PROTEIN-ASSOCIATED DNA BREAKS AS INDICATOR OF TOPOISOMERASE INHIBITION

点击翻译按钮获取中文摘要

DNA topoisomerase I and II (top 1 and top 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, and their selectivity for cancer cells. We have studied the cellular effects of several new topoisomerase inhibitors including the top 2 inhibitors, azatoxin (NCI patent) and hydroxyrubicin, the various top 1 inhibitors presently in clinical trials, and the dual top 1 and top 2 inhibitor, intoplicine (in early clinical trials in Europe). Also, we have obtained evidence that top 2 poisons differ from each other by the distribution of cleavage sites in the human c-myc gene in the case of amsacrine and etoposides. Thus, it appears that preferential gene damage by top 2 inhibitors may contribute to their differential anti cancer activity. 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 and Chinese hamster DC3F fibroblasts die by G2 block after 24-48 hours. We have set up an in vitro assay to reconstitute the nucleosomal DNA fragmentation using isolated nuclei and cytoplasmic extracts and have studied various ways to either stimulate (using protein kinase inhibitors, such as staurosporine) or inhibit apoptosis in HL-60 cells (spermine, Zinc, poly[ADPribose] polymerase inhibitors). Apoptosis is relevant to cancer chemotherapy because its occurrence may explain the hypersensitivity of leukemia to chemotherapy and drug hematological toxicity. Hence, new approaches aimed at triggering or suppressing apoptosis may provide new therapeutic strategies and help reduce drug side effects.