Characterization of DNA methylation patterns in tumorigenesis and the use of DNA methylation analysis to gauge toxic potential.

摘要

DNA methylation is an important epigenetic mechanism controlling patterns of genetic expression. In promoter regions of several genes, methylcytosine binding proteins decrease gene expression by inhibiting the binding of transcription factors and/or by facilitating chromatin condensation. Additionally, DNA methylation silences transposable elements, contributing to genomic stability. Aberrant patterns of methylation have been implicated in cancer and in certain neurological, immunological, and developmental disorders. To test the hypothesis that the ability to maintain normal patterns of methylation is inversely related to susceptibility to cancer and perhaps other toxic outcomes, I have characterized patterns of DNA methylation associated with carcinogenesis, as well as those elicited by treatment with cytolethal and noncytolethal concentrations of model compounds. I demonstrate that differences in the ability to maintain GC-rich patterns of methylation might, in part, underlie the differences in tumor susceptibility between the relatively tumor-sensitive C3H/He and B6C3F1 (C57/BL6 X C3H/He) mice compared to the relatively tumor resistant C57/13L6 strain. I also describe alterations in global, GC-rich and gene-specific methylation status in the promotion stage of carcinogenesis using an initiation-promotion SENCAR mouse skin model in which mice were initiated with dimethylbenz( a)anthracene and promoted with various doses of cigarette smoke condensate for different amounts of time. Notably, increases in GC-rich methylation were observed in a dose-and time-dependent, reversible manner during the promotion stage. Threshold levels of CSC necessary to detect changes in GC-rich methylation patterns following 9 wks promotion were predictive of those required for a marked increase in tumor number following 29 wks of promotion. Increased methylation in the promoter region of the tumor suppressors MGMT and p16 was observed only in tumor tissue, and this is the first report describing how reversible alterations in methylation correlate inversely with the expression of the HoxA5 tumor suppressor gene. Overall, these alterations in methylation status could contribute to the clonal expansion of increasingly abnormal cells in the promotion stage. Additionally, since alterations in gene expression due to changes in DNA methylation could potentially contribute to a number of toxic outcomes, I examined the global and GC-rich methylation status of rat hepatoma cells treated with cytolethal and non-cytolethal concentrations of model compounds not previously known to alter methylation. The observation that 2/4 of these compounds affected methylation indicates that chemically-mediated changes in DNA methylation might be more prevalent than commonly assumed. When used in conjunction with cytolethality and genotoxicity data, DNA methylation analysis of these compounds provided a basis for the more rational ranking of these compounds based on estimated toxic potential. Overall, the results of these studies support the view that DNA methylation may be viewed as a secondary mechanism underlying carcinogenesis and perhaps other toxic outcomes, and that DNA methylation assessment can enhance the ability to gauge the toxic potential of chemicals.