1. Volume 123, Issue 2, Pages 542-553 (August 2002)
Helicobacter pylori impairs DNA mismatch repair in gastric epithelial cells 
Jae J. Kim, Hong Tao, Emilia Carloni, Wai K. Leung, David Y. Graham, Antonia R. Sepulveda 
Gastroenterology 
Volume 123, Issue 2, Pages (August 2002)
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2. Fig. 1
Decreased hMSH2 protein levels after coculture with H. pylori. Western blot analysis of protein extracts of AGS cells cocultured with H. pylori (ATCC 43504) for 4, 24, and 48 hours at a concentration of 1 × 104 bacteria per cell. The same amounts (10 μg) of protein from AGS cells grown in similar conditions but not infected with H. pylori were harvested at experimental time 0 and used as control (C). The same blot was probed with anti-actin antibody as control.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Western blot analysis of proteins extracted from AGS cells cocultured with H. pylori. (A) Proteins extracted from AGS cells cultured for 24 hours that were not cocultured with H. pylori were used as control and run in lane C. AGS cells were exposed to 1 × 102, 1 × 103, or 1 × 104 bacteria per AGS cell. The same amount (10 μg) of protein was run in each lane. Antibodies against human MMR proteins (hMLH1, hPMS1, hPMS2, hMSH2, or hMSH6) were used to identify the corresponding proteins as indicated in A. The same Western blots were incubated with anti-PCNA and anti-actin antibodies as controls. (B) Protein levels in each band were determined by densitometry using Quantity One version 4.0 software (Biorad). The ratios of levels of each MMR protein in the control sample C relative to PCNA or actin (MMR/PCNA or MMR/actin) were taken as 100%, representing baseline relative levels (BRL) of the MMR proteins in cells without H. pylori infection. The relative decrease in MutS (S) and MutL (L) proteins was determined by comparing the BRL in control noninfected cells with the MMR/PCNA or MMR/actin ratios obtained when cells were cocultured with 104 H. pylori per AGS cells for 24 hours. The resulting values were plotted in B. All DNA MMR proteins decreased to <20% relative to PCNA and to <10% relative to actin when H. pylori were used in cocultures with AGS cells. C shows that the decrease in MMR protein levels caused by H. pylori infection of AGS cells was dose dependent. As shown in a typical experiment, levels of all DNA MMR proteins dramatically decreased as the number of H. pylori in coculture with AGS cells was increased from none to 1 × 102, 1 × 103, and 1 × 104 H. pylori organisms in coculture, resulting in MMR protein levels decreasing to <10% when 104 H. pylori were used to infect AGS cells.
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4. Fig. 3
Effect of H. pylori on DNA MMR protein levels in epithelial cell lines of different lineage. The H. pylori strain ATCC was cocultured with each of the various cell lines for 24 hours at 104 H. pylori organisms per cell. hMSH2 was examined by Western blot using 10 μg of protein extract from cells cocultured with H. pylori (Hp) or not exposed to the organisms (C). The gastric cancer–derived cell lines AGS, KATO-III, NCI-N87, SNU-1, SNU-16, SNU-601, SNU-638, SNU-668, and SNU-701 were examined. The colon cancer cell line HCT-116 and the cervical cancer cell line HeLa were also exposed to H. pylori in cocultures using similar conditions. Western blot analysis showed that H. pylori caused a decrease in the levels of hMSH2 MMR proteins in all cell lines examined, whereas the levels of actin, used as control, did not significantly change after exposure to H. pylori.
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5. Fig. 4
Effect of H. pylori cagA status on DNA MMR protein levels. AGS cells were exposed to the cagA-positive H. pylori strain ATCC and to the cagA-negative H. pylori isolate 401C. The cells were cocultured with either H. pylori strain for 24 hours using 1 × 104 H. pylori organisms per cell. Western blot analysis of hMSH2, in the upper panel, shows that relative to control nonexposed AGS cells (C), both the cagA-positive and cagA-negative strains caused a reduction in the levels of hMSH2. B shows the Western blot analysis of hMSH2 and hMLH1 after exposing AGS cells for 24 hours to 1 × 101, 1 × 103, or 1 × 104 cagA-negative H. pylori strain 401C.
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6. Fig. 5
Bacterial species-related effects on DNA MMR. AGS cells were exposed to H. pylori, C. jejuni, or E. coli for 24 hours using 1 × 102 organisms per cultured cell. Western blot analysis was performed with 10 μg of protein extracted from AGS cells that were not exposed to any organisms (C) or that were exposed to H. pylori (Hp), C. jejuni (Cj), or E. coli (B). Baseline relative levels were determined using actin as the internal control. Densitometric quantitation represented in the graph on the left showed that both H. pylori and C. jejuni caused a >50% decrease in the levels of both hMLH1 and hMSH2, whereas E. coli caused a smaller decrease in hMLH1 levels but did not decrease the levels of hMSH2.
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7. Fig. 6
Heat sensitivity of H. pylori factors cause impaired DNA MMR protein levels in AGS gastric carcinoma cells. H. pylori ATCC water extracts were obtained, and the supernatant (S) and pellet (P) fractions were tested separately by incubating the resulting bacterial products with AGS cells for 24 hours. The supernatant (sb) and pellet (PB) fractions of H. pylori were boiled and mixed with AGS cells and incubated in otherwise similar conditions. Protein extracts from AGS cells that were not exposed to H. pylori were used as control (C). Western blot analysis of hMSH2, hMLH1, and actin were performed using 10 μg of protein extracted from AGS cells treated in the different conditions. Only the nonboiled pellet fraction of H. pylori caused a significant decrease in the levels of hMSH2. The decrease in hMLH1 levels was similar to the decrease in hMSH2 levels after exposure to nonboiled H. pylori pellet (P), whereas a less marked decrease in hMLH1 levels was associated with exposure to supernatant (S) and no significant change in hMLH1 levels was caused by boiled supernatant (SB) and boiled H. pylori pellets (BP).
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8. Fig. 7
Decreased RNA levels of hMSH2 and hMSH6 in AGS cells after exposure to H. pylori. (A) Northern blot analysis of hMLH1, hMSH2, and hMSH6 RNA in AGS cells after coculture with H. pylori for 4, 24, or 48 hours. AGS cells were not exposed to H. pylori (C). (B) Quantitative representation of hMLH1, hMSH2, and hMSH6 RNA levels from a typical Northern blot after AGS cells were cocultured with H. pylori. (C) Quantitative representation of hMLH1, hMSH2, and hMSH6 RNA levels after coculture with H. pylori detected by semiquantitative RT-PCR. RNA from AGS cells exposed to H. pylori for 48 hours (AH48H) and AGS cells without exposure to H. pylori (C) was used for RT-PCR amplification. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control for both Northern blot and RT-PCR.
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9. Fig. 8
Detection of hMLH1 and hMSH2 by immunofluorescence in AGS cells. To determine whether the effect of H. pylori on MMR protein expression was reversible after H. pylori were removed from the medium, the AGS cells infected with H. pylori for 24 hours (24 h-Hp) were rinsed thoroughly with sterile PBS and allowed to grow for 48 hours after H. pylori removal (48 h-R). Control uninfected AGS cells were grown over the same time period as the infected AGS cells (24 h-Control and 48 h R-Control). No-pAB is a control for the immunostain performed in the absence of primary antibody. PCNA was used as a control nuclear stain.
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10. Fig. 9
H. pylori–induced decrease in hMLH1 and hMSH2 protein levels is reversible. In this experiment, AGS cells were cocultured with H. pylori for 48 hours. To determine whether the effect of H. pylori on MMR protein expression was reversible after H. pylori were removed from the medium, the cells were rinsed thoroughly with sterile PBS and allowed to grow for 48 hours on the same plate. Uninfected control AGS cells were grown over the same time periods. C-0, uninfected AGS cells at time point 0, corresponding to AGS cells in culture for 24 hours; C-48, uninfected AGS cells in culture for an additional 48 hours; 48 h-Hp, AGS cells cocultured with H. pylori for 48 hours. After the 48-hour coculture with H. pylori, the organisms were thoroughly removed by serial washings and the cells were cultured for an additional 48 hours in the absence of H. pylori (Post Hp-R). Control uninfected AGS cells grown for the same period of time as Post Hp-R (C-R).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions