This grant, first, emphasizes discovering aberrantly silenced and DNA hypermethylated colon cancer genes and defining their function in colon cancer progression. A second emphasis is to dissect the chromatin alterations which may initiate and/or maintain this gene silencing with the translational goal of better understanding colon cancer risk and fostering new prevention and treatment strategies. The first goal involves a microarray approach, based on blocking chromatin events associated with abnormal cancer gene silencing, to screen for the hypermethylated genes. This, already, has facilitated discovery of important genes and we will now improve the sensitivity to facilitate total genomic coverage including using new findings from studies outlined below. For the second emphasis, we study HCT116 cells genetically deleted for DNA methyltransferases (DNMT's) to determine how these enzymes co-operate to establish and/or maintain, promoter DNA hypermethylation and gene silencing. Such studies and siRNA studies of other cell lines reveal that colon cancer cells are surprisingly independent from the major """"""""maintenance"""""""" DNA methyltransferase (DNMT), DNMT1, for sustaining gene hypermethylation and silencing. This enzyme plus DNMTSb must be deleted to abolish virtually all total and promoter specific DNA methylation and cause re- expression of aberrantly silenced genes. We are replacing DNMT's in such cells to establish gene re- methylation models and then identify how DNMT's are complexed and targeted to genes. We have recently determined that the class III HDAC, SIRT1, helps maintain the silencing of the aberrantly silenced genes. Drug induced, dominant negative mediated, and SiRNA blocks of SIRT1 function all induce these genes to re-express without any loss of DNA methylation and synergize with DNA demethylation class I and II HDAC inhibitors to do so. SIRT1, mediates cellular response to environment, nutrient status, and stress, and increases longevity of lower organism and mammalian cells thus providing an important potential link between aging, gene hypermethylation, and cancer. We will determine whether SIRT1 contributes to initiation of aberrant DNA methylation and how it participates in the silencing of cancer genes by determining specific molecular interactions with other chromatin components of gene silencing. Relevance to Public Health: Our studies then provide, in terms of public health, discovering better means to understand and predict the risk of colon cancer. Our findings are suggesting new means, by reactivating abnormally silenced genes, for constructing prevention and treatment strategies for this disease. This proposal seeks to identify genes that are silenced by aberrant methylation and that contribute to the tumorigenic phenotype of colon cancer, and to characterize the mechanisms by which the DNMTS and HDACs function to control gene silencing in colon tumor formation. Strengths of the work include the expertise of the investigator, the reagents already generated, and exciting discoveries made during the last funding period. Weaknesses include the descriptive nature of the work, although the addition/improvement of aim three begins to address this. ? ? ? ?
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