 cells were more sensitive to VP-16 (4- fold) or VM-26 (1.5 fold) than VA-13 or HT-29 cells. The surprising and yet unexplained result of this computation is that VA-13 and HT-29 cells are relatively more sensitive to VM-26 than to VP-16 (Table 2). Similar experiments were performed in isolated nuclei to eliminate drug transport as a mechanism for differential cell sensitivities (Figure 4). The production of DPC in nuclei reflected the different sensitivities exhibited by the intact cells although less strikingly. It is, therefore, likely that the differences between cell lines are due to nuclear differences in levels of topoisomerase II associated with chromatin, the level being higher in L1210 cells than in VA-13 or HT-29. The nuclei experiments showed also that both drugs and more specifically VP-16 were less potent in isolated nuclei than in whole cells. Having established that the three cell lines studied varied in their sensitivities to VP-16 and VM-26, it was interesting to determine whether these differences would be reflected in differential cytotoxicities. Figure 5 shows the cytotoxicities of VP-16 and VM-26 in the three cell lines. Comparison of the two panels shows that VM-26 was uniformly 10 fold more cytotoxic than VP-16. In addition, L1210 cells were more sensitive than VA-13 and HT-29 to either drug. How- ever, this difference in sensitivity was less pronounced for VM-26 than for VP-16. These results are in agreement with the protein-linked DNA strand break results (Figures 2 and 3 and Table 2) and suggested that a correlation might exist TABLE 1.—Alkaline Elution Methodology“ v-Irradiation of cells or nuclei Lysis solution Elution solution Assay prior to elution Filter (pH 10) 5 ml (pH 12.1) DNA single-strand breaks None PC SDS-ProkK Pry,NH,OH + 0.1% SDS (SSB) DNA frank breaks None PVC LS-10 PryNH,OH DNA-protein crosslinks 3000 rad PVC LS-10 Pr,NH,OH (DPC) 9 LS-10 lysis solution=2 M NaCl, 0.2% sarkosyl, 0.04 M NaEDTA, pH 10; PC=polycarbonate (Nuclepore filters); PryNH4OH= Tetrapropylammonium hydroxide-EDTA, pH 12.1; PVC=polyvinylchloride (Millipore filters) or polyvinylchloride-acrylic copolymer (Gelman filters); SDS =sodium dodecyl sulfate; SDS-ProK lysis solution=29% SDS, 0.1 M glycine, 0.025 M Na,EDTA, pH 10, 0.5 mg/ml proteinase K. 118 NCI MONOGRAPHS, NUMBER 4, 1987 L1210 VA13 (rad-equivalents) nN T 1 XQ ~N. J 1 Y T Frequency of DNA Single Strand Breaks (#—e) and Frank Breaks (=—a) T | T B a HT29 \ Frequency of DNA-Protein Crosslinks (0) (rad-equivalents) 0 50 0 50 100 150 0 50 100 150 VP-16 Concentration (1M) FIGURE 2.—Frequency of DNA SSBs and DPCs as a function of VP-16 concentration in three cell lines. T° : L1210 VA13 HT29 g 8a 2 oo . + + . > a= : 29 ¥ S « £ 2000 5” l= » 2000 g ® 0 gs > / 2 Bo s w 2 L ~ “ J 4 / 3 g a 2 g or 7 < @ =e E54 7 # gt 5% EL 1,000 + ot 7 ot 7 41,000 2 * & ¥ o o, 22 Se / 7 8 oO 0 L / s 7 Pe 0 x / 1 7 E 2 4 € |S 3 / 2 o a — / - ” m - _ 2 _ _ 22 pe 0 < 0 1 2 3 40 1 2 3 40 1 2 3 4 VM-26 Concentration (nM) FIGURE 3.—Frequency of DNA SSBs and DPCs as a function of VM-26 concentration in three cell lines. between the DNA-protein crosslink frequency and cyto- toxicity. Figure 6 clearly shows that such a correlation exists for the cell lines and epipodophyllotoxins studied. When DNA-protein crosslink frequencies (expressed in rad- equivalents) were plotted against the log of the survival fractions, a straight line was generated. This occurred for both drugs. These regression lines are defined in the legend of Figure 6. It would thus be interesting to examine whether VP-16- and VM-26-induced cytotoxicity as a func- tion of DNA-protein crosslink frequency for cell types not yet examined would give the same relationship. TABLE 2.—Comparison Between the Potencies of VP-16 and VM-26 to Induce Protein-Linked Strand Breaks in L1210, VA-13, and HT-29 cells? L1210 Cells VA-13 Cells HT-29 Cells VP-16 15° 65 64 VM-26 1.7 27 2.4 4 The data are extrapolated from Figures 2 and 3. 5 Drug concentration (uM) producing 1000 rad-equivalent SSB or DPC. DNA TOPOISOMERASES IN CANCER THERAPY DISCUSSION VP-16 and VM-26 have been shown to inhibit mamma- lian DNA topoisomerase II by inducing the enzyme to bind covalently and to cleave DNA (11). In the presence of puri- fied enzyme, VM-26 was approximately 10-fold more potent than VP-16 (§). The DNA effects of both drugs in cultured mammalian cells have also been shown to be com- patible with topoisomerase II inhibition (/3). However, oL1210 1 AVA13 = HT29 3 2,000 (rad-equivalents) 50 100 150 0 1 2 3 4 5 © VP-16 Concentration (uM) VM-26 Concentration (uM) Frequency of DNA-Protein Crosslinks FIGURE 4.—Frequency of DPCs in the nuclei derived from three cell lines as a function of VP-16 or VM-26 concentration. 119 previous studies had raised the possibility that VP-16 could induce some free-radical-mediated DNA strand breaks in cultured L1210 cells (3). The present data exclude this pos- sibility, at least under the conditions used, since no frank breaks could be detected in three different cell lines, includ- ing L1210. Our results are in agreement with a topoisom- erase II-mediated DNA damage because the DNA breaks were quantitatively equal to the DNA-protein crosslinks and were all protein-associated (Figure 1). Earlier work performed in our laboratory sought whether a correlation existed between cytotoxicity and DNA strand breaks mediated by three different classes of intercalator in L1210 cells. No clear relationship emerged (14,15). There followed a study of Pommier et al. (/6) which demon- strated no correspondence of cytotoxicity with single- strand breaks for either 5-ID or m-AMSA in V79 Chinese hamster cells, but a strong one for double-strand breaks. Wozniak et al. (3) have reported that cytotoxicity in L1210 cells increased with VP-16 dose and Long et al. (4) showed that in A549 human adenocarcinoma the cytotoxic potency of epipodophyllotoxin congeners correlated with their DNA breaking activity. It is important to mention, however, that at higher con- centrations, for both drugs, no further increase in break frequency was detectable using the SSB method (see Table 1), while it was detectable using the DPC assay. Since at all doses examined, no protein-free breaks were present in any cell or nucleus, it became clear that the DPC assay reflected the true total break frequency occurring in the cell by virtue of the protein-adsorbing filter used. Because the filter binds proteins, all protein-associated double strand breaks, which are produced at high VP-16 or VM-26 concentrations, were stabilized and did not elute into the lysis fraction as was the case for the SSB assay (17). Possibly this explains why our earlier work saw variable correlation between cytotoxicity and break frequency when total breaks were measured with the SSB assay. A loss of DNA by elution into the lysis frac- tion may have occurred in the SSB assay (17). While it is of interest to note, from our work, that differ- ent cell lines vary considerably in their sensitivity to VP-16 and VM-26, much more striking is the fact that a given break frequency once achieved will consistently correlate with a quantum of cell kill for a given class of topoisom- erase II inhibitors. Although this study cannot rule out other mechanisms of demethylepipodophyllotoxin-mediated cytotoxicity, there is little doubt that VP-16 and VM-26 alter topoisom- 0 = HT29 S AVA13 S al eo L1210 gs HT29 i bi 7 [re HT29 i = VA13 : -2r VA13 + “ L1210 L1210 8 £2 sl 1 i 0 50 100 150 1 2 3 4 5 VP-16 Concentration (nM) VM-26 Concentration (nM) FIGURE 5.—Log survival fractions for three cell lines as a function of VP-16 or VM-26 concentration. 120 VP-16 c S g S uw ® 2 eg 2 1 3 ® = HT29 2 AVA13 = =3 elLi210 = 1,000 3,000 1,000 3,000 DNA-Protein Crosslink Frequency (rad-equivalents) FIGURE 6.—Log survival fractions as a function of DPC frequencies. For VP-16: log SF=1.007(DPC)— 0.137 (correlation = 0.96). For VM-26: log SF=1.079(DPC)— 0.319 (correlation= 0.93). erase II via a DNA-enzyme covalent stable intermediate. The utility of the DPC assay for studying the potency of topoisomerase II inhibitors is obvious. Large numbers of drugs and their congeners can be tested rapidly and corre- lated with their cytotoxic potencies. REFERENCES (1) LoikE JD, HorwITZ SB: Effect of podophyllotoxin and VP- 16-213 on microtubule assembly in vitro and nucleoside transport in HeLa cells. Biochemistry 15:5435-5443, 1976. (2) KALWINSKY DK, Look AT, DUCORE J, et al: Effects of the epipodophyllotoxin VP-16-213 on cell cycle traverse, DNA-synthesis, and DNA strand size in cultures of human leukemic lymphoblasts. Cancer Res 43:1592-1597, 1983. (3) Wozniak AJ, Rose WE: DNA damage as a basis for 4- demethylepipodophyllotoxin-9-(4,6-O-ethylidene-B-D- glucopyranoside) (etoposide) cytotoxicity. Cancer Res 43:120-124, 1983. (4) LoNG BH, MUSIAL ST, BRATTAIN MG: Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP16-213 and VM-26: A quantitative structure-activity relationship. Biochemistry 23:1183-1188, 1984. (5) CHEN GL, YANG L, Rowe TC, et al: Non-intercalative antitumor drugs interfere with the breakage-reunion reac- tion of mammalian topoisomerase II. J Biol Chem 259:13560-13566, 1985. (6) Wozniak AJ, GLISSON BS, HANDE KR, et al: Inhibition of etoposide-induced DNA damage and cytotoxicity in L1210 cells by dehydrogenase inhibitors and other agents. Cancer Res 44:626-632, 1984. (7) SzmiGIERO L, KOHN KW: Mechanisms of DNA strand breakage and interstrand cross-linking by diaziridinylben- zoquinone (Diaziquone) in isolated nuclei from human cells. Cancer Res 44:4453-4457, 1984. (8) SzMIGIERO L, ERICKSON LC, EWIG RA, et al: DNA strand scission and cross-linking by diaziridinylbenzoquinone (Diaziquone) in human cells and relation to cell killing. Cancer Res 44:4447-4452, 1984. (9) POMMIER Y, MATTERN MR, SCHWARTZ RE, et al: Changes in deoxyribonucleic acid linking number due to treatment of mammalian cells with the intercalating agent 4'-(9- acridinylaminomethanesulfon)-m-anisidide. Biochemistry 23:2927-2932, 1984. (10) FiLipskl J, KoHN KW: Ellipticine induced protein asso- ciated DNA breaks in isolated L1210 nuclei. Biochim Bio- phys Acta 698:280-286, 1982. (11) KoHN KW, EWIG RAG, ERICKSON LC, et al: Measurements NCI MONOGRAPHS, NUMBER 4, 1987 of strand breaks and cross-links by alkaline elution. In DNA Repair. A Laboratory Manual of Research Proce- dures (Friedberg EC, Hanawalt PC, eds). New York: Marcel Dekker, 1981, pp 379-401. (12) POMMIER Y, ZWELLING LA, MATTERN MR, et al: Effects of dimethyl sulfoxide and thiourea upon intercalator-induced DNA single-strand breaks in mouse leukemia (L1210) cells. Cancer Res 43:5718-5724, 1983. (13) Ross W, ROWE T, GLISSON B, et al: Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleav- age. Cancer Res 44:5857-5860, 1984. (14) ZWELLING LA, KERRIGAN D, MICHAELS S: Cytotoxicity and DNA strand breaks by 5-iminodaunorubicin in mouse leukemia 11210 cells: Comparison with Adriamycin and 4’-(9-acridinylamino)methanesulfon-m-anisidide. Cancer DNA TOPOISOMERASES IN CANCER THERAPY Res 42:2687-2691, 1982. (15) ZWELLING LA, MICHAELS S, KERRIGAN D, et al: Protein- associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl- 9-hydroxyellipticinium. Biochem Pharmacol 31:3261-3267, 1982. (16) POMMIER Y, ZWELLING LA, CHEN-SONG K-S, et al: Correla- lations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxic- ity in Chinese hamster cells. Cancer Res 45:3143-3149, 1985. (17) LoNG BH, MUSIAL ST, BRATTAIN MG: Single- and double- strand DNA breakage and repair in human lung adeno- carcinoma cells exposed to etoposide and teniposide. Cancer Res 45:3106-3112, 1985. 121 Structure-Activity Relationships of Podophyllin Congeners That Inhibit Topoisomerase II Byron H. Long "* ABSTRACT — Various analogs of etoposide have been studied and compared in different tests in order to identify which tests best correlate with antitumor activity. These tests included DNA breakage assays using standard alkaline elution procedures as a means of studying topoisomerase II inhibition in intact cells, cyto- toxicity studies in naturally sensitive and resistant human carci- noma cell lines, in vitro assays of the effect of the different con- geners on topoisomerase II activity, and a preliminary evaluation of the ability of etoposide and teniposide to induce resistance. As in previous studies, a direct correlation was seen between double strand DNA breakage and cytotoxicity but not between single strand DNA breakage and cytotoxicity. Analogs with blocked 4’-hydroxyl groups were poor antitumor agents but were still cap- able of inhibiting topoisomerase II as evidenced by the production of DNA breaks. However, this DNA breakage was qualitatively different from that produced by VP16. None of the analogs were able to overcome either naive or acquired drug resistance. The dihydroxy analog of VP16, a possible bioactivated analog, was much less potent and possibly less stable than VP16. A model is proposed for the inhibition of topoisomerase II by demethylepi- podophyllotoxins that may explain the relationship between dou- ble strand DNA breakage and cytotoxicity.—NCI Monogr 4:123-127, 1987. INTRODUCTION Etoposide (VP16) is an exciting new antitumor agent with a broad spectrum of activity (1,2), especially against testicular (3) and small cell lung carcinomas (SCLC) (4). Teniposide (VM26) has a similar spectrum of action (1) but is about 10 fold more potent in vitro (5,6) and also has promising activity against SCLC (7). These drugs produce a drug dose dependent induction of protein associated DNA breaks in human cells (5,6) that are rapidly reversible upon removal of drug from cells (6). An accurate struc- ture-activity relationship (SAR) was obtained for DNA breakage by using available congeners from Dr. H. Sti- helin at Sandoz Pharmaceuticals (5). The protein associa- tion with the DNA breaks, their rapid reversibility, and the strict SAR lead us to consider topoisomerase II as a possi- ble target for these drugs. My laboratory and others have been able to show that these breaks are a result of the inhibition of topoisomerase II by these drugs, resulting in the stabilization of the transient complex formed between the enzyme molecules and their substrate, DNA (8-11). I Pharmaceutical Research and Development Division, Bristol-Myers Co., Wallingford, CT. * Reprint requests: Byron H. Long, Ph.D., Pharmaceutical Research and Development Division, Bristol-Myers Co., P.O. Box 5100, Walling- ford, CT 06492-7660. Studies have demonstrated a direct correlation between cytotoxicity, DNA breakage, and topoisomerase II inhibi- tion activities among the podophyllotoxin analogs lacking antimicrotubule activities (9,10). Furthermore, a direct correlation was shown to exist between double strand DNA breakage and cytotoxicity but not between single strand DNA breakage and cytotoxicity produced by VP16 and VM26 in 4 different human lung carcinoma cell lines (12). More recently, a 3’,4’-didemethylepipodophyllotoxin-(4,6- O-ethylidine-B-D-glucopyranoside) (diOHVP) and the cor- responding oxidized orthoquinone form (diQVP) have been described (1/3), and novel podophyllotoxin and alpha- peltatin analogs have been synthesized and evaluated for topoisomerase II inhibition activity (14). The early SAR investigations have been well reviewed and no better compounds have been discovered to date (15-17). Nevertheless, it may be possible to synthesize more active analogs than etoposide and teniposide. Therefore, it will be important that the methods are in place to identify and characterize promising lead compounds. In this report, various analogs of etoposide were studied in different assays in order to evaluate these tests as tools intended to predict the antitumor activity of an analog and to better understand the mechanism by which topoisomerase II inhibitors produce cytotoxicity. In addition, these studies will allow us to better understand the relation between topoisomerase II inhibition and the antitumor activity dis- played by this novel chemotype. METHODS Cell culture, radioactive labeling, and alkaline elution.— Human colon tumor cell line HCT116, human lung adeno- carcinoma cell line A549, human small cell lung carcinoma lines SW900 and SW1271 were grown and maintained in plastic culture flasks in McCoy’s 5A tissue culture medium containing 10% heat-inactivated fetal bovine serum, peni- cillin, and streptomycin and supplemented with pyru- vate. Cell populations HCT116(VM)35, HCT116(VP)34, A549(VM)28, and A549(VP)28 were derived by challenge of parental cells and selection for resistance by weekly 1 hr exposures to the inducing drug. For alkaline elution stud- ies, drug-treated cells containing [“C]DNA and cells containing [PH]DNA that were exposed on ice to 300 rad of gamma radiation to introduce random single strand DNA breaks were layered over polycarbonate filters (Nucle- pore), washed twice with 10 ml of cold phosphate buffered saline, and lysed on the filters at room temperature by addi- tion of SDS-EDTA lysis solution followed by lysis solution containing 0.5 mg/ml proteinase K (E. Merck, Darmstadt, 123 W. Germany) as previously described (5,6). DNA elutions were conducted at pH 12.1 to assess single strand DNA breaks and at pH 9.6 to assess double strand DNA breaks. Cytotoxicity assay.—Cytotoxic effects of the drugs on the different cell lines were determined by assessing inhibi- tion of cell proliferation in 25 cm? flasks 5 to 9 days follow- ing a | hr drug exposure, as previously described (1/2). Cells surviving drug treatment were released from flasks with trypsin-EDTA, fixed with formaldehyde, and counted with a Coulter counter when the control flasks or dishes were generally 809% confluent. Counts for drug-treated cells were expressed as percentage of the average number of untreated control cells from four flasks or dishes for each experiment. At least 3 separate experiments, each based on 5 drug con- centrations run in duplicate, were conducted for each drug. Generally, standard deviation of the six or more values converted to percentage of control value for each drug con- centration did not exceed 10% and probit plots of the mean values vs. log concentration yield correlation coefficients for linearity of greater than 0.95, as determined by linear regression analysis. A variance of plus or minus 10% in cell numbers expressed as percentage of control cells usually translated into a standard deviation for the ICs, value of less than 209% of the mean value. RESULTS The chemical structures of the podophyllin analogs under investigation are presented in Fig. 1 and 2. In addi- tion, two other previously described congeners, epipodo- phyllotoxin-8-D-glucopyranoside (EPG) and 4’-demethyl- epipodophyllotoxin-B-D-glucopyranoside (DMEPG), were included in this study (5). It should be noted that five of these congeners contain groups that block the 4-hydroxyl group. Topoisomerase II inhibition activities of these congeners were evaluated by the indirect means of assessing DNA breakage in intact cells rather than the direct means of measuring the enzyme inhibition in vitro, because both the quantity and quality can be easily measured by alkaline elution techniques. This approach has yielded us valuable information about the drug action. When tested for the induction of single strand DNA breaks, those congeners with blocked 4’ positions were all moderately to substantially less potent than their corre- sponding free 4-hydroxyl congeners but all were still able to induce some DNA breakage, nevertheless. However, no production by MeVP, BeVP, and EPG of double strand DNA breaks was detected at concentrations as high as 300 uM and only relatively low levels were produced by diOHVP, diQVP, EPBG, and MeVM (B. Long, manu- script in preparation). Little correlation was observed when cytotoxicity was plotted against single strand DNA breaks (Fig. 3). In fact, some analogs could induce almost 20 single strand DNA breaks without producing appreciable toxic- ity. A much better correlation was observed between dou- ble strand DNA breaks and cytotoxicity (Fig. 3), a result similar to that previously observed for VP16 and VM26 in four sensitive and naively resistant cell lines (12). These results indicate that both subunits of topoisomerase II need to be inhibited in order for a cytotoxic event to occur. A model to explain this observation will be presented below. It is interesting that both the dihydroxyl and the diqui- none analogs of VP16 produce greater cytotoxicity per 124 H R 0 0 Q HO OHN-0_ H / 0 Wl 0 yf 1 Oo H4CO i OR” OR’ DRUG R R’ R” VP16-213 HC- “H -CH, VM26 {)- H OH, DMEPBG {)- -H -CH, MeVP HE- -CH, -CH, MeVM {)- -CH, CH EPBG ) -CH, -CH, BeVP HL- : HO -CH, diOHVP H,C- “H -H FIGURE 1.—Analogs of VP16. double strand DNA break than the other congeners (Fig. 3). It has been proposed that VP16 may require bioactiva- tion to these active intermediates, which are then capable of alkylating DNA and protein (13,18,19). If alkylation of DNA were a factor, then formation and repair of these lesions would be slower than the inhibition of topoisom- erase II and the reversal of inhibition following removal of drug from cells. Figure 4 shows to the contrary that the DNA breakage induced by diOHVP forms more rapidly upon exposure of cells to drug and disappears with equal or H "Co 0 0 HO OHN-0_ H TI 0 0 30 | Oo H4CO y 0 oO FIGURE 2.—Orthoquinone form of VP16 (diQVP). NCI MONOGRAPHS, NUMBER 4, 1987 1 1 1 FIGURE 3.—Comparison of cytotoxicity with single and double strand DNA breakage. 0 10 20 30 40 0 1000 2000 SINGLE STRAND DNA BREAKS (BREAKS/108NUCLEOTIDES) INHIBITION OF CELL PROLIFERATION ® VM O MeVM =m DMEPBG 0 EPBG Aa VP X BeVP v DMEPG + diOHVP * diQMP greater rates than the breakage produced by VP16. Alkyla- tion induced breakage should continue to increase with continued incubation, since the drug concentration is not rate limiting. This was not observed for the DNA breakage produced by diOHVP, which actually decreased to 70% of its 1 hr maximum after 4 hr of continued incubation (Fig. 4). These observations are compatible with greater meta- bolic inactivation of diOHVP than VP16. A comparison of the abilities of the different analogs to overcome naive resistance to VP16 is presented in Table 1. Of the two SCLC lines, SW900 is quite resistant and SW1271, like the human lung adenocarcinoma line A549, is sensitive to VP16 (12). Neither VM26 nor DMEPBG show any appreciable advantage over VP16 in these three cell lines when the ratios are compared and thus, when the po- tency differences are taken into consideration. It has been shown quite early that increased potency of an analog has no advantage in antitumor activity, in that ortho-chloro- benzylidine analog, the most potent analog reported by Keller-Juslén et al. (20), had very little antitumor activity. Interestingly, MeVM and EPBG, the more potent analogs blocked in the 4’ position, and diOHVP and diQVP are significantly more cytotoxic than their 4-demethyl analogs in A549 cells relative to SW1271 cells, yet no difference was T T T T 44M VP16 20 50M diOHVP Single Strand DNA Breaks/108 Nucleotides 1 0 1 2 3 4 Time (HR) © FIGURE 4.—Formation and disappearance of single strand DNA breaks produced by VP16 and diOHVP in A549 cells. Cells were incubated continuously for various lengths of time or for 1 hr with drug then incubated in the absence of drug for various lengths of time. Single strand DNA breaks were assessed as described in Methods. DNA TOPOISOMERASES IN CANCER THERAPY 3000 4000 DOUBLE STRAND DNA BREAKS (RAD EQUIVALENTS) seen in SW900 cells relative to SW1271 cells. This hyper- sensitivity of A549 cells to these analogs presently escapes explanation. The results in Table 1 show that none of the tested analogs are able to overcome the naive resistance of SWI00 cells to VP16 and VM26. When the analogs were evaluated against the acquired resistance developed in human lung and colon carcinoma cell lines and a mouse melanoma cell line, no changes in the ratios of resistant relative to parental cells were observed in the A549 cell lines, but DMEPBG produced a noticeable reduction in the ratio observed for the colon cell lines (Table 2). The present work with a selected group of ana- logs has demonstrated that the analog DMEPBG may have a slight advantage over VP16 in overcoming acquired resistance. DISCUSSION In this work, demethylepipodophyllotoxin, epipodophyl- lotoxin, and dihydro and diquinone analogs of VP16 and VM26 have been studied in order to extend an earlier struc- ture-activity relationship study (5) and evaluate the ability of different biological and biochemical tests to predict anti- tumor activity. Furthermore, information gained from these analogs has been used to better understand how inhi- bition of topoisomerase II results in cytotoxicity. Previous studies have shown a direct correlation between cytotoxic- TABLE 1.—Ability of Analogs to Overcome Naive Drug Resistance Median cytotoxic concentrations A549 SW900 Drug SWI1271 A549 SW900 SWI271 SWI271 VPI16 72 uM 8.4 uM 50 uM 1.2 6.9 VM26 0.5 0.5 -22 1.0 4.4 DMEPBG 0.6 0.4 2.6 0.7 4.3 DMEPG 250 MeVP >100 MeVM 8.4 3.0 32 0.36 3.8 EPBG 5.8 8.1 24 0.26 4.1 EPG >100 diOHVP 200 36 >300 0.18 — diQVP 270 72 >300 0.27 — 125 TABLE 2.—Ability of Analogs to Overcome Acquired Drug Resistance Median cytotoxic concentrations A549(VM) AS549(VP) Drug A549 A549(VM)28 AS549(VP)28 A549 A549 Lung VP16 84 uM 75 uM 70 uM 8.9 8.3 VM26 0.5 42 4.3 8.4 8.6 DMEPBG 0.4 31 3.6 7.8 9.0 Median cytotoxic concentrations HCT116(YM HCT116(VP) Drug HCT116 HCT116(VM)34 HCT116(VP)35 HCTI116 HCT116 Colon VP16 10 uM 110 uM 86 uM 11.0 8.6 VM26 0.8 7.4 5.6 9.2 7.0 DMEPBG 0.7 5.4 5.0 7.7 7.41 Median cytotoxic concentrations BI6(VM) BI6(VP Drug B16 B16(VM)22 B16(VP)26 B16 B16 Mouse melanoma VPI16 38 uM 21 uM 9.0 uM 55 24 VM26 0.3 2.0 0.7 6.6 23 DMEPBG 0.2 1.7 0.2 8.5 1.0 ity, DNA breakage, and topoisomerase II inhibition (5,8,9). The tests under evaluation here included a compari- son of cytotoxicity vs. either single or double strand DNA breakage (Fig. 3), an evaluation of the ability of the analogs to overcome either naive or acquired resistance (Tables 1 and 2), and an investigation of the role drug metabolism may play in the activity or inactivity of an analog. These results indicate that the free 4’-hydroxyl group is not essential for topoisomerase II inhibition activity, as was previously believed (5), but is important for potency and antitumor activity. However, increased potency toward topoisomerase inhibition does not appear to correlate with increased antitumor activity (20). It has been suggested that VP16 may produce cytotoxicity through bioactivation (13,18,19). However, when the proposed metabolic inter- mediates were tested for cytotoxicity, they were much less potent than VP16, raising the possibility that increased sus- ceptibility of an analog to metabolic alteration may actu- ally lead to inactivation, such as is suggested from Figure 4. Finally, none of the analogs studied were able to overcome either naive or acquired resistance to VP16. No one yet knows how the active podophyllin analogs interact with DNA and/or topoisomerase II to produce cytotoxicity. However, information can be gained not only from the analog studies presented here but also from com- parison with what is known of the prokaryote topoisom- erase II, gyrase. Because of the structural similarities between the podophyllins and the quinolone family of anti- bacterial agents, specifically oxolinic and nalidixic acids, it may be likely that VP16 forms hydrogen bonds with the single strand DNA regions produced at the cleavage site by the enzyme, similar to that reported by Shen and Pernet for the inhibition of gyrase by the quinolone antibiotics (21). This interaction (Fig. 5) may temporarily prevent religation of the DNA. The observation that cytotoxicity correlates more directly with double strand DNA breakage than single strand DNA breakage produced by the various analogs of VP16 and VM26 (Fig. 3) confirms similar obser- vations for VP16 and VM26 in sensitive and resistant cells (12) and intercalators in Chinese hamster cells (22). These data support the hypothesis in that both subunits of topo- 126 isomerase II must be inhibited for a cytotoxic event to occur (12,22) and suggest that two molecules of drug may be required to inhibit both enzyme subunits. It may be that analogs with blocked 4’-hydroxyl groups can inhibit one subunit but are less likely to inhibit the second subunit. The unknown cytotoxic event mentioned above may actually be the deletion, insertion, and/or recombination of DNA sequences by heterologous exchange of topoisomerase II subunits, similar to that described by Ikeda ef al. to explain the observed genetic recombination events that occurred in vitro when gyrase was inhibited by oxolinic acid (23). An illustration of how genetic recombination could be facili- tated by heterologous subunit exchange is presented in Figure 6. This heterologous exchange of subunits could only occur when both of the enzyme subunits are inhibited 5 3 FIGURE 5.—Model depicting the possible interaction of active podophyllin drugs with single strand DNA regions at the cleavage site of topoisom- erase IL. It is quite possible that the hypothesized interaction of the drug with single stranded DNA occurs via hydrogen bonds between appro- priate groups on the drug molecule and bases of the DNA molecules. The actual number of drug molecules interacting at the cleavage site is unknown. Also shown is the hypothesized orientation of a DNA inter- calating drug to DNA molecule. NCI MONOGRAPHS, NUMBER 4, 1987 GENOMIC DNA lp SV40 DNA WITHIN CELLS O-O- FIGURE 6.—Heterologous subunit exchange model proposed to describe the actual cytotoxic event produced by the inhibition of topoisomerase II by active podophyllin drugs through the heterologous interaction and subsequent recombination of subunits from two proximally located topoisomerase II enzymes. All four involved subunits must be tran- siently inhibited by drug for an exchange to occur. Such an exchange of subunits would result in deletion and recombination events. 2 by drug, thus explaining the observed cytotoxicity and DNA breakage data (Fig. 3). Such a model is supported by the observation of Singh and Gupta that both VP16 and VM26 are mutagenic (24). However, more specific experi- ments will need to be conducted to confirm or refute this hypothesis. REFERENCES (1) IsseLL BF: The podophyllotoxin derivatives VP16-213 and VM26. Cancer Chemother Pharmcol 7:73-80, 1982. (2) IsseLL BF, RUDOLPH AR, LOUIE AC: Etoposide (VP-16- 213): An overview. In Etoposide (VP-16): Current Status and New Developments (Issell BF, Muggia F, Carter SK, eds). New York: Academic Press, 1984, pp 1-14. (3) BUNN PA: The role of chemotherapy in small-cell lung cancer. In Etoposide (VP-16): Current Status and New Developments (Issell BF, Muggia F, Carter SK, eds). New York: Academic Press, 1984, pp 141-161. (4) WILLIAMS SD, BIRCH R, LOEHRER PJ, et al: Testicular cancer: Role in chemotherapy. In Etoposide (VP-16): Cur- rent Status and New Developments (Issell BF, Muggia F, Carter SK, eds). New York: Academic Press, 1984, pp 225-232. (5) LONG BG, MUSIAL ST, BRATTAIN MG: Comparison of cyto- toxicity and DNA breakage activity of congeners of podo- phyllotoxin including VP16-213 and VM26—A quantita- tive structure-activity relationship. Biochemistry 23:1183- 1188, 1984. (6) LoNG BH, MusiAL ST, BRATTAIN MG: Single- and double- strand DNA breakage and repair in human lung adeno- carcinoma cells exposed to etoposide and teniposide. Cancer Res 45:3106-3112, 1985. (7) BORK E, HANSEN M, DOMBERNOWSKY P, et al: Teniposide (VM-26), an overlooked highly active agent in small-cell lung cancer. Results of a phase II trial in untreated patients. J Clin Oncol 4:524-527, 1986. (8) LoNG BH, MINOCHA A: Inhibition of topoisomerase II by VP16-213 (etoposide), VM26 (teniposide), and structural congeners as an explanation for in vivo DNA breakage DNA TOPOISOMERASES IN CANCER THERAPY and cytotoxicity. Proc Am Assoc Cancer Res 24:321, 1983. (9) MINOCHA A, LONG BH: Inhibition of the DNA catenation activity of type II topoisomerase by VP16-213 and VM26. Biochem Biophys Res Commun 122:165-170, 1984. (10) LoNG BH, BRATTAIN MG: The activity of etoposide (VP16- 213) and teniposide (VM26) against human lung tumor cells in vitro: Cytotoxicity and DNA breakage. In Etopo- side (VP16-213): Current Status and New Developments (Issell BF, Muggia F, Carter SK, eds). New York: Aca- demic Press, 1984, pp 63-86. (11) CHEN GL, YANG L, ROWE TC, et al: Nonintercalative anti- tumor drugs interfere with the breakage-reunion of mam- malian DNA topoisomerase II. J Biol Chem 259:13560- 13566, 1984. (12) LoNG BH, MusIAL ST, BRATTAIN MG: DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide. Cancer Res 46:3809-3816, 1986. (13) SINHA BK, MYERS CE: Irreversible binding of etoposide (VP-16-213) to deoxyribonucleic acid and proteins. Bio- chem Pharmacol 33:3725-3728, 1984. (14) THRUSTON LS, IRIE H, TANI S, et al: Antitumor agents. 78. Inhibition of human DNA topoisomerase II by podophyl- lotoxin and a-peltatin analogues. J Med Chem 29:1547- 1550, 1986. (15) DOYLE TW: The chemistry of etoposide. In Etoposide (VP16-213): Current Status and New Developments (Issell BF, Muggia F, Carter SK, eds). New York: Academic Press, 1984, pp 15-32. (16) JARDINE I: Podophyllotoxins. /n Anticancer Agents Based on Natural Product Models (Cassady JM, Douros JD, eds). New York: Academic Press, 1980, pp 319-351. (17) STRIFE RJ: High performance liquid chromatographic assay and structure-activity relationships of the epipodophyllo- toxin anticancer drug VP16-213 (Etoposide) and VM26 (Teniposide). Ann Arbor, MI: University Microfilms, 1980, pp 1-46. (18) SINHA BK, TRUSH MA, KALYANARAMAN B: Microsomal interactions and inhibition of lipid peroxidation by etopo- side (VP-16, 213): Implications for mode of action. Bio- chem Pharmacol 34:2036-2040, 1985. (19) VAN MAANEN JMS, DE RUITER C, KOOTSTRA PR, et al: Inactivation of X 174 DNA by the ortho-quinone deriva- tive or its reduction product of the antitumor agent VP 16-213. Eur J Cancer Clin Oncol 21:1215-1218, 1985. (20) KELLER-JUSLEN C, KUHN M, VON WARTBURG A, et al: Syn- thesis and antimitotic activity of glycosidic lignan deriva- tives related to podophyllotoxin. J Med Chem 14:936-940, 1971. (21) SHEN LL, PERNET AG: Mechanism of inhibition of DNA gyrase by analogues of nalidixic acid: The target of the drugs is DNA. Proc Natl Acad Sci USA 82:307-311, 1985. (22) POMMIER Y, ZWELLING LA, KAO-SHAN C-§, et al: Correla- tions between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxic- ity in Chinese hamster cells. Cancer Res 45:3143-3149, 1985. (23) IKEDA H, AoKI K, NAITO A: Illegitimate recombination mediated in vitro by DNA gyrase of Escherichia coli: Structure of recombinant DNA molecules. Proc Natl Acad Sci USA 79:3724-3728, 1982. (24) SINGH B, GUPTA RS: Comparison of the mutagenic re- sponses of 12 anticancer drugs at the hypoxanthine- guanine phosphoribosyl transferase and adenosine kinase loci in Chinese hamster ovary cells. 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RE I Wop oot EAE Lane I Pn = ! un ou gil Airgas Bal Wr fn ba Bp sD ee Ce, BEE TERA pI wate of wo fs ries my Wy fr La Rid EX ou oS Ap: RAR a fu aX a “i Arh J » ih ll A igen ai - oo ng fp pee 0 Pa I if i I ah 4 Lo a oi el , i 1 uf } rid Al ‘e » . ] Hl a a . . 5 ae nity Tafa d a gh Ti 3 " 5 or ik a di sv » : JS - a = y, ) es Eo fom URE | 2a, 3 a Bi rt) ay whet te sl | why ne a cr i: CRE te © EER a a oh x Ar bl ped x al Eee Hap is n r I Pons, ni Riis mi x ) o tei] og HEE “i 5, gE a A i id es he . 4] | I = J EE v LI - IL ul y E k a ei SEE a at Eee . : kn HY) SHIA 38 ia Joa a ind 8 ed a: Ai pal PNT cei fo : hp 5 ) Inernllte ln. - Satetegtu . my STN) Lt, mar, BI Spa 200% TR Treatment Strategies in Relation to Drug Action Franco M. Muggia?* and Gordon McVie? Focus on newly established common mechanisms of drug action and resistance have already had a major impact on drug development. In more subtle ways they are influencing the design and interpretation of clinical trials, although mechanisms have not been precisely defined. Eventually the type of drugs being introduced will un- doubtedly directly determine the targets for initial study. This chapter assesses the impact of the topoisomerase 11 activation mechanism specifically on our clinical studies and speculates on future implications for both drug devel- opment and trial design. The drugs with toxicities mediated by topoisomerase 11 include several classes of DNA intercalating agents and the semisynthetic derivatives of epipodophyllotoxin. The evi- dence for such mechanisms has been presented in several chapters; moreover, behavior of resistant lines and cross resistant patterns incriminate failure of this abortive cellu- lar repair mechanism in the eventual loss of cytotoxic activ- ity. Since these phenomena are implicated in the action of two major groups of clinically useful drugs represented by doxorubicin and etoposide, it is worth examining whether there are discernible clinical counterparts for these labora- tory observations. We shall examine strategies in specific diseases for such evidence. SMALL CELL LUNG CANCER Clinical Observations The activity of etoposide in this disease was discovered mostly in trials where patients had not been pretreated with doxorubicin-containing combinations (7). Significantly lower to absent response rates were observed in subsequent years when the use of doxorubicin-containing regimens became the rule. Vincristine, by contrast, was shown to be active in spite of much prior treatment (2). A combination of etoposide-doxorubicin and methotrexate (VAM) when alternated with procarbazine, vincristine, cyclophospha- mide, and CCNU (POCC) was superior to POCC alone, but ineffective when used on crossovers (3). More recently, use of etoposide together with doxorubicin (and cyclo- I' Supported in part by Public Health Service grant CA-14089 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. 2 Division of Medical Oncology, University of Southern California Comprehensive Cancer Center, Los Angeles. 3 The Netherlands Cancer Institute, Amsterdam. * Reprint requests: Franco M. Muggia, M.D., Division of Medical On- cology, University of Southern California Comprehensive Cancer Center, 1441 Eastlake Ave., Los Angeles, CA 90033. phosphamide) has resulted in improved survival for these patients (4,5), and also use of etoposide (with cisplatin) at crossover has resulted in survival advantages (6). Schedule dependence has been observed for etoposide in several of these trials (7,8) (Table 1). Teniposide has also been shown active and its activity related to duration of prior chemo- therapy (9). Interpretation Biological studies in man have not yet given indication of whether these tumors commonly develop the multidrug re- sistance to natural products (10). Notoriously resistant cell lines have been documented and have been characterized as variants commonly associated with c-myc amplification (11). On the other hand, topoisomerase II-mediated mech- anisms may be operative in the above clinical observa- tions. Both drugs (doxorubicin and VP-16) appear to have at least an additive effect, but if etoposide is withheld until failure of the initial doxorubicin-containing regimen then no response is likely. Schedule dependency of etoposide and therapeutic advantage of doxorubicin-etoposide com- binations are noteworthy. One might speculate about en- zyme induction as a mechanism for such findings. S-phase specificity for etoposide cytotoxicity has been shown and may be implicated in its activity against this particular his- tologic type of lung cancer. NON-HODGKIN'S LYMPHOMAS Clinical Observations Regimens against high grade lymphomas have included doxorubicin as part of initial therapy together with vincris- tine, cyclophosphamide, and steroids. More recently, regi- mens have claimed superior results by adding other drugs (bleomycin, methotrexate, etoposide, or procarbazine), by intensifying their administration, and by non-crossresistant alternating schedules (12). A striking series reported by Jacobs et al, however, suggested nearly equivalent results could be achieved with etoposide alone, or etoposide and doxorubicin (/3) but not etoposide and cyclophosphamide (Table 2). In second line regimens, etoposide is definitely less effective than in the untreated patient (1/4). Neverthe- less, daily X 5 schedules of etoposide every 2 weeks (15) were considerably more effective. Combinations of etoposide with ifosfamide, methylglioxalbisguanylhydrazone (Methyl GBG), and methotrexate (MIME) have been heralded as second-line regimens with curative potential in high grade lymphomas (16) (Table 3). In second or third line regimens, other intercalating agents such as mitoxantrone (/7) and to a lesser extent m-AMSA (18) show occasional efficacy. 129 TABLE 1.—Small Cell Lung Cancer: Etoposide Schedule Study Schedule PR/ Total Median survival? 500 mg/m? 24 h iv 2/20 167 days 100 mg/m? 24 h ivX§ 14/18 294 days @ All patients crossed over to CAV (8). TABLE 2.—First Line Lymphoma: VP-16 Alone or in Combination (/3) Regimen Dose E Other drugs % CR Total E 60 mg/m?X 5 — 55.77 EC Same Cytoxan 29, 41 EA Same Adriamycin 62, 75 Interpretation Initial strategies for lymphomas have not adequately explored whether addition of etoposide alone to doxo- rubicin-containing regimens might lead to superior results, as it appears in SCLC. Schedule dependence for etoposide is suggested also in salvage studies in these diseases. The activity of the MIME regimen is of interest since it is possi- ble that polyamine depletion potentiates the effects of topoisomerase Il-reactive drugs (19). Residual activity for new DNA intercalating drugs is of interest but scanty information exists on factors associated with response. In low grade lymphomas, activity of all these drugs appears to be considerably less, a fact that reinforces the notion that topoisomerase Il-reactive agents have enhanced activity against proliferating cells. HODGKIN'S DISEASE Clinical Observations Trials by Santoro, Bonadonna, and co-workers have established the doxorubicin and vinblastine containing reg- imen ABVD as very effective in MOPP failures and equally effective if not superior as initial therapy. The alternating regimens of MOPP/ABVD and “hybrid” variants have also manifested considerable activity in this disease and were superior to MOPP alone in one trial (20). McElwain and co-workers have tested an etoposide + vincristine-contain- ing regimen (OPEC) which also shows considerable activity against Hodgkin’s disease. The etoposide in this regimen is given as 200 mg/m? orally for 5 days (21). This regimen has been administered to failures from other regimens and also alternating with the chlorambucil-vinblastine-prednisone— procarbazine regimen (Ch1VPP). The overall disease con- trol reported is 87%. More recently they have utilized a regimen referred to as HOPE-Bleo as salvage therapy from Ch1VPP. This regimen includes the same dose schedule of etoposide as OPEC plus doxorubicin 40 mg on day 1, and vincristine 1.4 mg/m? on days 1 and 8. The Milan group has also utilized etoposide together with prednimustine and CCNU as third line salvage with an appreciable complete remission rate (22) (Table 4). Salvage regimens containing doxorubicin for MOPP failures utilized by others have been employed with varying success. Other DNA interca- lating drugs such as m-AMSA have also shown activity against this disease (18,23). Interpretation As in the lymphomas, etoposide shows considerable activity after failure of doxorubicin-vinca combinations. This implies residual sensitivity to topoisomerase-dependent mechanisms and is consistent with the higher cleavage effi- ciency of etoposide over doxorubicin in experimental sys- tems (38). As front line treatment etoposide regimens utiliz- ing daily X5 schedules do appear to fare at least as well as other regimens. LEUKEMIAS Clinical Observations The use of anthracyclines in high risk acute lymphocytic leukemias (ALL) and in nonlymphocytic leukemias (ANLL) is commonplace, but sequencing studies are rare. In ANLL, the regimen usually consists of anthracycline on days 1 to 3 and cytosine arabinoside (ara-C) on days 1 to 7 (24). The group at St. Jude’s has successfully utilized the epipodo- phyllotoxin teniposide (VM-26) combined with ara-C for salvage regimens in ALL. Combinations of VM-26, predni- sone and vincristine were also quite active (25). In relapsed ANLL, etoposide followed by azacytidine has also been tested with success when high doses of etoposide (200 mg/m? iv X 3) were used (26), but ineffective at lower doses (75 to 100 mg/m?X3) (26,27) (Table 5). Drug sensitivity testing of untreated ANLL patients at this institution indi- cates a very close relationship in response to etoposide and anthracyclines. m-AMSA and mitoxantrone have also shown activity in anthracycline-refractory leukemias (28, 29), and have been combined with high dose ara-C in active salvage regimens (30,30a). Interpretation Dose intensity of drugs used in leukemia is generally higher than in other tumors. It is difficult to state whether these salvage drugs work better in these situations than would reinduction with the initial inducing agent. Although ara-C pretreatment potentiates topoisomerase-mediated DNA cleavage by m-AMSA, other biochemical mecha- TABLE 3.—Salvage Lymphoma: VP-16(E) or VM-26(T) Author Reference Regimen Dose E/T Other drugs % CR Total Chiuten (40) T 70 mg/m? qw — 17, 22 Grossberg 41) TC 60 mg/m? qw CCNU 14, 28 Bender (15) E 60 mg/m?X5 — 5.42 Cabanillas (16) IME 100 mg/m? X 3 Ifos, MTX 38, 62 Cabanillas (16) AIE 70 mg/m? X 3 AMSA, Ifos 24, 39 Cabanillas (16) MIME 100 mg/m? X 3 MGBG, Ifos, MTX 24, 63 130 NCI MONOGRAPHS, NUMBER 4, 1987 TABLE 4.—Hodgkin’s Disease: VP-16(E) Regimens Author Reference Regimen Dose E po Other drugs % CR Total McElwain 1) OPEC 200 mg/m? d1-5 VCR, Pred, Chl 89, —1 McElwain 21 OPEC/ChlVPP ” plus VLB, Pro 76, 87 McElwain 21) HOPE-Bleo 200 mg/m? d1-4 Adria, VCR, Pred 48, 76° Bonadonna (22) CEP 100 mg/m? d1-5 CCNU, Pred 35, 60°¢ 4 All 9 patients previously treated with Ch1VPP. b All 29 patients previously treated with various regimens. ¢ All 40 patients previously treated with MOPP/ABVD. nisms may also be operative at least in the ara-C synergy with mitoxantrone, i.e., higher levels of ara-CTP (31), and scheduling has not been shown to be crucial. MISCELLANEOUS TUMORS Clinical Observations Breast cancer is particularly responsive to doxorubicin but not strikingly to etoposide, although little experience in previously untreated patients is available (32). On the other hand, germ cell tumors are considerably more responsive to etoposide than doxorubicin and certainly more than other DNA intercalating drugs (33). The reported synergy of cisplatin with etoposide in clinical studies is derived from observations on these malignancies as well as from SCLC. In sarcomas, only doxorubicin has reproducible activity (34). Interpretation Knowledge of factors involved in disease susceptibility has lagged behind other aspects of chemotherapeutic action. Why doxorubicin and not other topoisomerase- active drugs is effective against sarcomas requires explana- tion and could implicate mechanisms for cytotoxicity other than topoisomerase Il-mediated pathways. In addition, answers to these questions must be sought in future studies of molecular pharmacology. GENERAL IMPLICATIONS FOR FUTURE STRATEGIES Drug activity in a disease may provide insight into mech- anisms of cytotoxicity (35). In turn, this may lead to studies aimed at exploring synergy or optimizing drug regimens. For example, the schedule dependence of etoposide, and possible synergy between etoposide and doxorubicin, etop- oside and cisplatin, and between various topoisomerase- reactive drugs and ara-C may be mediated by these mecha- nisms (Table 6). In addition, the activity of the MIME TABLE 5.—Refractory Acute Leukemia: Non-anthracycline Regimens TABLE 6.—Examples of Possible Drug Synergy Mediated via Topoisomerase II Activation Etoposide + etoposide Etoposide + doxorubicin Etoposide + cisplatin Doxorubicin + cisplatin VM-26 + ara-C 27-64% CR (ALL)? (165 mg/m?X 4) (300X4) VP-16 + aza-C 1 CR/16 (ANLL)? (100X3) (150X3) vs 11 CR/24 with 2X dose AMSA + ara-C 30 CR/40 (ANLL)? 4 See (25). b See (30). DNA TOPOISOMERASES IN CANCER THERAPY regimen could point to synergy between methyl GBG and etoposide. These interactions should be explored further. Other possible synergistic interactions are suggested from the various laboratory studies exploring topoisom- erase-reactive mechanisms. For example, estrogens may activate these mechanisms in receptor positive cells. Gluco- corticoids and other steroids are similarly capable of open- ing up chromatin sites for possible enhanced binding of DNA-intercalating drugs in susceptible cells (36). Clinical studies may provide the leads for such drug synergy. Finally, how resistance to topoisomerase Il-reactive drugs develops is an area of great interest. Inconsistencies between uptake of doxorubicin and resistance have led to some questioning of the relevance of the multidrug resis- tance P-glycoprotein-mediated efflux as the mechanism for such resistance (37). The existence of other seemingly independent mechanisms such as enhanced detoxification of activated oxygen species (39) could explain variable cross resistant patterns to DNA intercalating drugs, vinca alkaloids, and epipodophyllotoxin derivatives. Of addi- tional interest are some clinical observations suggesting return of susceptibility after interruption of treatment in certain instances. Clearly a new dimension in clinical con- cepts of drug efficacy and failure has been opened, and in turn, this will lead to new therapeutic strategies. REFERENCES (I) RADICE PA, BUNN PAJ, IHDE DC: Therapeutic trials with VP-16-213 and VM-26: Active agents in small cell cancer, non-Hodgkin’s lymphomas, and other malignancies. Cancer Treat Rep 63:1231-1239, 1979. (2) DOMBERNOWSKY P, HANSEN HH, SORENSON PL, et al: Vincristine in the treatment of the small cell anaplastic carcinoma of the lung. Cancer Treat Rep 60:239-292, 1976. (3) DANIELS JR, CHAK LY, SIKIC BI, et al: Chemotherapy of small cell carcinoma of lung: A randomized comparison of alternating and sequential combination chemotherapy programs. J Clin Oncol 2:1192-1199, 1984. 131 (4) HANSEN M, OSTERLIND K, SORENSON §S, et al: Cyclic al- ternating chemotherapy in small cell bronchogenic carci- noma. Proc ASCO 2:C-784, 1983. (5) RupoLPH A, EINHORN L, WAMPLER G, et al: Cytoxan, Adriamycin, etoposide (CAE) versus cytoxan, Adria- mycin and vincristine (CAVI) in the treatment of small cell lung cancer. Proc Am Assoc Cancer Res 21:92, 1983. (6) GRECO FA, PEREZ C, EINHORN LH, et al: Combination chemotherapy with or without concurrent thoracic radio- therapy in limited-stage small cell lung cancer: A phase III study. Proc ASCO 5:176, 1986. (7) CAVALLI F, SONTAG RW, JUNGIF, et al: VP-16-213 mono- chemotherapy for remission induction of small-cell lung cancer. A randomized trial using three dosage schedules. Cancer Treat Rep 62:473-476, 1978. (8) SLEVIN ML, CLARK PI, OSBORNE RJ, et al: A randomized trial to evaluate the effect of schedule on the activity of etoposide in small cell lung cancer. Proc ASCO 5:175, 1986. (9) GIACCONE G, DONADIO M, BANARDI G, et al: Teniposide (VM26) in small cell lung cancer (SCLC): Influence of time from prior chemotherapy on response and survival. Unpublished data. (10) KORTNER N, RIORDAN JR, LING V: Cell surface p-glyco- protein associated with multi-drug resistance in mamma- lian cell lines. Science 222:1284-1288, 1983. (11) CARNEY DN, MITCHELL JB, KINSELLA TJ: In vitro radia- tion and chemotherapy sensitivity of established cell lines of human small cell lung cancer and its large cell morpho- logical variants. Cancer Res 43:2806-2811, 1983. (12) FISHER RI, DEVITA VT JR, HUBBARD SM, et al: Diffuse aggressive lymphomas: Increased survival after alternat- ing flexible sequences of Promace and MOPP chemo- therapy. Ann Intern Med 98:304-309, 1983. (13) Jacoss P, KING HS, DENT DM, et al: VP16-213 in the treatment of stage III and IV diffuse large cell lym- phoma. Cancer 56:1006-1013, 1985. (14) ScHMOLL H: Review of etoposide single agent activity. Cancer Treat Rev 9(suppl A):21-30, 1982. (15) BENDER RA, ANDERSON T, FISHER RI, et al: The activity of the epipodophyllotoxin VP16 in the treatment of combination chemotherapy resistant non-Hodgkin’s lymphoma. Am J Hematol 203-209, 1978. (16) CABANILLAS F, HAGEMEISTER FB, BODEY GP, et al: IMVPI16: An effective regimen for patients with malig- nant lymphomas who have relapsed after initial combi- nation chemotherapy. Blood 57:693-698, 1982. (17) CoLTMAN CA JR, COLTMAN TM, BALCERZAK SP: Mitox- antrone in refractory non-Hodgkin’s lymphoma. A Southwest Oncology Group Study. Semin Oncol 11: 50-53, 1984. (18) CABANILLAS F, LEGHA SS, BODEY GP, et al: Initial expe- rience with AMSA as single agent treatment against malignant lymphoproliferative disorders. Blood 57:614- 616, 1981. (19) ZWELLING LS, KERRIGAN D, MARTON LJ: Effect of difluo- romethyl ornithine, an inhibitor of polyamine biosynthe- sis, on the topoisomerase II-mediated DNA scission pro- duced by 4’-(9-acridinylamine)methanesulfon-m-anisidide in L1210 cells. Cancer Res 45:1122-1126, 1985. (20) SANTORO A, BONADONNA G, BONFANTE V, et al: Alternat- ing drug combinations in the treatment of advanced Hodgkin’s disease. N Engl J Med 306:770-775, 1982. (21) MCELWAIN TJ, SELBY P: Etoposide combination for the treatment of Hodgkin’s disease. In Etoposide (VP-16): Current Status and New Developments (Issell BP, Mug- gia FM, Carter SK, eds). New York: Academic Press, 1984, pp 293-299. 132 (22) BONADONNA G: Chemotherapy strategies to improve the control of Hodgkin’s disease. The Richard and Hinda Rosenthal lecture. Cancer Res 42:4309-4320, 1982. (23) LoUIE AC, CARELL F, ROZENCWEIG M: New agents for Hodgkin’s and non-Hodgkin’s lymphoma. /n Malignant Lymphomas and Hodgkin’s Disease. Experimental and Therapeutic Advances (Cavalli F, Bonadonna G, Rozen- cweig M, eds). Boston: Martinus Nijhoff, 1985, pp 493-520. (24) OMURA GA: Sequencing of cytosine arabinoside and dau- norubicin in acute myelogenous leukemia. Cancer Treat Rep 60:629-635, 1986. (25) RIVERA G, DAHL GV, MURPHY SB, et al: VM26 therapy in children with drug refractory lymphocytic leukemia. In New Approaches in Cancer Therapy (Cortes Funes H, Rozencweig M, eds). New York: Raven Press, 1982, pp 59-64. (26) DAHL GV, Look AL, RIVERA G: VP 16-213 plus 5-azacytidine for remission induction of refractory acute nonlymphocytic leukemia. In ibid, pp 65-72. (27) VAN EcHO DA, LICHTENFELD KM, WIERNIK PH: Vinblas- tine, 5-azacytidine and VP 16-213 therapy for previously treated patients with acute nonlymphocytic leukemia. Cancer Treat Rep 61:1499-1602, 1977. (28) ARLIN ZA, SKLAROFF RB, GEE TS: Phase I and II trial of 4’(9-acridinylamino)methanesulfon-m-anisidide in pa- tients with acute leukemia. Cancer Res 40:3304-3306, 1980. (29) Pacciucct PA, OHNUMA T, CULTNER J, et al: Mitoxan- trone in patients with acute leukemia in relapse. Cancer Res 43:3919-3922, 1983. (30) HINES JD, MAzzA JJ, OKEN MM, et al: High dose cytosine arabinoside (ara-C) and m-AMSA in relapsed acute non-lymphocytic leukemia (ANLL). Proc ASCO 2:173, 1983. (30a) ARLIN Z: Current status of amsacrine (AMSA) combina- tion chemotherapy programs in acute leukemia. Cancer Treat Rep 67:967-970, 1983. (31) PLUNKETT W, HEINEMANN V, MURRAY D, et al: Mitoxan- trone-induced DNA damage in leukemia cells is enhanced by treatment with high dose ara-C. In Proceedings of the Fifth NCI-EORTC Symposium on New Drugs in Can- cer Therapy, Amsterdam, Oct 1986, Abstr No. 930. (32) VAN EcHO DE, TCHEKMEDYAN NJ, AISNER J: Salvage chemotherapy in breast cancer: The possible role of etoposide in combination. /n Etoposide (VP-16): Current Status and New Developments (Issell BF, Muggia FM, Carter SK, eds). New York: Academic Press, 1984, pp 261-272. (33) HAINSWORTH JD, GRECO FA, WILLIAMS SD, et al: VP-16 in the treatment of refractory germinal neoplasms. In ibid, pp 243-252. (34) BRAMWELL VHC, MOURIDSEN HT, MULDER JH, et al: Carminomycin vs Adriamycin in advanced soft tissue sarcomas: An EORTC randomized phase II study. Eur J Cancer Clin Oncol 19:1097-2003, 1983. (35) RIBSON CN, HARRIS AL, HICKSON ID: Isolation and char- acterization of Chinese hamster ovary cell lines sensitive to mitomycin-C and bleomycin. Cancer Res 45:5303- 5309, 1985. (36) SHEPHERD R, HARRAP KR: Modulation of the toxicity and antitumor activity of alkylating drugs by steroids. Br J Cancer 45:413-420, 1982. (37) SIEGFRIED JM, TRITTON TR, SARTORELLI AC: Compari- son of anthracycline concentration in S180 cell lines of varying sensitivity. Eur J Cancer Clin Oncol 49:1133- 1141, 1983. NCI MONOGRAPHS, NUMBER 4, 1987 (38) TEWEY KM, ROWE TC, YANG L: Adriamycin-induced DNA damage mediated by mammalian topoisomerase II. Science 226:466-468, 1984. (39) MYERS CE, CowAN KH, SINHA BK, et al: Circumvention of multidrug resistances. In Proceedings of the Fifth NCI-EORTC Symposium on New Drugs in Cancer Ther- apy, Amsterdam, Oct 1986, Abstr No. 303. DNA TOPOISOMERASES IN CANCER THERAPY (40) CHIUTEN DF, BENNETT JM, CREECH RH, et al: VM-26, a new anti-cancer drug with effectiveness in malignant lymphoma: An Eastern Cooperative Oncology Group Study (EST 1474). Cancer Treat Rep 63:7-11, 1979. (41) GROSSBERG H, OPFELL R, GLICK J, et al: Treatment of advanced refractory lymphoma with teniposide and lomustine. Cancer Treat Rep 71:215-216, 1987. 133 Priority Announcement Service Available for Future Issues If you would like to be notified when additional NCI Monographs are issued, please complete the form below and mail to the address on the form. (You will also be notified when other cancer-related publications are issued.) 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CardNo. [I I ITT TTT TIT TI TITITIT1] PLEASE PRINT OR TYPE Charge orders may be telephoned to the GPO = Expiration Date ly RTT SURE Company or Personal Name Cl For Office Use Only rn a HERE AR Quantity Charges SL TROL ONL OO LJ OO J OOO OL Publications Street address Sper So Charges — Ltr r rrr rrr LL] LLL | | A — Lettre r rrr ues (or Country) grit Balance Due 000 TO JE VO J JN OO OO OO COOL OO JO QT Geo —— Refund JNCI Monographs NCI MONOGRAPHS, NUMBER 4, 1987 Bru |. il off hi ki Eo - gh ingle 2K Toy sat gh oi EFS td Tage rh iy ye B i EL ToT a le es i | ed joy 5, yl pa wh = = ol Ti ae we = i Ea i mmr - = | n Te FEC = tis Cl ae, te Ri re The International Cancer Information Center of the National Cancer Institute announces publication of the first issue of the new NCI Monographs: Proceedings of the NIH Consensus Development Conference on Adjuvant Chemotherapy and Endocrine Therapy for Breast Cancer. During the past decade, systemic therapy has been added to local-regional treatment of breast cancer to prevent recurrence and improve survival of patients. This volume of NCI Monographs presents data from many scientists and clinical investigators, who participated in an NIH consensus development conference that was held September 9-11, 1985. Based on these presentations, important conclusions were drawn with respect to improvements in relapse-free survival, appropriate patients for therapy, and the toxic effects of these treatments. Taken together, these data represent one of the most significant advances in the treatment of human solid tumors and should be of interest to all involved in the management of patients with cancer. Copies of this volume of NCI Monographs (Number 1, 1986) are available from the U.S. Government Printing Office ($8.50 per copy domestic, $10.65 foreign). To order, request Stock No. 017-042-00190-1. 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This volume of NCI Monographs contains the proceedings of a symposium held at The Johns Hopkins Hospital (Baltimore, MD), which focused on the use of antibodies in cancer diagnosis and treatment. The meeting represents the beginning of dialogue among investigators who have done research in the disciplines of physics, radiobiology, immunol- ogy, and pharmacology and those who have clinical expertise in the fields of nuclear medicine and radiation oncology. The work reported at this meeting involves use of dosimetry, low-dose-rate radiation, and antibody fragments, as well as pilot studies of isotope linkage, chemical cytotoxic antibodies, and diagnostic and therapeutic programs. These presenta- tions demonstrate the promise of antibody therapy, which can deliver a broad range of cytotoxic agents, often without the acute side effects of other types of therapy. 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Tad E37 y oh ay =o = ILI CG ONCOLOGY OVERVIEW PREPUBLICATION NOTICE FROM THE NATIONAL CANCER INSTITUTE ONCOLOGY OVERVIEWS are specialized bibliographies with abstracts, each referencing 150-500 recent publications on a cancer topic of high current interest, drawn from over 3,000 sources. Leading researchers in the field covered by each OVERVIEW review and select the most relevant and significant abstracts for each topic. This peer-review selection process results in a tightly focused quick reference to the most recent cancer literature. Fifteen OVERVIEWS are pub- lished each year. The 1987 titles are listed below and are available for between $4.00 and $7.00 each. OT-SERIES (Diagnosis and Therapy) Diagnosis and Treatment of the Acquired Immunodeficiency Syndrome Biological Markers of Central Nervous System Neoplasms Diagnosis and Treatment of Esophageal Neoplasms Cancer Prevention Diagnosis and Therapy of Basal Cell Carcinoma of the Skin Recent Advances in the Systemic Therapy of Genitourinary Malignancies Diagnosis and Therapy of Large Bowel Neoplasms Childhood Nervous System Neoplasms OK-SERIES (Carcinogenesis) Potential Occupational Causes of Cancer: II. The Urinary Tract Dioxins and Dibenzofurans in Carcinogenesis, 1980-86 Radioprotectors: Experimental Studies and Clinical Applications Oncogene Protein Products Chemopreventive Agents Translocation and Amplification of Oncogenes OB-SERIES (Virology, Inmunology and Biology) Viral Etiology of Cancer: II. Papillomaviruses Viral Etiology of Cancer: III. Hepatitis B Virus Idiotypes, Anti-Idiotypes and Network Regulation Transforming Growth Factors and Other Autocrine Growth Factors If you would like to be notified when these and other cancer-related publications are available, please complete the form below and mail to the appropriate address on the form. Priority Announcement Request Form Please put me on your free Priority Announcement List (N-569) to be notified when new ONCOLOGY OVER- VIEWS and other cancer publications are issued. If you are located in the U.S. or its possessions, return (Name) this form to: Superintendent of Documents Mail Stop: SSOM Washington, DC 20401-9374 (Address) If you are located outside the U.S., return this form to: Oncology Overview Coordinator ICRDB, Bldg 82/Rm 103 (City, State, ZIP Code) Bethesda, MD 20892 NCI MONOGRAPHS, NUMBER 4, 1987 147 Y ad Bie Wales In = hod; si x Ai 'y xr a fe Hh + ha he FRG LS “ . But pi » 5d die so hi ame YOU: Experience and Expertise PDQ: State-of-the-Art Cancer Treatment Information YOU and PDQ: The Right Combination for Your Patients A gastroenterologist wants to enter his Dukes C colon cancer patient onto a protocol. By searching PDQ, he finds that a clinical trial is being performed in his city. A surgeon wants treatment information for a premenopausal woman with Stage II breast cancer; her tumor is estrogen and progesterone receptor negative. PDQ provides standard and investigational treatment options. A pediatrician wants the prognosis for a child diagnosed as having Wilms’ tumor; he thinks it is curable. He learns what the curative regimens are by searching PDQ. PDQ, the cancer treatment database from the National Cancer Institute, backs up your expertise with Prognostic, stage, and treatment information for all major types of cancer across the country. More than 1,000 protocol summaries that are open to patient accrual. User-friendly menus for easy access to up-to-date information. To learn more about PDQ, write the National Cancer Institute, International Cancer Information Center, R.A. Bloch Building, Bethesda, Maryland 20892 or telephone (301) 496-7403. NCI MONOGRAPHS, NUMBER 4, 1987 149 MN NN FE MN Cen - Fe ER | le - so . - E - . B # a = . = ia I = : - In In . a Bn - : : : fi B - : u = i NCI Monographs 1 Introduction and Overview 3 DNA Topoisomerases: From a Laboratory Curiosity to a Subject in Cancer Chemotherapy 7 Molecular Genetic Analysis of Topoisomerase II Gene From Drosophila melanogaster 11 DNA Topoisomerase Activity Is Required as a Swivel for DNA Replication and for Ribosomal RNA Transcription 17 Association of Topoisomerase I With Transcriptionally Active Loci in Dresophila 23 Structural Aspects of Mammalian DNA Replication: Topoisomerase II 31 Regulation of DNA Topoisomerases During Cellular Differentiation 37 Involvement of Intracellular ATP in Cytotoxicity of Topoisomerase II-targetting Antitumor Drugs 41 In Vivo and In Vitro Stimulation by Antitumor Drugs of the Topoisomerase II-induced Cleavage Sites in c-myc Proto-oncogene 49 Camptothecin Inhibits Hsp 70 Heat-Shock Transcription and Induces DNA Strand Breaks in Asp 70 Cenes in Drosophila 55 Topoisomerase Inhibitors Can Selectively Interfere With Different Stages of Simian Virus 40 DNA Replication a 61 Topoisomerase II as a Target of Anticancer Drug Action in Mammalian Cells ! NIH Publication No. 87-2943 Number 4 1987 73 Role of Proliferation in Determining Sensitivity to Topoisomerase II-active Chemotherapy Agents 79 Topoisomerase II as a Target of Antileukemic Drugs 83 Topoisomerase Alterations Associated With Drug Resistance in a Line of Chinese Hamster Cells 89 Mediation of Multi-drug Resistance in a Chinese Hamster Ovary Cell Line by a Mutant Type II Topoisomerase 95 Elevated Topoisomerase II Activity and Altered Chromatin in Nitrogen Mustard-resistant Human Cells 99 Heat Shock Proteins: Role in Thermotolerance, Drug Resistance, and Relationship to DNA Topoisomerases 105 DNA Topoisomerase II as a Potential Factor in Drug Resistance of Human Malignancies 111 Metabolic Activation of N-Acylanthracyclines Precedes Their Interaction With DNA Topoisomerase 11 117 Protein-linked DNA Strand Breaks Produced by Etoposide and Teniposide in Mouse L1210 and Human VA-13 and HT-29 Cell Lines: Relationship to Cytotoxicity 123 Structure-Activity Relationships of Podophyllin Congeners That Inhibit Topoisomerase II 129 Treatment Strategies in Relation to Drug Action U. C. BERKELEY LIBRARIES «WA _<0L0938074 Te Tp