Helicobacter pylori infection introduces DNA double-strand breaks in host cells

Abstract

Gastric cancer is an inflammation-related malignancy related to long-standing acute and chronic inflammation caused by infection with the human bacterial pathogen Helicobacter pylori. Inflammation can result in genomic instability. However, there are considerable data that H. pylori itself can also produce genomic instability both directly and through epigenetic pathways. Overall, the mechanisms of H. pylori-induced host genomic instabilities remain poorly understood. We used microarray screening of H. pylori-infected human gastric biopsy specimens to identify candidate genes involved in H. pylori-induced host genomic instabilities. We found upregulation of ATM expression in vivo in gastric mucosal cells infected with H. pylori. Using gastric cancer cell lines, we confirmed that the H. pylori-related activation of ATM was due to the accumulation of DNA double-strand breaks (DSBs). DSBs were observed following infection with both cag pathogenicity island (PAI)-positive and -negative strains, but the effect was more robust with cag PAI-positive strains. These results are consistent with the fact that infections with both cag PAI-positive and -negative strains are associated with gastric carcinogenesis, but the risk is higher in individuals infected with cag PAI-positive strains.

FIG 1

Analysis of ATM activation caused by infection with Helicobacter pylori. (A to C) Immunohistochemistry of gastric mucosa. The tissue section was stained with anti-phospho-S1981 ATM antibody. (A) Pyloric region of stomach from an uninfected individual; (B) pyloric region from an individual with gastritis; (C) pyloric region from an individual with intestinal metaplasia. (D) Immunofluorescent staining of activated ATM in vitro. AGS cells were infected with H. pylori. After 24 h of incubation, cells were fixed and stained for phospho-S1981 ATM (Phos.-ATM; red) and phospho-Ser139 histone (γ-H2AX; green). Nuclei were stained with DAPI (blue). (E) Quantification of ATM activation and appearance of γ-H2AX after infection with wild-type H. pylori after 24 h. (F) ATM-deficient fibroblasts were infected with H. pylori and stained for γ-H2AX (green) and H. pylori (red). (G) ATM-proficient fibroblasts were infected with H. pylori and stained for γ-H2AX (green) and H. pylori (red).

FIG 2

Analysis of γ-H2AX induced by infection with H. pylori. (A) Immunofluorescent staining of γ-H2AX in vitro. AGS cells were infected with H. pylori strains of the indicated genotypes and stained for γ-H2AX (green) and H. pylori (red). cag PAI, mutants negative for PAI; cagA, strain with a mutation in cagA; vacA, strain with a mutation in vacA; vacA cag PAI, strain that has a mutation in vacA and that is negative for cag PAI. (B) Percentage of γ-H2AX-positive cells after infection with H. pylori. The percentages of γ-H2AX-positive cells were determined by counting more than 200 cells. The means and SEs were determined from four independent experiments. (C) Western blot analysis of phospho-S1981 ATM and γ-H2AX. AGS cells were infected with H. pylori strains of the indicated genotypes and stained for phospho-S1981 ATM and γ-H2AX. ATM and tubulin were loading controls. (D, E) Analysis of DSBs by pulsed-field gel electrophoresis. (D) DSBs after infection with wild-type and cag PAI-negative strains. Intact DNA was stacked in the well, while broken DNA migrated into the gel. DNAs of various sizes (from 500 kb to at least 5.7 Mb) were compacted in one band, indicated as broken DNA. (E) Quantification of DSBs from the results of pulsed-field gel electrophoresis. *, statistically significant difference by Student's t test (P < 0.05). (F) Western blot analysis of γ-H2AX. AGS cells were infected with wild-type and cag PAI-negative strains and analyzed for the appearance of γ-H2AX.

FIG 3

Analysis of DNA damage responses after infection with H. pylori. (A) Cell cycle analysis after infection with H. pylori. H. pylori strains of the indicated genotypes were infected, and the cell cycle state was analyzed with PI staining in a flow cytometer. Mitomycin C (MMC) was used as a control. (B) Analysis of ATM-CHK2-dependent cell cycle checkpoint. Cells were either treated with mitomycin C or infected with H. pylori, and the phosphorylation states of ATM and CHK2 were analyzed with antibodies that recognize the specific phosphorylated antigens. (C) Microarray analysis of H. pylori-infected AGS cells. Cells were infected with H. pylori strains of the indicated genotypes, and gene expression profiles related to DNA repair, DNA damage responses, and cell cycle control were compared. babA, strain with a mutation in babA. (D) Western blot analysis of RAD51. Accumulation of the RAD51 protein was analyzed at 24 h after infection with wild-type H. pylori and H. pylori mutants of the indicated genotypes. Actin was used as a loading control.