In Vivo DNA Repair Mechanisms and Carcinogenesis
The maintenance of the genome is an important barrier to carcinogenesis, and one of the major cellular pathways involved in genome maintenance is DNA repair. There are several types of DNA repair pathways, but one type, the base excision repair (BER) pathway repairs the majority of endogenous DNA. Understanding how 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 BER deficiencies are explored. The first study involves inflammation-mediated carcinogenesis and the decrease of BER capacity under inflammatory conditions observed in some studies (Theme IA). The types of lesions repaired by the BER pathway are often the result of inflammatory processes, and, in fact, chronic inflammatory diseases are often pre-malignant conditions and confer high risk for cancer development. This highlights the importance of BER in inflammatory diseases as a critical "tumor suppressor" pathway. In an animal model of inflammation-mediated hepatocarcinogenesis, the Long Evans Cinnamon (LEC) rat, we observed a significant decrease in BER activity in liver tissues during acute hepatitis and determined through extensive gene expression microarray analysis that early carcinogenic changes occurred during acute hepatitis. 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, which was consistent with the observations in LEC tissues. Furthermore, 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, N6-ethenoadenine (eA), associated with pre-malignant inflammatory disease and the relationship of sequence-dependent deficiencies to the location and pattern of mutation sites in 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. We determined the mechanism of slow repair at mutation sites was codon-specific slow turnover of the BER enzyme, N-methylpurine DNA glycosylase (MPG), the enzyme that initates repair of eA. Finally, one of the disadvantages inherent in the study of BER mechanisms is the lack of appropriate in vivo methodology to study the pathway in a physiologically relevant manner. Therefore, 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-localization and fluorescence resonance energy transfer (FRET), and was able to reliably monitor functional APE1 interaction with AP-site DNA. Importantly, the method was also able to distinguish specific and non-specific interaction over time.
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