Mitomycin C (MC) is an antibiotic and antitumor agent widely used in clinical cancer chemotherapy. Its mode of action has been the subject of intensive study and speculation for more than 20 years, since the discovery that it induced covalent crosslinking of DNA. Recently we elucidated the basic chemistry of the activation of MC and its ultimate reaction products with DNA including the MC-DNA crosslink. Our objective is to relate each of the three major reaction products to the biological effects of MC, i.e. to determine (a) which is more effective for antitumor and cytotoxic activities, (b)-why is it more effective and (c) how to enhance the formation of such effective reactions in the cell. The broad and long-term objective is to understand the molecular basis of the antitumor activity of the mitomycins and make this knowledge applicable to new drug design and treatment protocols in cancer chemotherapy. First, the three adducts of MC and DNA will be examined for their location at specific DNA sequences, using footprinting techniques as well as Ba131 digestion as a tool. Effects of each adduct on DNA conformation will be assessed by examining the conformations of oligonucleotides modified at a single site by each of the three adducts, as models. Assay of adducts formed in cell cultures will be used as probe for (i) effect of intracellular 02 on crosslinking efficiency of MC, (ii) relative cytotoxicity of the three adducts, (iii) kinetics of removal of adducts by cellular repair. Excision repair of adducted DNA will be studied in an in vitro system, using single-site adducted DNA fragments. Two MC analogs, more effective than the parent MC will be examined for the molecular basis of their greater effectiveness by comparing their activation and DNA- reactivity with those of MC.
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