Mitomycin C (MC) is a natural antitumor antibiotic used in the clinic for cancer chemotherapy. MC generates six different DNA adducts in tumor cells. One of these is a DNA interstrand cross-link, as a result of bifunctional alkylation of the complementary DNA strands by MC, while the others are adducts of the drug, linked to only one strand of DNA (monoadducts and an intrastrand crosslink). The broad, long-range objective of the present proposal is to elucidate the relationship of each of the six adducts to the cytotoxicity/antitumor activity of MC. In addition, the putative toxic role of reactive oxygen species (ROS), generated by redox cycling of MC, will be examined using Fanconi's Anemia (FA) cells.
The first aim of the proposal is to determine whether the known hypersensitivity of FA cells to MC is primarily due to ROS generation or to DNA adduct formation by the drug. These studies will better define the biochemical basis of the unique sensitivities of Faneoni's anemia cells, as well as providing insights into the activities of the mitomycins. The other specific aims serve to detect and characterize biochemical responses unique to the different DNA adducts of MC. In this context we shall investigate differential rates of repair of the adducts in living EMT6 tumor cells in cell culture. Differential modes of nucleotide excision repair of cross-link and monoadducts will be investigated in subcellular systems by constructing 91-mer oligonucleotide duplexes modified site-specifically with the various MC adducts, and using these as substrates for uvrABC excinuclease, or for the repair enzymes present in a mammalian whole cell extract. The same substrates will be used to search for """"""""adduct binding proteins"""""""", specific for the cross-link, in cell extracts. Differential inhibition of lesion bypass by DNA polymerases by the different mitomycin monoadducts will also be investigated, using synthetic adduct-template-primer complexes as substrates. These latter experiments are aimed at identifying the basis for the unusually low cytotoxicity of the monoadducts 5 and 6. This research will lead to a better understanding of the molecular and biochemical basis of the antitumor activity and toxicology of MC and, more generally, of other DNA cross-linking agents, a major class of drugs currently used in cancer chemotherapy.
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