The molecular mechanisms by which H. pylori (Hp) increases gastric cancer (GC) risk are vastly unknown. Direct interaction of Hp organisms in co-culture with gastric epithelial cells causes a marked decrease in the levels of the main DNA mismatch repair (MMR) proteins MLH1 and MSH2 and microsatellite instability (MSI)-type mutations in a reporter gene. Up to 30% of GC show MSI-High, gastric mucosa with chronic gastritis and intestinal metaplasia frequently show MSI-mutations, and patients with MSI-positive GC are more likely to have active Hp gastritis. These data lead to our hypothesis that Hp might cause mutation accumulation in the stomach epithelium by impairing DNA MMR, representing a pathway of GC development and explaining at least in part how Hp infection increases GC risk.
Specific aim one : To determine the degree of MMR deficiency required for MSI mutation accumulation, to characterize the spectrum of mutational targets and mutations and to identify the fundamental mechanisms underlying the reduced levels of MLH1 and MSH2 proteins induced by Hp in cultured gastric epithelial cells (GEC). GEC and increasing numbers of Hp will be co-cultured. The levels of MLH1 and MSH2 proteins associated with MSI development will be determined. MSI mutation accumulation will be determined in GEC repeatedly exposed to Hp organisms using GFP reporter vectors by western and FACS analyses. We will determine the spectrum of mutations at polyCA and polyA repeats. Transcription rates, mRNA and protein stability of MSH2 and MLH 1 will be measured after Hp infection.
Specific aim two : To characterize the alterations of MLH1 and MSH2 and level and frequency of MSI mutation accumulation during H. pylori gastritis in humans. Gastric epithelium will be obtained by laser capture microdissection using biopsies from Hp infected individuals before and after Hp eradication and from control non-infected patients. Protein and mRNA levels of MLH1 and MSH2 will be determined by western and Taqman analysis. Mutations in the epithelium of Hp infected individuals will be evaluated by examining a recommended panel of microsatellite markers and gene targets of MSI-mutagenesis. The MSI and MLH1 and MSH2 protein and mRNA levels will be evaluated before and after Hp eradication to test whether the changes induced by Hp are reversible. The long-term goals and impact of this study are to understand the Hp bacterial-host interaction mechanisms that lead to increased risk of GC. Characterization of MMR alterations and target gene mutations may become useful as a molecular tool for surveillance and GC risk assessment of patients with chronic Hp infection. Knowledge gained from this study is likely to support the indication for Hp eradication in H. pylori-infected patients to prevent GC. ? ? ? ? ?
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