The long-term goal of the proposed studies is to elucidate critical cellular mechanisms involved int he antitumor activity of mitomycin C (MC), a natural product widely employed in clinical cancer chemotherapy, and to translate this information into the improved use of MC and its analogs against cancer. Four intracellular variables will be examined with regards to their effects on the cytotoxicity and DNA-adduct-forming ability of MC in tumor cells: (i) The nature of the enzymes that reductively activate MC; (ii) the glutathione level in the cell; (iii) low intracellular pH; and (iv) monofunctional (decarbamoyl mitomycin C) versus bifunctional mitomycin (MC) as the administered agent. All of the variables will be studies in the EMT66 mouse mammary tumor cell line. Complete MC-DNA adduct patterns formed in the cells will be determined using [3H]-labeled MC and analyzing the radiolableed adducts by HPLC, by a method developed previously in our laboratoy. Analogous analyses will be conducted in cell-free system using purified MC-activating enzymes and EMT6DNA, to evaluate intrinsic (cell-independent) effects of the above variables on DNA adduct patterns. The structures of unknown MC-DNA adducts will be determined. Cytotoxicity of MC to cells with enhanced levels of gluthathione as well as the cytotoxicity of the mitomycin metabolie 2, 7-diaminomitosene will be studied in conjunction with the DNA-adduct analyses. This work is expected to result I identifying in intact cells (i) critical MC-activating enzynes and modulation of their activity by cellular microenvironmental conditions and (ii) the relative cytotoxic importance of MC-DNA cross-links versus MC-monoadducts. The knowledge obtained should contribute to improving the use of the mitomycins in the treatment of cancer.
