KI RAS ACTIVATION AND ADDUCT FORMATION BY CYCLOPENTA PAH

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The long-term goals of this research are to gain insight into mechanisms by which chemically-induced DNA lesions are processed into mutations that may ultimately lead to tumorigenesis. This specific project aims to relate formation of DNA adducts by cyclopentaPAH to activation of the Ki- ras protooncogene in strain A/J mouse lung tumors. The cyclopentaPAH are biologically active non-alternant PAH, formed in incomplete combustion processes, that appear to be activated by metabolic pathways differing from those inferred for alternant PAH with bay-region features. The cyclopentaPAH tested to date are all potent tumorigens in A/J mouse lung, and many of the resulting tumors carry an unusual GGT to CGT transversion in codon l2 of Ki-ras not seen with alternant PAH. The availability of nucleosides covalently modified by cyclopentaPAH enables us to begin to correlate specific adducts with mutations, and renders feasible the generation of site-specifically modified DNA sequences for use as templates to investigate replication and processing of chemically-induced lesions, proceeding as follows: I. Tumor Biology: Evaluate the relative carcinogenic potencies of four structurally-related cyclopentaPAH, cyclopenta [cd) pyrene, benz [j) aceanthrylene, benz [l) aceanthrylene and aceanthrylene, in A/J mouse lung; verify the presence of activated Ki-ras in tumor tissue; examine the resulting mutational spectra, particularly with respect to the abundance of the unique GGT to CGT mutation in codon 12 of Ki-ras. II. Metabolism and Activation of CyclopentaPAH: Identify and quantitate ultimate active metabolites found in A/J mouse lung, and verify consistency with the DNA adducts formed. Determine the identity and stereochemistry of adducts formed, and their quantitative relationship to administered dose and observed tumorigenic potency. III. Chemistry of Adducts: Synthesize and characterize the putative major adducts of cyclopentaPAH with deoxyadenosine and deoxyguanosine, prepare 3'-phosphate nucleotides to use as chromatography standards, and 3'- phosphoramidite-5'-DMT adduct derivatives for synthesis and characterization of modified Ki-ras oligonucleotide sequences. IV. Molecular Biology of Site-Specifically Modified Ki-ras sequences: Investigate mutational spectra resulting from replication on Ki-ras templates modified by cyclopentaPAH adducts. Characterize the relative transforming efficiencies of the major cyclopentaPAH-induced mutations of Ki-ras codon 12, and correlate with mutational spectra.