In Vivo Dna Repair Mechanisms and Carcinogenesis

Understanding how DNA repair deficiencies predispose cells to damage accumulation and mutation is particularly important to understanding the carcinogenic process. In the first theme (Theme I) of this work, two types of base excision repair (BER) deficiencies are explored. In the first study (Theme IA), we observed a significant decrease in BER activity in liver tissues during acute hepatitis in an animal model of inflammation-mediated hepatocarcinogenesis, the Long Evans Cinnamon (LEC) rat. We hypothesized that decreased BER during acute hepatitis induced oncogenic mutations and pursued the development of an appropriate cell culture model to test this hypothesis. We developed a hepatocyte cell line from the LEC rat during acute hepatitis (LEC-AH) and a tumor cell line from a LEC rat with HCC (LEC-T). The LEC-AH cell line exhibited decreased BER and increased DNA damage, and we determined that the LEC-T cell line harbored an activating mutation in codon 12 of the K-ras oncogene which was characteristic of deficient BER. The LEC-AH cell line characterization and the mutation observed in LEC-T cells provide an appropriate model in which to perform lesion mapping and establish a link between deficient BER during acute hepatitis and oncogenic mutation. The second study examines the role of mutation site-specific deficiencies in repair of an adduct, 1, N-ethenoadenine (eA) within the tumor suppressor gene p53 (Theme IB). We determined that eA at frequently mutated codons in p53 was dramatically impaired compared to eA at non-mutation sites due to slow turnover of the BER enzyme, Nmethylpurine DNA glycosylase (MPG). Finally, the second theme (Theme II) describes the development of a fluorescence microscopy-based technique to monitor interactions between DNA repair proteins and DNA adducts in vivo. We utilized the interaction of a well-known DNA repair protein, human apurinic apyrimidinic endonuclease 1 (APE1) with its substrate, the abasic (AP) site, as a model. The method developed borrowed from techniques generally used to detect protein-protein interactions, such as co-