PAX6 methylation and ectopic expression in human tumor cells.

The mechanisms underlying the de novo methylation of CpG islands in human cancer remain almost completely unknown. We used a methylation-sensitive arbitrarily primed polymerase chain reaction (Ms AP-PCR) technique to scan genomic DNA for differential methylation patterns and identified a 550 bp band that was hypermethylated in a majority of colon and bladder cancer cells. This band corresponded to a CpG-rich region in exon 5 of PAX6, which is a highly conserved transcription regulatory factor involved in embryogenesis. Interestingly, exon 5 was very frequently methylated in solid tumors compared with adjacent normal tissues, 17 out of 27 (63%) in bladder, and colon, but the promoter remained unmethylated in all these cases. This methylation was not effective in blocking transcription since ectopic expression of PAX6 was seen in several tumors and cell lines with extensive exon 5 methylation. De novo methylation of the promoter was only seen in tumor cell lines and was associated with gene silencing since treatment with 5-aza-2'-deoxycytidine restored expression to the cells and resulted in a less methylated promoter. Thus, ectopic expression and hypermethylation of exon 5 of PAX6 demonstrate that methylation within a transcribed region, as opposed to promoter methylation, does not block gene expression. Copyright 2000 Wiley-Liss, Inc.