Characterization of Mutational Spectra, Mechanisms of Toxicity and Homologous Rec

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The goal of this project is to characterize mechanisms of mutagenicity, apoptosis and homologous recombination resulting from DNA damage induced by NO, with particular emphasis on how specific types of damage may contribute to tumor initiation and development. The ability of macrophages and neutrophils to overproduce NO and reactive oxygen species is well-established, as is the ability of these reactive species to induce lethal and mutagenic DNA damage. Work in Dr. Tennebaum's Project has established that the chemistry of cellular damage induced by NO, although complex, may result from only two fundamental processes: (1) reaction with oxygen to form N203, a nitrosating agent; and (2) reaction with superoxide to form peroxynitrite (ONOO-). This project is designed to test the hypothesis that mutations, alterations in apoptotic signaling and homologous recombination result from DNA damage produced by NO and ONOO- in chemically-defined systems as well as by activated phagocytes. Our first specific aim will be to determine effects of dose and dose-rate on mutagenicity and mutation spectra induced by NO and ONOO- in the supF gene of pSP189 replicated in human cells. Second, we shall determine relationships among NO and ONOO- dose and dose-rate, DNA damage, mutagenesis and apoptosis in cultured human cells. Cells to be used include human lymphoblastoid TK6 cells and human colon adenocarcinoma HCT116 cells; the pivotal role of p53 in modulating responses will be evaluated by parallel studies in isogenic p53 knockout cells. Our third line of investigation will focus on relative contributions of NO and reactive oxygen species generated by activated macrophages to mutagenesis of endogenous and exogenous genes in co-cultivated target cells. In the fourth specific aim, we propose to identify the DNA lesion(s) giving rise to mutations observed in NO-treated cells. Oligonucleotides will be synthesized containing modified bases formed by NO, inserted into the genome of M 13 bacteriophage and replicated in E. coil. The type and amount of genetic change, if any, will be characterized, and genetic requirements for mutagenesis by specific lesions will be defined. Preliminary studies revealed that homologous recombination was a critical determinant of NO-induced lethality in E. coll. In the final specific aim, we therefore propose to determine relationships between NO-induced DNA lesions and homologous recombination in mammalian cells, inasmuch as LOH, an important contributor to tumorigenesis can result from aberrant recombination events.