Mitomycin C (MC) is an antibiotic and antitumor agent widely used in clinical cancer chemotherapy. It is mode of action has been the subject of intensive study and speculation for almost 30 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 four 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 mitomycin and make this knowledge applicable to new drug design and treatment protocols in cancer chemotherapy. The proposed work has the following specific aims: (1) Characterization of MC-DNA adducts on the structural level: (i) NMR study of mitomycin monoadduct an mitomycin intrastrand cross-link adduct. This involves chemical synthesis of oligonucleotide-MC (Columbia University). (ii) Gel electrophoresis of multimers of oligonucleotide-MC adducts to detect bending of DNA at the MC adduct sites. (iii) Determination of the structure of a newly discovered guanine-MC adduct by spectroscopic microscale methods (masspec, NMR). (2) Adduct structure-activity relationships: This involves chemical construction of oligonucleotides selectively modified by one of the four major adducts of MC at a single site. The constructs are ligated into longer sequences and applied to the following: (a) Effect of the different adducts on primer extension by DNA polymerases. (b) Effect of template modification on transcription by T7 RNA polymerase. (c) Action of urvABC excision endonuclease in vitro on the adducted constructs. (d) In vivo effects on survival and mutagenesis: The adducted oligonucleotides will be incorporated into a """"""""shuttle vector"""""""" plasmid system and the resulting recombinant plasmid will be used to transfect COS cells or transform E. coli directly. Replicated plasmids will be isolated from mutant cells and analyzed for genomic sequence changes. (3) Analysis of MC-DNA adducts formed in intact cells. [3H]-labelled MC is used to treated mammalian cells in culture, followed by isolation of such analyses are to determine (a) the effect of hypoxia on adduct formation; (b) relative cytotoxicities of monofunctional and bifunctional adducts; (c) intracellular repair of MC- DNA adduct lesions; (d) different efficiency of repair of cross-link and monoadducts; (e) effect of hypoxia on repair; (f) effect of intracellular glutathione level on adduct patterns; (g) adduct patterns in MC-resistant mutant cell lines.
