PROTEIN-ASSOCIATED DNA BREAKS AS INDICATOR OF TOPOISOMERASE INHIBITION

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DNA topoisomerases are major targets for cancer chemotherapy. Topoisomerase I is concentrated in nucleoli and topoisomerase II is a major component of the nuclear scaffold for interphase nuclei and of the metaphase chromosome scaffold. Topoisomerase II activity is required for chromosome segregation during mitosis and both enzymes are involved in DNA metabolism. Camptothecin inhibits topoisomerase I, and DNA intercalators (amsacrine, anthracyclines) and demethylepipodophyllotoxins (VP-16 & VM-26) inhibit topoisomerase II. Enzyme inhibition results from enzyme-linked DNA breaks, which are believed to be the initial cytotoxic lesions of the drugs. However, the breaks reverse quickly upon drug removal. This prompted us to determine the cytotoxic lesion(s) induced by the topoisomerase-linked DNA breaks. We have found that the topoisomerase I-DNA complexes induced by camptothecin probably kill rapidly proliferative cells by interacting with DNA replication complexes. We have also determined that topoisomerase-mediated DNA breaks are not toxic in cells which have been depleted of calcium. We have confirmed our previous finding that pleiotropic resistant cell lines selected by exposure to adriamycin are cross-resistant to other topoisomerase II inhibitors and have both increased P-glycoprotein and modified topoisomerase II. In addition, we have found that cells selected for resistance to vincristine have increased P-glycoprotein without drug-resistant topoisomerase II. Finally, we found evidence that two forms of DNA topoisomerase I are regulated by signal transduction pathways including protein kinase C.