Several cancer chemotherapeutic agents, including radiomimetic drugs and topoisomerase inhibitors induce specific types of terminally blocked DNA double-strand breaks, and these lesions have been implicated in both cytotoxic and mutagenic effects of these compounds. In addition, the radiomimetic compounds, like ionizing radiation itself, induce a global genomic instability in a fraction of the progeny of treated cells. The first major goal of the proposed studies is to determine the stringency and the nature of an apparent linkage between specific double-strand break misrepair events and the acquisition of genomic instability. Toward this end, genomic instability, as judged by chromosome rearrangements as well as other criteria, will be quantitatively assessed in various classes of mutants induced in CHO cells by the radiomimetic agents bleomycin and neocarzinostatin, and by the topoisomerase II inhibitor amsacrine. The second major goal of the studies is to determine the possible role of DNA-PK in the repair of double-strand breaks induced by these three agents, with particular emphasis on whether DNA-PK play an active role in enforcing the fidelity of the repair. To this end, mutations induced by bleomycin in CHO cell sublines deficient in this kinase will be compared to existing mutants generated in parental CHO cells, mutants which apparently arise by inaccurate end-joining of double-strand breaks. Repair by end-joining will also be examined in vitro, using a system based on Xenopus egg extracts and synthetic substrates bearing defined, chemically modified DNA ends, again with emphasis on defining the biochemical role of DNA-PK in the end-joining process, particularly as it relates to repair fidelity. The relationship between double- strand break repair, repair fidelity, and acquired genomic instability should have important implications for the overall response of normal and tumor cells to double-strand cleaving agents, and may in the long term aid in efforts to manipulate cellular responses so as to increase the efficacy and reduce the carcinogenicity of these agents.
Showing the most recent 10 out of 84 publications
