Several candidate genes were consistent with previous reports, such as BTG4, FLI1, TWIST1, ADHFE1, UNC5C, and SPG20. We validated the methyla tion status in the promoter CpG islands of candidate genes by QMSP for the investigation of large contiguous CpG sites, the results of which were concordant with the array results for most genes except FLI1. Among the various CIMP markers in CRC, 18 CIMP markers were selected for the validation of methylation status and methylation based therapeutic targets in CRC. ADAMTS1, CHFR, DAPK1, IGF2, IGFBP3, NEU ROG1, SFRP1, TAC1, THBD, and WRN were also hyper methylated in our chip data. In our QMSP results of CIMP markers, DAPK1, TAC1, THBD, APC, CACNA1G, and CDKN2A were not significantly methylated in CRC tissues.

These discrepan cies may be due to differences full report in the epigenomes of tu mors or patient ethnic background. The hypermethylation of AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 have not been previously reported in CRC. AKR1B1, aldo keto reductase family 1, member B1, catalyzes the reduction of aldehydes including the aldehyde form of glucose. It was reported to be down regulated in endometrial cancer and gastric cancer. The product of CHST10, carbohydrate sulfotransferase 10, is known to inhibit the invasiveness of melanoma cells. ELOVL4 gene product, elongation of very long chain fatty acids 4, is responsible for the biosynthesis of fatty acids. Hypermethylation of ELOVL4 was re ported in hepatocellular carcinoma and pancreatic adenocarcinoma by genome wide methylation analysis. SOX5 is a member of the SOX family of transcription factors.

It is well known that SOX5 regulates embryonic development and determines cell fate. STK33, serine threonine kinase 33, is located on chromosome 11p15. 3, a gene rich re gion that has been associated with human diseases and malignancies. ZNF304, zinc finger protein 304, com bines two conserved domains, class II AU rich elements selleck inhibitor and a Krüppel associated box, and is associated with the regulation of lymphocyte activation. DNA methylation mediated silencing of gene expres sion can be restored by demethylation agents such as 5 aza dC. DNA methyltransferase inhibitor, 5 azacytidine, may act as an inducer of cell differentiation by causing de methylation and re expression of genes silenced by hypermethylation.

5 Azacytidine was approved in 2004 by the US Food and Drug Administration for treat ing myelodysplastic syndrome, and 5 aza dC as a 5 azacytidine analog was widely used in DNA methylation studies. Vincristine is a microtubule inhibitor and is commonly used for chemotherapy in pediatric acute lymphoblastic patients. Several anticancer drugs are associated with drug induced DNA hypermethylation in human lung adenocarcinoma and rhabdomyosarcoma cells. Interestingly, the methylated cytosine was re duced after treatment with concentration of vincristine less than 100 umole but it was induced after treatment with higher than 1000 umole in human lung adenocar cinoma cells.