DNA-damaging agents constitute an important class of antineoplastic drugs, most of which are mutagenic in vitro systems. It is highly likely that the mutagenic properties of these agents contribute substantially to some of the adverse effects of chemotherapy, such as acquisition of drug resistance in tumors, and development of second cancers in long-term survivors. The ultimate objective of the proposed research is an understanding of the mechanisms by which specific forms of drug-induced DNA damage eventually lead to genetic alterations involved in malignancy and drug resistance. Such an understanding would be useful in the selection and development of potentially less mutagenic and carcinogenic drugs. In pursuit of this objective, a more immediate goal is the determination of the types of mutations produced by certain drugs in model systems, and identification of the initial DNA lesions responsible for those mutations. Mutagenesis by bleomycin and neocarzinostatin has been examined in detail in a prokaryotic system based on the cI gene of lambda phage. Proposed experiments involving enzymatic repair and other in vitro modifications of drug-damaged DNA are designed to test two alternative hypotheses suggested by these studies (i) that a specific class of oxidized apurinic/apyrimidinic sites, i.e., those accompanied by closely opposed breaks in the complementary strand, are primarily responsible for base substitution mutagenesis and (ii) that secondary reactions of the aldehyde moiety in the oxidized apurinic/apyrimidinic sites play a critical role. Other studies will be directed toward determining whether similar mutational mechanisms occur in shuttle vectors replicated in mammalian cells and whether bleomycin specifically induces activation of the c-Ha- ras-1 oncogene at codon 12, as predicted from its mutational specificity in the prokaryotic system. Nitrogen mustard also strongly mutagenic in the lambda cI gene, and similar approaches will be employed to determine mutational specificity and identify mutagenic DNA lesions, in both E. coli and mammalian models. Finally, studies on the mechanisms of drug-induced deletion mutations will begin with the cloning and sequencing of bleomycin- induced deletions in the aprt gene in CHO cells.
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