Comprehensive DNA Methylation and Extensive Mutation Analysis of Gastric Neuroendocrine Carcinoma

Mechanisms of the progression from Barrett's esophagus to esophageal adenocarcinoma (EA) are not fully understood.Acid reflux may play an important role in this progression.We have previously shown that acid exposure increases H 2 O 2 production and cell proliferation, and decreases cell apoptosis, effects which are blocked by knockdown of an NADPH oxidase NOX5-S.However, which downstream proteins mediate acid-induced and NOX5-S-dependent decrease in cell apoptosis are not fully understood.Silencer of death domain (SODD), also known as BCL2-associated athanogene 4, is known to be involved in the survival of cancer cells and related to aggressiveness of different cancers.In this study we examined the role of SODD in acid-induced decrease in cell apoptosis.Human genomic microarray analysis showed that knockdown of NOX5-S in FLO cells caused about 16-fold decrease in the expression of SODD.SODD mRNA levels were significantly higher in EA cell line FLO and OE33 than in normal esophageal squamous epithelial cell line HET-1A or Barrett's cell line BAR-T.Acid treatment significantly upregulated SODD expression and increased SODD promoter activity in FLO cells.Acid-induced increase in SODD expression and promoter activity were significantly decreased by knockdown of either NOX5-S or NF-κB1 p50 in FLO cells.One potential NF-κB binding element was identified in the DNMT1 promoter region: GGGACACCCT (positions -310 to -300 from ATG).Chromatin immunoprecipitation (ChIP) assay showed that SODD DNA was detectable in the immunoprecipitated chromatin samples of FLO cell lysate by PCR using a pair of primers covering the above potential NF-κB-binding site, suggesting that NF-κB may bind to the SODD promoter.The gel shift mobility assays showed that one prominent complex was detectable with IRdye 700-labelled SODD oligonucleotide, containing the NF-κB binding site GGGACACCCT.Competition experiments with a high concentration of unlabeled (cold) SODD oligonucleotide significantly reduced binding.The addition of the mutant SODD oligonucleotide had less effect on binding.We conclude that acid-induced decrease in cell apoptosis may depend on sequential activation of NOX5-S, NF-κB1 p50 and SODD in FLO cells.NF-κB1 p50 may directly bind to the SODD promoter and promote the expression of SODD.