Mitomycin C is a clinically significant antineoplastic antibiotic used widely in combination chemotherapy for the treatment of patients with advanced breast cancer and cervical ovarian cancers. The mechanism of mitomycin C action in vivo remains an enigma. First, research has shown that mitomycin C acts principally as an electrophilic trapping agent that prevents efficient bonding to the target DNA. Second, recent studies have raised questions whether in vitro mitomycin C-DNA bonding investigations accurately reflect in vivo processes. We propose a multidisciplinary program to provide information concerning mitomycin C-DNA transformations. First, we will determine the key interactions that precede the initial bonding of the activated drug to DNA and examine if bonding is catalyzed by DNA. UV-visible, viscometric, and high-filed NMR studies will give information on the mechanism of drug-DNA binding and bonding and provide a solution structure of the precovalent complex. Information concerning the structural elements necessary for the drug-DNA recognition process will be obtained with specifically modified mitomycins and genomic DNAs, using the UVRABC assay. Second, we will determine if in vivo drug-DNA bonding is efficient and sequence selective, if these processes follow the same bonding rules found for in vitro transformation, and if chromatin structure and DNA-mediated processes affect mitomycin bonding. These fundamental questions are still unanswered because of the lack of suitable monitoring techniques. We advance the combined use of the UVRABC assay and the ligation-mediated polymerase chain reaction to follow mitomycin-DNA processes in cultured mammalian cells. Third, we will determine the activation and bonding pathways for two new classes of mitomycin agents to learn if novel routes exist for the efficient and selective bonding of mitomycins to DNA. Members of both classes of compounds are undergoing clinical trials. Knowledge from these studies will contribute to our understanding of the mitomycins and serve as the basis for the design of new DNA site-specific anticancer drugs.
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