[CANCER RESEARCH 47. 2050-2055, April 15, 1987] Topoisomerase I(-mediated DNA Breaks and Cytotoxicity in Relation to Cell Proliferation and the Cell Cycle in NIH 3T3 Fibroblasts and L1210 Leukemia Cells Judith Markovits,1 Yves Pommier,2 Donna Kerrigan, Joseph M. Covey, Eugene J. Tilchen, and Kurt W. Kohn Laboratory of Molecular Pharmacology, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892 ABSTRACT The DNA intercalato!-, 4'-{9-acridinylamino)methanesuIfon-/n-anisi- dide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase Il-mediated protein-linked DNA strand breaks. This func tion of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia LI 210 cells stop proliferation when they reach a certain density. Nuclei were isolated from proliferative or quiescent cells and then treated with drug for 30 min. DNA modifications were assayed by alkaline elution. We found that the frequencies of m-AMSA- or VP-16-induced DNA-protein links were higher in nuclei from exponentially growing than in those from quiescent cells in both the 3T3 and the L1210 lines. Drug-induced protein- associated DNA breaks were also studied as a function of the cell cycle in 3T3 cells that had been arrested by contact inhibition in medium containing 1% calf serum and then stimulated to proliferate by replating at a lower cell density in medium containing 10% serum. In these synchronized cells, a large peak of [3H]thymidine incorporation occurred 18-30 h after replating. The yield of DNA-protein cross-links produced by 30-min drug treatments of nuclei isolated at various times after growth initiation increased concomitantly with the peak of the DNA synthesis. The topoisomerase II activity of nuclear extracts, as measured by kine- toplast DNA decatenation followed a similar pattern. Using colony- forming assays, we also observed that m-AMSA and VP-16 were most cytotoxic in proliferative cells and during DNA synthesis. These results suggest that alkaline elution measurement of m-AMSA- or VP-16- induced protein-linked DNA breaks reflects the association of topoisom erase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time. INTRODUCTION The diverse activities of eukaryotic type II topoisomerases have been well reviewed (1-3). These enzymes play an impor tant role in the organization and behavior of chromosomal DNA. It was demonstrated that type II topoisomerase is a major component of mitotic chromosome scaffold and the nuclear matrix (4, 5). Using different topoisomerase II pheno- types (mutants) of yeast, Holm et al. (6) demonstrated the indispensability of yeast topoisomerase II for cell growth. Re cent studies have shown that one of the essential roles of eukaryotic topoisomerase II is its ability to unknot and deca- tenate chromosomal DNA, thus enabling the segregation of intertwined, newly replicated DNA molecules during mitosis (7). However, it is unlikely that topoisomerases act only in the mitotic phase of the cell cycle in mammalian cells. It is strongly suggested that these enzymes are also present during the G, and the S phases of the cell cycle. Topoisomerases also control, by their relaxing effects on supercoils, the superhelical density of DNA in dm imat in. perhaps in conjunction with mammalian Received 8/25/86; revised 12/19/86; accepted 1/13/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Present address: Laboratoire de Pharmacologie Moléculaire, LA 147 CNRS, U 140 INSERM. Institut Gustave Roussy, 94805 Villejuif, France. 1 To whom requests for reprints should be addressed, at Laboratory of Molec ular Pharmacology, National Cancer Institute, NIH. Building 37, Room 5AI9, Bethesda, MD 20892. type I topoisomerase (1-3). The initiation step of replication of bacteriophage T4 may also depend upon the phage-encoded type II topoisomerase (8). Duguet et al. (9) reported that topoisomerase II activity increased in regenerating rat liver. This result suggested a possible correlation between the activity of mammalian topoisomerase II and cell proliferation. Surpris ingly, the studies of Tricoli et al. (10) showed no differences in topoisomerase II activity (measured by the ATP-dependent catenation reaction with PM2 DNA) in proliferating versus non-proliferating mouse embryo film¡blast(C3H10T'/2) cells, nor in the G,, S, and the M phases of synchronized C3H10T'/2 cells. Sullivan et al. (11) found that topoisomerase II DNA cleavage activity was severalfold higher in exponential phase versus plateau phase CHO3 cells but not strikingly different in HeLa or LI210 cells. Further evidence has been accumulating that mammalian type II topoisomerases, which transiently break and rejoin double-stranded DNA, are targets of many DNA intercalative antineoplastic drugs, such as Adriamycin, daunomycin, the dlipiidnes. and m-AMSA, and of the nonintercalative epipo- dophyllotoxins VP-16 and VM-26 (teniposide) (12-16). These drugs trap topoisomerase II on the DNA within DNA-enzyme complexes. The complexes consist of topoisomerase II homo- dimers bound to DNA double- or single-strand breaks in such a way that the breaks are bridged by the enzyme, and one or both subunits are covalently linked to the 5'-ends of the DNA. DSB, SSB, and DNA-protein cross-links can be easily detected in mammalian cells by alkaline elution (17). These lesions have been characterized as drug-induced protein-linked DNA strand breaks (16, 18-20). A correlation has been observed between the induction of such lesions by these drugs and their cytotoxic effects (21-26). We used the alkaline elution method to examine the formation of protein-linked DNA breaks by topoisomerase II inhibitors as a function of the proliferation state of the cells, and more specifically as a function of the cell cycle in synchronized cells. We also investigated whether the cytotoxicity of these drugs correlated with the production of DNA lesions in different phases of the cell cycle. MATERIALS AND METHODS Drug and Chemicals. m-AMSA (NSC 249992) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treat ment, NCI. VP-16 (VP-16-213, NSC 141540) was a generous gift from Bristol-Myers Co. (Syracuse, NY). m-AMSA and VP-16 were dissolved in dimethyl sulfoxide at a concentration of 10 HIM.m-AMSA stock solutions were kept frozen at -20°Cfor no longer than 15days, while VP-16 stock solutions were prepared immediately before use. [methyl- 3The abbreviations used are: CHO, Chinese hamster ovary; m-AMSA, 4'- (9-acridinylamino)methanesulfonate-m-aniside; DPC, DNA-protein cross-link; DSB, double-strand break; HBSS, Hanks' balanced salt solution; MEM, minimal essential medium; SSB, single-strand break; dThd, thymidine; VP-16, etoposide (VP-16-213). 2050 Research. on November 24, 2021. © 1987 American Association for Cancer cancerres.aacrjournals.org Downloaded from