To identify CpG islands differentially methylated in pancreatic
adenocarcinoma, we used methylated CpG island amplification (MCA) coupled with representational difference analysis. Of 42 CpG islands identified by MCA/representational difference analysis, 7 CpG islands [methylated in
carcinoma of the pancreas (MICP)] were differentially methylated in a panel of eight
pancreatic cancer cell lines compared with normal pancreas. In a larger panel of 75 pancreatic
adenocarcinomas, these 7 MICPs (ppENK,
Cyclin G, ZBP, MICP25, 27, 36, and 38) were methylated in 93, 3, 9, 15, 48, 19, and 41% of
cancers, respectively, by methylation-specific PCR but not in any of 15 normal pancreata. In
pancreatic cancer cell lines, methylation of ppENK, a gene with known growth suppressive properties, was associated with transcriptional silencing that was reversible with
5-aza-2'-deoxycytidine treatment. Relationships between the methylation patterns of pancreatic
adenocarcinomas and their clinicopathological features were also determined. Larger
pancreatic cancers and those from older patients (P = 0.017) harbored more methylated loci than smaller
tumors and those from younger patients (P = 0.017). ppENK, MICP25, and 27 were variably methylated in normal gastric, duodenal, and colonic mucosae. These data indicate that aberrant methylation of ppENK and its transcriptional repression is a common event in pancreatic
carcinogenesis.