Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5' regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5' regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription-PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5' CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.