Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells
Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells
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DOI：
10.1111/j.1523-5378.2006.00408.x
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发表时间：
2006-08-01
期刊：
影响因子：
4.4
通讯作者：
Sepulveda, Antonia R.
中科院分区：
文献类型：
--
作者：
Yao, Yuan;Tao, Hong;Sepulveda, Antonia R.
背景：幽门螺杆菌胃炎增加胃癌风险。微卫星不稳定型突变继发于DNA错配修复缺陷。幽门螺杆菌胃炎在微卫星不稳定性阳性胃癌患者中更为常见，并且独立于炎症的幽门螺杆菌可以降低DNA错配修复蛋白水平，这提出了幽门螺杆菌可能在感染期间导致突变的假设。材料和方法：采用绿色荧光蛋白报告载体（pEGFP-CA13）检测突变。将转染pEGFP-CA13的胃癌AGS细胞与幽门螺杆菌或大肠杆菌共培养。检测绿色荧光蛋白（GFP）阳性细胞数量，Western blot检测GFP、hMSH2、hMLH1蛋白水平。通过甲基化特异性聚合酶链反应测定幽门螺杆菌对hMLH1 CpG甲基化状态的影响。结果：幽门螺杆菌共培养AGS细胞中GFP水平和GFP阳性细胞数显著升高，hMLH1和hMSH2水平下降。幽门螺杆菌共培养诱导hMLH1启动子的低水平CpG甲基化。与幽门螺杆菌共培养的细胞序列分析显示，与未与幽门螺杆菌共培养的细胞相比，移码突变和点突变的数量增加（p = .03和p = .001）。结论：这是第一个报告显示幽门螺杆菌可能导致基因组突变的积累，独立于潜在的炎症。这与DNA错配修复减少有关，并且至少部分与hMLH1启动子的CpG甲基化有关。这些数据支持这样一种观点，即慢性胃炎期间胃上皮细胞幽门螺杆菌诱导的突变和表观遗传改变可能导致与幽门螺杆菌感染相关的胃癌风险增加。
Background: Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection.Materials and Methods: Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction.Results: GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively).Conclusions: This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.