Identification of epigenetically silenced genes in human pancreatic cancer by a novel method 'microarray coupled with methyl-CpG targeted transcriptional activation' (MeTA-array)

Identification and characterization of epigenetically silenced genes is important for cancer research, because information from hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses and genes frequently methylated in a tumor-specific manner can be used as tumor markers. Here, we describe the identification of transcriptionally silenced hypermethylated genes in pancreatic cancer cells by using a novel method called "microarray coupled with methyl-CpG targeted transcriptional activation" (MeTA-array for short), which can effectively reactivate genes containing the stringent criteria of CpG islands at promoter regions. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2 and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE as a control, were examined with this method, and 19 genes were upregulated twofold or more in all the three cancer cell lines after MeTA; 16 of these 19 genes have not been detected previously when using a conventional DNA demethylating agent, 5-aza-2'-deoxycytidine. Among these 16 genes, CSMD2, SLC32A1, TMEM204 and TRH were further analyzed by methylation-specific PCR, and we found that 90% (19/21) of CSMD2, 100% (21/21) of SLC32A1, 95% (20/21) of TMEM204 and 100% (21/21) of TRH were methylated in our series of pancreatic cancer cell lines. Furthermore, CSMD2, SLC32A1 and TRH were also hypermethylated in primary pancreatic cancers in a tumor-specific manner. These results suggest that MeTA-array is a highly efficient method for identifying methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.