| Aberrant DNA methylation is found in virtually every type of human neoplasm and methylation of tumor suppressor genes is associated with the inappropriate transcriptional silencing of the genes. Silencing of hMLH1 by aberrant hMLH1 promoter methylation accounts for majority of microsatellite instable sporadic colon cancers. We have previously shown hMLH1 silencing is biallelic and actively maintained. To test whether the underlying mechanism is dominant or recessive at the cellular level, we characterized cells with methylated hMLH1 that had received a transfected hMLH1 promoter reporter construct, a transferred exogenous human chromosome 3 bearing wild-type hMLH1, or an entire exogenous human genome by somatic cell fusion to cells bearing unmethylated hMLH1 alleles. In no instance did we observe methylation and silencing of newly introduced unmethylated hMLH1 promoters. Conversely, in somatic cell hybrids between cells that silenced hMLH1 and cells that do not, we saw no evidence for demethylation and reactivation of hMLH1 expression, either. Rather, in these somatic cell hybrids, all hMLH1 alleles maintained the parental phenotype, either methylated or unmethylated, characteristic of their cells of origin. We conclude that the methylation status of hMLH1 in colon cancer is chromosomal autonomous, and is likely dependent on still unknown cis chromosomal marks.; The second part of my graduate study was focused on identifying and characterizing new methylation target genes in colon cancer. Through the application of restriction landmark genome scanning, we found a new gene, SLC5A8, and showed it is a candidate tumor suppressor gene whose silencing by aberrant methylation is a common and early event in human colon neoplasia. In normal colon mucosa SLC5A8 is unmethylated, and SLC5A8 transcript is expressed. In contrast, SLC5A8 is aberrantly methylated in 59% of primary colon cancers and 52% of colon cancer cell lines. SLC5A8 expression is silenced in methylated cells, and can be reactivated by a demethylating drug, 5-azacytidine. Wild type SLC5A8 suppresses colony growth selectively in transfected SLC5A8-deficient cells. SLC5A8 methylation is detectable among aberrant crypt foci, which define the microscopic earliest abnormality of colonic epithelial cells. Furthermore, by structural homology and functional testing, we found that SLC5A8 encodes a new sodium solute symporter. |
