Intragenic DNA Methylation Concomitant with Repression of ATP4B and ATP4A Gene Expression in Gastric Cancer is a Potential Serum Biomarker

  • Published : 2012.11.30


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.


  1. Berdasco M, Esteller M (2010). Aberrant epigenetic landscape in cancer: how cellular identity goes awry. Dev Cell, 19, 698-711.
  2. Brenet F, Moh M, Funk P, et al (2011). DNA methylation of the first exon is tightly linked to transcriptional silencing. PLoS One, 6, e14524.
  3. Chow DC, Forte JG (1995). Functional significance of the beta-subunit for heterodimeric P-type ATPases. J Exp. Biol, 198, 1-17.
  4. Edwards JR, O'Donnell AH, Rollins RA, et al (2010). Chromatin and sequence features that define the fine and gross structure of genomic methylation patterns. Genome Res, 20, 972-80.
  5. Feng S, Jacobsen SE, Reik W (2010). Epigenetic reprogramming in plant and animal development. Science, 330, 622-7.
  6. Hong SJ, Kang MI, Oh JH, et al (2009). DNA methylation and expression patterns of key tissue-specific genes in adult stem cells and stomach tissues. J Korean Med Sci, 24, 918-29.
  7. Judd LM, Andringa A,Rubio CA, et al (2005). Gastric achlorhydria in H/K-ATPase-deficient (Atp4a(-/-)) mice causes severe hyperplasia, mucocystic metaplasia and upregulation of growth factors. J Gastroenterol Hepatol, 20, 1266-78.
  8. Kakei N, Ichinose M, Tatematsu M, et al (1995). Effects of long-term omeprazole treatment on adult rat gastric mucosa -enhancement of the epithelial cell proliferation and suppression of its differentiation. Biochem Biophys Res Commun, 214, 861-8.
  9. Kim TY, Lee HJ, Hwang KS, et al (2004). Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest, 84, 479-84.
  10. Lee TL, Leung WK, Chan MW, et al (2002). Detection of Gene Promoter Hypermethylation in the Tumor and Serum of Patients with Gastric Carcinoma. Clin Cancer Res, 8, 1761-6.
  11. Lo YM (2001). Circulating Nucleic Acids in Plasma and Serum: An Overview. Ann NY Acad Sci, 945, 1-7.
  12. Maunakea AK, Nagarajan RP, Bilenky M, et al (2010). Conserved role of intragenic DNA methylation in regulating alternative promoters. Nature, 466, 253-7.
  13. Morley GP, Callaghan JM, Rose JB, et al (1992). The mouse gastric H,K-ATPase beta subunit. Gene structure and coordinate expression with the alpha subunit during ontogeny. J Biol Chem, 267, 1165-74.
  14. Rajkumar T, Vijayalakshmi N, Gopal G, et al (2010). Identification and validation of genes involved in gastric tumorigenesis. Cancer Cell Int, 10, 45.
  15. Scarff KL, Judd LM, Toh BH, Gleeson PA, van Driel IR (1999). Gastric H(+),K(+)-adenosine triphosphatase beta subunit is required for normal function, development, and membrane structure of mouse parietal cells. Gastroenterology, 117, 605-18.

Cited by

  1. Divide and conquer: subproteomic approaches toward gastric cancer biomarker and drug target discovery vol.11, pp.4, 2014,
  2. Acid-base transport in pancreatic cancer: Molecular mechanisms and clinical potential vol.92, pp.6, 2014,
  3. Aberrant Methylation of the 1p36 Tumor Suppressor Gene RIZ1 in Renal Cell Carcinoma vol.16, pp.9, 2015,
  4. Role of Circulating Cell-Free DNA in Cancers vol.19, pp.6, 2015,
  5. Network-based integration of mRNA and miRNA profiles reveals new target genes involved in pancreatic cancer pp.08991987, 2018,
  6. Differential expression profiles of long noncoding RNAs in synchronous multiple and solitary primary esophageal squamous cell carcinomas: A microarray analysis pp.07302312, 2018,
  7. Identify gene expression pattern change at transcriptional and post-transcriptional levels pp.2154-1272, 2019,