H. pylori Infection Is Associated with DNA Damage of Lgr5-Positive Epithelial Stem Cells in Human Stomach
Takeshi Uehara, Yao Yuan, Michael Feldman, Amy Ziober, Hiroyoshi Ota, Antonia Sepulveda. University of Pennsylvania, Philadelphia, PA; Shinshu University, Matsumoto, Japan
Background: The G-protein coupled receptor Lgr5 is expressed in gastric antral epithelial stem cells and in cancer tissues. Mutagenesis of the epithelial stem cell genome has been proposed to underlie gastric cancer (GC) development. 8-hydroxydeoxyguanosine (8OHdG) is a DNA modification induced by reactive oxygen species that can be used to determine levels of DNA injury at the individual cell level. Studies have shown that H. pylori and associated inflammation are main GC risk factors, and this may be related to increased mutagenesis of the gastric epithelium. The aim of our study was to determine whether Lgr5-positive gastric epithelial stem cells are susceptible to mutagenesis associated with H. pylori infection.
Design: Lgr5 and 8OHdG expression was characterized in non-neoplastic gastric antral mucosa of gastrectomy specimens from 24 patients (16 with GC: 9 Hp positive (Hp+), 7 Hp negative (Hp-); and 8 without GC, all Hp-). To determine the extent of mutagenesis in Lgr5 positive epithelial cells (Lgr5+), expression of nuclear 8OHdG, a marker of DNA mutagenesis, was determined in co-stains with Lgr5 by immunofluorescence. Primary Lgr5 rabbit polyclonal and 8OHdG monoclonal mouse antibodies, followed by fluorescence labeled antibodies were used. Quantification of 8OHdG was done by spectral image analysis using a Nuance Trio microscope and CRI imaging software. In each case, at least 10 Lgr5+ and 10 Lgr5- cells from contiguous glandular cells were scanned.
Results: Overall the 8OHdG fluorescence maximum intensity (FMI) in Lgr5+ (mean 313.1, SD 143.1) was significantly higher than in Lgr5- cells (mean 286.5, SD 134.9)P=.03. In the group of GC cases in which both Hp+ and Hp- cases were available, the 8OHdG FMI (mean 263.6, SD 126.9) in Lgr5+ was significantly higher than in Lgr5- cells (mean 215.0, SD 106.5), P=.012 in Hp+ cases but not in Hp- cases (8OHdG FMI mean 342.8, SD 148.0 in Lgr5+ and mean 329.4, SD 134.8, in Lgr5- cells, P=.41).
Conclusions: These data suggest that DNA damage occurs in Lgr5+ gastric epithelial stem cells and that these cells may be more susceptible to oxidative stress than other gastric glandular cells. This is supported by the finding that 8OHdG accumulation was higher in Lgr5+ (stem) cells as compared to Lgr5- cells in patients with active H. pylori infection but not in those without H. pylori infection. These data support a role for H. pylori infection in mutagenesis of gastric stem cells which may underlie H. pylori associated gastric carcinogenesis.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 253, Wednesday Afternoon