Inverse association between CDH1 promoter methylation level and CDH1 mRNA expression level in human cell lines. (A) Detection of the CDH1 mRNA transcripts in 6 human cell lines by reverse transcription (RT)-polymerase chain reaction (PCR) analysis. mRNA transcripts of the GAPDH, a housekeeping gene, were also amplified as an internal control. M, 100-bp DNA ladder. (B) Map of the CpG sites in the CDH1 promoter. The positions of the CpG sites are indicated by vertical lines. Vertical arrows indicate the location of the -348_-347insA, -161C>A, and -73A>C genetic polymorphisms. +1, transcription start site. (C) Determination of the methylation status of the CpG sites in the CDH1 promoter in 6 human cell lines by bisulfite sequencing analysis. Ten subcloned promoter fragments were sequenced in each cell line. Each horizontal row represents a single allele. The positions of the CpG sites are numbered at the top of the column. Methylated CpG sites are shown as black boxes and unmethylated CpG sites as white boxes.