PROTEIN-ASSOCIATED DNA BREAKS AS INDICATOR OF TOPOISOMERASE INHIBITION

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DNA topoisomerase I and II (top 1 and 2) are major targets for cancer chemotherapy. Topoisomerase poisons act by stabilizing enzyme-linked DNA breaks which can be detected as protein- associated DNA breaks in drug-treated cells. The goal of this project is to study drug interactions with cellular DNA and to elucidate the antitumor mechanisms of top 1 and 2 poisons, their selectivity for cancer cells, and the functions of topoisomerases in normal and cancer cells. We have studied the cellular effects of several new topoisomerase inhibitors including the top 2 inhibitor, azatoxin (NCI patent), and the various top 1 inhibitors presently in clinical trials. Also, we are studying the differential sensitivity and resistance of the cell lines from the NCI cell Screen to identify the parameters that are best correlated with cytotoxicity and that could be used in the clinic to predict and monitor the response to topoisomerase inhibitors. We and others have recently described apoptosis as a mode of cell death, especially in malignant hematopoietic cells exposed to topoisomerase inhibitors. Interestingly, HL-60 cells die in interphase by apoptosis within 3 hours after drug treatment, while human colon carcinoma HT-29 cells die by G2 block after 24-48 hours. We have studied the changes in cell cycle-related protein kinases/ phosphatase in HL-60 cells treated with topoisomerase I inhibitors. We find that cyclin B/cdc2 kinase become down regulated as cells undergo DNA fragmentation and apoptosis. We are using our in vitro assay that reconstitutes the internucleosomal DNA fragmentation during apoptosis in HL-60 cells to elucidate the biochemical pathways involved. New approaches aimed at triggering or suppressing apoptosis may provide new therapeutic strategies and help reduce drug side effects.