[1665] Acid Increases P16 Methylation Via Activation of NADPH Oxidase NOX5-S in Barrett's Esophageal Adenocarcinoma Cells.

Weibiao Cao, Jie Hong, Jose Behar, Li Juan Wang, Jack Wands, Ronald A DeLellis, Murray Resnick. Rhode Island Hospital and Warren Alpert Medical School of Brown University, Providence

Background: Gastroesophageal reflux disease(GERD) complicated by Barrett's esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE (intestinal metaplasia) to EA are not fully understood. Inactivation of the tumor suppressor gene p16 may play an important role in this progression. Hypermethylation of the p16 promoter is an important mechanism for inactivating p16. In this study we examined whether acid increases methylation of the p16 gene promoter by activating NADPH oxidase NOX5-S in an EA cell line OE33.
Design: P16 mRNA and methylation of p16 promoter were measured by real-time PCR. Transfection of NOX5 siRNA and plasmid was carried out by using Lipofectamine 2000 and Amaxa-Nucleofector-System respectively.
Results: P16 methylation was significantly increased in OE33 cells when compared with Barrett's cell line BAR-T and squamous cell line HET-1A. Acid treatment significantly increased methylation of p16 promoter and decreased the p16 mRNA level. OE33 cells expressed NADPH oxidase NOX5-S. Acid-induced increase in H2O2 production was significantly reduced by knockdown of NOX5-S. Knockdown of NOX5-S significantly increased p16 mRNA, inhibited acid-induced down-regulation of p16 mRNA. Knockdown of NOX5-S also blocked the acid-induced increase in p16 methylation. Conversely, overexpression of NOX5-S and exogenous H2O2 significantly decreased p16 mRNA and increased p16 methylation.
Conclusions: NOX5-S is involved in acid-induced hypermethylation of the p16 gene promoter and in downregulation of p16 mRNA. It is possible that acid reflux in BE patients may activate NOX5-S. NOX5-S-derived ROS in turn increases p16 promoter methylation and down-regulates p16 expression, thereby contributing to the progression from BE to EA. Supported by NIH NIDDK R01 DK080703.
Category: Pathobiology

Wednesday, March 2, 2011 9:30 AM

Poster Session V # 221, Wednesday Morning

 

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