PROJECT 3. A continuing programmatic goal is elucidation of mechanisms through which reactive nitrogen species (RNS) and reactive oxygen species (ROS) contribute to increased cancer risks. Project 3 addresses this goal by testing the hypothesis that damage to DMAand other cellular macromolecules by RNS from NO- producing macrophages and/or ROSfrom neutrophils either drives cells into apoptosis or inhibits apoptosis and enhances mutation. We employ models enabling mechanistic studies of DNA damage, genotoxicity, mutagenicity and cell death. Project 3 also defines DNA lesions with biological properties that explain mutagenic and lethal endpoints in cellular- and organism-level systems. Association of inflammatory bowel disease with risk of colon cancer is well-documented, and evidence also clearly associates high frequency of iNOS-containing tumor cells with poor survival of stage III melanoma patients. Thus, our first specific aim is to elucidate mechanisms underlying relationships among dose and dose-rate, DNA damage, mutagenesis and apoptosis induced in human colon carcinoma cells and human melanoma cells by exposure to RNS and ROS. The pivotal role of p53 in modulating responses will be evaluated by parallel studies in closely related p53-mutant and p53-null cells. Second, we shall characterize effects of dose and dose-rate on mutagenic potency and mutation spectra induced by RNS and ROS in the gpt reporter gene in three settings: (a) in pSV2gpt-transformed CHO AS52 cells exposed in vitro to NO* under controlled conditions;(b) in AS52 cells co-cultivated with activated RAW264.7 mouse macrophages and/or HL60 cells;and (c) integrated into the genome of Rag 2-1- IL10-/- mice developing inflammation-related colon adenocarcinoma.
Our third aim concerns genetic prioritization of DNA adducts as biomarkers of lethal and mutagenic endpoints. Adducts already nominated for potential as inflammation-derived pre-mutagenic lesions will first be evaluated by insertion into oligodeoxynucleotides, ligation into a viral genome and replication in E. coli cells of differing repair proficiency. Secondly we shall use the novel tool of chemical-biological fingerprinting to accelerate functional linkage of DNA damage and mutational spectra to lesions responsible for specific biological endpoints. Mutagenicity corresponding to features of mutational spectra in the gpt gene damaged with RNS or ROS (aim #2), will be characterized structurally (in collaboration with Project 2).
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