Eighteen candidate genes and 11 CIMP markers were selected to identify the demethylating effects of vincristine. The methylation status of 29 genes was determined by PMR values. In normal colon cells, most genes were not affected by 5 aza dC and vincristine treatment. In con trast, 14 candidate genes and seven CIMP markers were significantly demethylated by 5 aza dC treatment in two CRC cell lines. In addition, 12 candidate genes and eight CIMP markers were signifi cantly demethylated by vincristine treatment in two more CRC cell lines. Restoration of mRNA expression by vincristine in DLD 1 cells The effect of methylation on mRNA expression was in vestigated by MSP and RT PCR analysis in 5 aza dC and vincristine treated DLD 1 and CCD18Co cells.

The methylation status of CHST10, ELOVL4, EYA4, FLI1, STK33, SOX5, and ZNF304 was decreased by treatment with 5 aza dC and vincristine selleckchem in DLD 1 cells, but were not changed in CCD18Co cells. The methylation status of CHST10, ELOVL4, EYA4, and ZNF304 was highly de creased by vincristine. The mRNA expres sion of AKR1B1, CHST10, ELOVL4, FLI1, STK33, SOX5, and ZNF304 was increased by treatment with 5 aza dC and vincristine in DLD 1 cells, but EYA4 mRNA expres sion was not detected. The mRNA expression levels of all genes were not affected by 5 aza dC treatment in CCD18Co cells. The methylation of AKR1B1 was not decreased significantly by treatment with 5 aza dC or vincristine, but the mRNA expression levels of this gene were increased.

These results sug gest that vincristine promotes the demethylation of CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304, and the methylation mediated silencing or down expres sion of these genes was restored by vincristine in DLD 1 cells to the same extent as 5 aza dC, as measured by mRNA expression. selleck chemical Discussion This study identified novel hypermethylated genes in CRC through a genome wide study. DNA hypermethylation leads to the downregulation and silencing of tumor sup pressor genes during the pathogenesis of various human cancers. Recently, genome wide array based studies have reported altered DNA methylation gene pro files in CRC. Oster et al. identified hypermethy lated FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1, and EYA4 genes in the adenomas and carcinomas of colorectal lesions. Kim et al. also reported 20 top ranking hypermethylated genes in CRC. Mori et al. identified several novel candidate CRC biomarkers including VSX2, BEND4, NPTX1, BTG4, and GLP1R. In our methylation chip array results, we dis covered 1,411 hypermethylation CpG sites that were lo cated in the promoter CpG islands of 597 genes, and selected 21 candidate genes for further study.