Progressive, non-random altered patterns of methylation in gene-specific and GC-rich regions of DNA underlie tumorigenesis

Epigenetics is broadly defined as processes that establish heritable states of gene expression without altering the DNA sequence. DNA methylation, (i.e. 5-methylcytosine content of DNA), is a well characterized epigenetic mark. Altered patterns of DNA methylation can lead to the aberrant expression of genes. My central hypothesis states that the ability to maintain patterns of methylation is inversely related to susceptibility to tumorigenesis. This hypothesis is tested in the context of the central paradigm for explaining events leading to tumorigenesis. The model describes a multi-stage and multistep (i.e. multi-mechanism) model for the development of precancerous lesions and their evolution into frank carcinomas. The stages defined by this model are initiation, promotion and progression. The promotion stage of tumorigenesis involves the step-wise accumulation of heritable changes which are critical for the selection and clonal expansion of initiated cells. Therefore changes in methylation which accumulate during the promotion stage are not a result of the neoplastic state, but key contributors to the process. With the development of a novel method for measuring changes in methylation in GC-rich regions of the genome with high reproducibility, I have characterized patterns of DNA methylation during the promotion stage of carcinogenesis in three separate model systems. Specifically I have demonstrated highly similar changes in methylation, predominantly hypomethylation, with three different promoting compounds. In addition, I observed hypomethylation of the promoter region of the Ha-ras oncogene with simultaneous stability of patterns of methylation in the promoter region of LINE-1 elements which are retrotransposable elements that comprise ∼30% of the mouse genome. These findings exemplify the selective nature of promoter-induced (i.e. phenobarbital) disruption and indicate that changes in methylation are not entirely random. Detection of hypomethylation, occurring simultaneously with hypermethylation, in GC-rich regions is demonstrated as a principal contributor to the development and progression of tumors. Consistent with the working model of carcinogenesis, regions of altered methylation are seen to persist from early time points to later precancerous and cancerous time points. This lends strong experimental support for the accumulation of critical changes in DNA methylation, both increases and decreases, during tumor promotion. The extent and frequency (i.e. more changes accumulating in a shorter period of time increases susceptibility) with which changes in methylation accrued seems to strongly relate to the relative susceptibilities of B6C3F1 and C57BL/6 mice to liver tumorigenesis, a key point supporting the overall hypothesis. (Abstract shortened by UMI.)...