Abstract
Based on our previous report on gastric cancer which documented ATP4A and ATP4B mRNA down- regulation in gastric tumors relative to normal gastric tissues, we hypothesized that epigenetic mechanisms could be responsible. ATP4A and ATP4B mRNA expression in gastric cancer cell lines AGS, SNU638 and NUGC-3 was examined using reverse transcriptase PCR (RT-PCR). AGS cells were treated with TSA or 5'-AzaDC and methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) analysis were performed. MSP analysis was on DNA from paraffin embedded tissues sections and plasma. Expression analysis revealed downregulation of ATP4A and ATP4B genes in gastric cancer cell lines relative to normal gastric tissue, while treatment with 5'-AzaDC re-activated expression of both. Search for CpG islands in their putative promoter regions did not indicate CpG islands (CGI) but only further downstream in the bodies of the genes. Methylation specific PCR (MSP) in the exon1 of the ATP4B gene and exon7 in ATP4A indicated methylation in all the gastric cancer cell lines tested. MSP analysis in tumor tissue samples revealed methylation in the majority of tumor samples, 15/19, for ATP4B and 8/8 for ATP4A. There was concordance between ATP4B and ATP4A down-regulation and methylation status in the tumour samples tested. ATP4B methylation was detectable in cell free DNA from gastric cancer patient's plasma samples. Thus ATP4A and ATP4B down-regulation involves DNA methylation and methylated ATP4B DNA in plasma is a potential biomarker for gastric cancer.
Highlights
Gastric cancer is one of the leading sites of cancer seen in the South Indian population and one of the leading causes of death from cancer
We analysed for DNA methylation in the respective intragenic regions of ATP4A and ATP4B genes which revealed DNA methylation in the exon 1 of ATP4B and exon7 of ATP4A genes
The methylation pattern was observed in the tumor tissue samples and was consistent with the repressed levels of mRNA expression of the genes which indicated that DNA methylation in the intragenic regions is involved in the down regulation of expression
Summary
Gastric cancer is one of the leading sites of cancer seen in the South Indian population and one of the leading causes of death from cancer. ATP4A, a 114-KDa subunit with an ATP and a cation binding site, is the catalytic unit of the membrane protein, while ATP4B, a 35-KDa heavily glycosylated subunit is involved in the proper folding and membrane localization of protein (Morley et al, 1992). ATP4B knockout mice show defects in the membrane integrity of the parietal cells consistent with this hypothesis (Scarff et al, 1999). In the instance of ATP4A knockout, adult mice exhibited achlorhydria, and hypergastrinemia, changes in the parietal cell membrane, and metaplsia of gastric mucosa, with no changes in parietal cell viability or chief cell differentiation (Judd et al, 2005). Inhibition of H,K-ATPase activity by drugs like omiprazole in mice is reported to cause epithelial cell proliferation and suppression of its differentiation (Kakei et al, 1995). H,K-ATPase is essential for normal functioning and homeostasis of gastric tissue
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