The overall objective of this research project is to discern mechanisms that can facilitate the aberrant expression of protooncogenes involved in carcinogenesis. Clearly, mutagenesis plays a role in carcinogenesis. However, carcinogenesis is more than mutagenesis, e.g., with the possible exception of tumor suppressor genes, a mutated gene must be expressed in order to affect a cell's phenotype. DNA methylation plays a key role in the regulation of gene activity, there is an inverse relationship between the percent of DNA-cytosine present as 5-methylcytosine (5MeC) and gene expression. There is a persuasive body of evidence indicating that differential methylation of DNA (i.e., 5-methylcytosine v. cytosine) is a determinant of chromatin structure and that the methyl group provides a chemical signal which is recognized by transacting factors that regulate transcription. Hypomethylation of a gene is necessary but not sufficient for its expression and, therefore, a hypomethylated gene can be considered to poses an increased potential for expression as compared to a hypermethylated gene. The proposed research project is designed to test the hypothesis that hypomethylation of DNA is a nongenotoxic mechanism underlying the aberrant expression of protooncogenes involved in carcinogenesis. Hypomethylation is a mechanism underlying the role of cell proliferation in carcinogenesis, and hypomethylation could possibly result from an enzymatic replacement of 5MeC with cytosine that is not linked to DNA replication. The testing of this hypothesis can serve as a focal point for a mechanism of action oriented approach for considering key aspects of carcinogenesis: aberrant gene expression, heritable epigenetic events, species to species extrapolation/unique species sensitivity, tumor promotion, and thresholds. Changes in the methylation status of a gene provide a mechanism by which its potential for expression can be altered in an epigenetic heritable manner, and it is expected that modifications in DNA methylation would result from threshold-exhibiting events. The experimental model will be liver tumorigenesis. We shall focus upon oncogenes (e.g., Ha-ras and raf) relevant to mouse liver tumorigenesis and employ the liver tumor prone B6C3F1 (C57BL/L6 plus x C3H/He cross) mouse and make relevant comparisons with the sensitive C3H/He paternal strain and the resistant C57BL/6 maternal strain. Hypomethylation of DNA is a very common feature of both animal and human cancer. Thus, the study of liver tumorigenesis in these three strains of mice provides an excellent model system that may have applicability to mechanisms of carcinogenesis (e.g., hypomethylation of DNA) that are not restricted to liver.
The specific aims are designed to place an emphasis on determining which changes in methylation might affect the expression of specific oncogenes, with emphasis upon an assessment of the methylation status of the 5'-flanking (promoter-containing) region of the oncogenes of interest (i.e., Ha-ras and raf) in relationship to specific cis elements to which trans-acting factors that can control gene expression may bind. Significance; A unique aspect of this research project is that it offers the potential to provide insight regarding molecular mechanisms that underlie promotion of carcinogenesis while at the same tie the results can provide the type of information that is required to take a more rational approach towards carcinogen risk assessment. Specifically, the research addresses the areas of dose- response relationships and species-to-species extrapolation issues.
