Topoisomerase II-mediated DNA breaks and cytotoxicity in relation to cell proliferation and the cell cycle in NIH 3T3 fibroblasts and L1210 leukemia cells.

The DNA intercalator, 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and the nonintercalator, etoposide (VP-16) produce topoisomerase II-mediated protein-linked DNA strand breaks. This function of topoisomerase II was investigated in relation to cell proliferation and cell cycle. Mouse fibroblasts NIH 3T3 and mouse leukemia L1210 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 raplating 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 kinetoplast 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 topoisomerase II with DNA. This association is increased during DNA replication, making the cells more vulnerable to m-AMSA and VP-16 at this time.

[1]  K. Kohn,et al.  Effects of the bifunctional antitumor intercalator ditercalinium on DNA in mouse leukemia L1210 cells and DNA topoisomerase II. , 1986, Cancer research.

[2]  M. Brattain,et al.  DNA breakage in human lung carcinoma cells and nuclei that are naturally sensitive or resistant to etoposide and teniposide. , 1986, Cancer research.

[3]  D. Brutlag,et al.  Multiple forms and cellular localization of Drosophila DNA topoisomerase II. , 1986, The Journal of biological chemistry.

[4]  Y. Pommier,et al.  Altered DNA topoisomerase II activity in Chinese hamster cells resistant to topoisomerase II inhibitors. , 1986, Cancer research.

[5]  T. Uemura,et al.  Mitotic spindle pulls but fails to separate chromosomes in type II DNA topoisomerase mutants: uncoordinated mitosis , 1986, The EMBO journal.

[6]  D. Sullivan,et al.  Proliferation dependence of topoisomerase II mediated drug action. , 1986, Biochemistry.

[7]  W. Ross,et al.  Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity. , 1986, Cancer research.

[8]  K. Kohn,et al.  Reduced formation of protein-associated DNA strand breaks in Chinese hamster cells resistant to topoisomerase II inhibitors. , 1986, Cancer research.

[9]  K. Kohn,et al.  Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II. , 1986, Biochemistry.

[10]  K. Kohn,et al.  Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage. , 1985, Biochemistry.

[11]  Y. Pommier,et al.  Correlations between intercalator-induced DNA strand breaks and sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster cells. , 1985, Cancer research.

[12]  D. Botstein,et al.  DNA topoisomerase II is required at the time of mitosis in yeast , 1985, Cell.

[13]  W. Earnshaw,et al.  Localization of topoisomerase II in mitotic chromosomes , 1985, The Journal of cell biology.

[14]  J. Bertram,et al.  DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells. , 1985, Experimental cell research.

[15]  L. Liu,et al.  Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. , 1984, The Journal of biological chemistry.

[16]  L. Liu,et al.  Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. , 1984, Science.

[17]  K. Kohn,et al.  Formation and rejoining of deoxyribonucleic acid double-strand breaks induced in isolated cell nuclei by antineoplastic intercalating agents. , 1984, Biochemistry.

[18]  Y. Pommier,et al.  Absence of swiveling at sites of intercalator-induced protein-associated deoxyribonucleic acid strand breaks in mammalian cell nucleoids. , 1984, Biochemistry.

[19]  M. Brattain,et al.  Comparison of cytotoxicity and DNA breakage activity of congeners of podophyllotoxin including VP16-213 and VM26: a quantitative structure-activity relationship. , 1984, Biochemistry.

[20]  L. Liu,et al.  Mechanism of antitumor drug action: poisoning of mammalian DNA topoisomerase II on DNA by 4'-(9-acridinylamino)-methanesulfon-m-anisidide. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. Fornace.  Recombination of parent and daughter strand DNA after UV-irradiation in mammalian cells , 1983, Nature.

[22]  M. Duguet,et al.  DNA topoisomerases from rat liver: physiological variations. , 1983, Nucleic acids research.

[23]  K. Kohn,et al.  Protein-associated deoxyribonucleic acid strand breaks produced in mouse leukemia L1210 cells by ellipticine and 2-methyl-9-hydroxyellipticinium. , 1982, Biochemical pharmacology.

[24]  Y. Pommier,et al.  The formation and resealing of intercalator-induced DNA strand breaks in isolated L1210 cell nuclei. , 1982, Biochemical and biophysical research communications.

[25]  K. Kohn,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. , 1981, Biochemistry.

[26]  P. T. Englund,et al.  Decatenation of kinetoplast DNA by topoisomerases. , 1980, The Journal of biological chemistry.

[27]  N. Cozzarelli.  DNA gyrase and the supercoiling of DNA. , 1980, Science.

[28]  K. Kohn,et al.  X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution. , 1979, Nucleic acids research.

[29]  K. Kohn,et al.  Qualitative and quantitative aspects of intercalator-induced DNA strand breaks. , 1979, Biochimica et biophysica acta.

[30]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[31]  B. Alberts,et al.  Studies on DNA replication in the bacteriophage T4 in vitro system. , 1983, Cold Spring Harbor symposia on quantitative biology.