Volume 138, Issue 5, Pages 1836-1844.e4 (May 2010) CpG Methylation and Reduced Expression of O6-Methylguanine DNA Methyltransferase Is Associated With Helicobacter pylori Infection  Antonia R. Sepulveda, Yuan Yao, Wen Yan, Dong Il Park, Jae J. Kim, William Gooding, Suhaib Abudayyeh, David Y. Graham  Gastroenterology  Volume 138, Issue 5, Pages 1836-1844.e4 (May 2010) DOI: 10.1053/j.gastro.2009.12.042 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Analyses of MGMT CpG island methylation in tissue samples before and after H pylori eradication. Panels A and B: The plot on panel A shows the levels (percentage of reference positive control) of MGMT methylation detected by SYBR Green real-time PCR (y-axis) tested in paired samples of gastric mucosa with active H pylori gastritis before H pylori eradication (B) and after H pylori eradication (A). The graph on panel B represents the mean methylation levels (y-axis: percentage of reference positive control) in the paired samples with active H pylori gastritis before H pylori eradication (B) and after H pylori eradication (A) (mean, 12.6%; standard deviation, 13.9; standard error of the mean, 2.84; and mean, 5.7%; standard deviation, 8.8; standard error of the mean, 1.8, respectively, P = .025). Panel C: Examples of qualitative analyses of CpG methylation detected by MSP (methylated [m] or unmethylated [u] MGMT CpG island). Examples of results from H pylori-positive (B) and H pylori-negative cases after H pylori eradication (A) are shown. Cases 311 and 332 show positive MGMT methylation before H pylori eradication and negative methylation status after H pylori eradication. Case 310 shows positive MGMT methylation in gastric mucosa before and after H pylori eradication. Case 20 does not show CpG methylation before or after H pylori eradication. Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 Effect of H pylori on MGMT protein, mRNA expression, and CpG methylation in AGS cells. (A) MGMT protein levels detected by Western blot after 1 (36 hours) or 2 cycles (total of 72 hours) of coculture of AGS cells with H pylori (ATCC 43504) with increasing levels of organisms in coculture from 0 to 100 to 1000 multiplicity of infection. (B) MGMT protein levels detected by Western blot from repeat experiments. (C and D) MGMT CpG methylation levels determined by SYBR Green quantitative real-time PCR after 1 or 2 cycles of coculture of AGS cells with H pylori organisms with increasing levels of organisms in coculture from 0 to 100 to 1000 multiplicity of infection. The end point amplification products with MGMT methylation-specific primers (Met) or control ACTB methylation specific primers after gel electrophoresis are shown in panel D. (E) MGMT mRNA levels in AGS cells and after H pylori coculture at 0, 100, or 1000 multiplicity of infection for 1 or 2 cycles of coculture, detected by SYBR Green quantitative real-time reverse transcription-PCR. Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effect of H pylori wild-type strain 60190 and isogenic mutant on MGMT protein in AGS cells. (A) Western blot analysis of protein from control AGS cells (0 H pylori) and after coculture with the wild-type (H pylori wt strain 60190) and isogenic cagA mutant cagA(−). (B) Quantitative analyses of repeat experiments. Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 MGMT expression in gastric mucosa by immunohistochemistry. Nuclear expression of MGMT is appreciated before eradication (B1 and B2) and after H pylori eradication (A1 and A2). B2 and A2 are the same images as represented in B1 and A1, respectively, after image analysis identification of nuclei with no expression (blue), with intermediate level of MGMT (green), and with high levels of expression (red). The surface epithelium in panels B2 and A2 is selected, highlighting increased numbers of surface cells with higher MGMT expression after H pylori eradication (panels A1 and A2). Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Expression of MGMT in gastric mucosa demonstrated by immunohistochemistry. (A) MGMT expression in nuclei of gastric mucosa surface epithelium before and after H pylori eradication. The percentages of surface epithelium nuclei with negative, intermediate, or high level MGMT expression by immunohistochemistry are represented in the y-axis. (B) MGMT expression in nuclei of gastric surface epithelium, glandular epithelium, and in lamina propria adjacent to the mucosal surface or in the deep glandular region of the mucosa. Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 1 MGMT protein levels detected by Western blot analysis after coculture of AGS cells with H pylori (ATCC 43504) for 4, 12, or 36 hours of coculture. Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions

Supplementary Figure 2 Bisulfite sequence analysis of MGMT CpG island. Panels A and B represent 41 and 40 base pairs of an 81 base pair region of the MGMT CpG island, displayed from the 5′ and 3′ end (left to right) to strand of the MGMT gene. The MGMT sequence WTX61657 without bisulfite modification, the same sequence assuming fully unmethylated status (UM) and fully CpG methylated status (Methylated), are represented. Bisulfite sequence analyses of DNA from 3 representative gastric biopsy cases (325B, 324B, and 337B) and from AGS cells cocultured with H pylori organisms are shown. Case 325B and 324B reveal extensive CpG methylation, while case 337B does not show CpG methylation. CpG methylation is shown in DNA sequences from cocultures with H pylori (Hp100 and Hp1000), but not from AGS cells alone (Hp0). Gastroenterology 2010 138, 1836-1844.e4DOI: (10.1053/j.gastro.2009.12.042) Copyright © 2010 AGA Institute Terms and Conditions