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 both the nuclear scaffold in interphase nuclei and the chromosome scaffold during mitosis. Topoisomerase II activity is required for chromosome segregation during mitosis and both enzymes are involved in DNA replication and transcription functions. Camptothecin inhibits topoisomerase I, and DNA intercalators (amsacrine, anthracyclines) and demethylepipodophyllotoxins (VP-16 & VM-26) inhibit topoisomerase II. The drugs act by stabilizing enzymelinked DNA strand breaks, and these may be the initial cytotoxic lesions produced by the drugs. However, the breaks reverse quickly upon drug removal, indicating that other processes are involved in the sequence of events leading to cell death. This project aims to elucidate these events. We have obtained evidence that the topoisomerase I-DNA complexes induced by camptothecin kill rapidly proliferating Chinese hamster DC3F and slowly proliferating human colon cancer cells by interacting with DNA replication complexes. We have also demonstrated that topoisomerase-mediated DNA breaks are not toxic in cells which have been depleted of calcium and that protein kinases can modulate cellular sensitivity to topoisomerase inhibitors. Further, it now appears possible that topoisomerase I is regulated by signal transduction pathways including protein kinase C. We have started mapping cleavage sites in the c-myc oncogene and find that m-AMSA selectively inhibits topoisomerase II by stabilizing cleavage complexes in the P2 promoter, a site most often used for c-myc transcription in tumor cells. A new project has been the development of camptothecin-resistant cells. We have now obtained a highly resistant cell line and are studying its topoisomerases.