Volume 141, Issue 5, Pages 1696-1708.e2 (November 2011) Spermine Oxidase Mediates the Gastric Cancer Risk Associated With Helicobacter pylori CagA  Rupesh Chaturvedi, Mohammad Asim, Judith Romero–Gallo, Daniel P. Barry, Svea Hoge, Thibaut de Sablet, Alberto G. Delgado, Lydia E. Wroblewski, M. Blanca Piazuelo, Fang Yan, Dawn A. Israel, Robert A. Casero, Pelayo Correa, Alain P. Gobert, D. Brent Polk, Richard M. Peek, Keith T. Wilson  Gastroenterology  Volume 141, Issue 5, Pages 1696-1708.e2 (November 2011) DOI: 10.1053/j.gastro.2011.07.045 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Induction of SMO in ImSt cells cocultured with H pylori strains at an MOI of 200. (A) Real-time polymerase chain reaction (PCR) (upper panel) and semi-quantitative reverse transcription PCR (lower panel) for SMO mRNA expression assessed at 6 h. (B) Upper panel, densitometry; lower panel, representative Western blot for SMO and β-actin. (C) Flow cytometric analysis of SMO protein. (D) Immunofluorescent detection of SMO; cells were stained for SMO and β-actin, detected with secondary antibodies conjugated with fluorescein isothiocyanate (green) and rhodamine (red), respectively. Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Examples of perinuclear and nuclear staining for SMO are denoted by white arrows. (E) Cells were cultured with isogenic mutants of 7.13. SMO mRNA expression was measured by real-time PCR after 6 h. For panels A, B, and E, n = 3 in duplicate; ***P < .001 vs control (Ctrl); ##P < .01 vs B128; §§P < .01, §§§P < .001 vs 7.13. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Induction of H2O2 and apoptosis in ImSt cells by H pylori and effect of SMO small interfering RNA (siRNA). Cells were cocultured with H pylori at an MOI of 200 for 24 h. (A) H2O2 in supernatants. (B) Summary data for apoptosis, and (C) representative dot plots; percent of cells in the Annexin V+/propidium iodide (PI)+ (late apoptosis) quadrants are indicated. (D) Levels of H2O2 in supernatants and (E) summary apoptosis data in cells transfected with scrambled (Scr) siRNA or SMO siRNA. In panels A, B, D, and E, n = 3 in duplicate. For panels A and B, **P < .01, ***P < .001 vs control; ##P < .01, ###P < .001 vs B128; and §§P < .01, §§§P < .001 vs 7.13. For panels D and E, *P < .05, **P < .01 vs uninfected cells transfected with scrambled siRNA; §P < .05, §§P < .01 vs cells transfected with scrambled siRNA and incubated with 7.13. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Induction of oxidative DNA damage in ImSt cells by H pylori and effect of SMO small interfering RNA (siRNA). Cells were cocultured for 24 h with H pylori at MOI 200. (A) Summary data for 8-oxoguanosine levels, in mean fluorescence units, measured by flow cytometry. (B) Representative histogram for 8-oxoguanosine levels. (C) Summary data for DNA damage in cells transfected with siRNA. (D) Representative histograms for 8-oxoguanosine levels in transfected cells. In panels A and C, n = 3 in duplicate. For panel A, *P < .05 vs control, #P < .05 vs B128, §P < .05 vs 7.13. For panel C, *P < .05, ***P < .001 vs uninfected ImSt cells transfected with scrambled siRNA; §P < .05, §§P < .01 vs cells transfected with scrambled siRNA and incubated with 7.13. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Ectopic expression of cagA in ImSt cells and effect on apoptosis and DNA damage. (A) Representative Western blot for CagA and β-actin in ImSt cells transfected with empty vector and CagA expression vector. (B–F) Cells were cotransfected with EGFP and cagA vectors, and data were analyzed from EGFP+ cells 24 h later. (B) SMO expression levels analyzed by flow cytometry. (C) Summary apoptosis data in cells ± the SMO inhibitor MDL 72527 (250 μM). (D) Representative dot plots of Annexin V vs propidium iodide PI for apoptosis measurement. (E) Summary data for oxidative DNA damage. (F) Representative histogram for 8-oxoguanosine levels. In panels B, C, and E, n = 2 in duplicate; **P < .01, ***P < .001 vs cells transfected with empty vector; in panels C, and E, §§P < .01 vs cells transfected with cagA vector. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Levels of SMO, DNA damage, and apoptosis in human antral gastric biopsies. (A) Left panel: SMO mRNA expression measured by TaqMan PCR; red diamonds indicate strains polymerase chain reaction–positive for cagA, but CagA protein-negative (Supplementary Figure 6A); right panel: scoring of SMO staining intensity in epithelial cells from immunohistochemistry analysis. (B–C) Immunohistochemistry for SMO. (D) Immunohistochemistry for 8-hydroxy-2′-deoxyguanosine. (E) Levels of apoptosis by in situ oligo ligation, imaged by DIC microscopy. (F) Gastric epithelial cells were isolated from gastric tissues and analyzed by flow cytometry. Left panel: levels of SMO; middle panel: levels of DNA damage; right panel: correlation (Pearson coefficient) between SMO and DNA damage levels. In panels A and F, each circle is a different patient; **P < .01, ***P < .001 vs uninfected normal tissues, §P < .05, §§P < .01, §§§P < .001 vs cagA-positive tissues. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Levels of SMO protein, DNA damage, and apoptosis in gerbils infected with 7.13 or 7.13 cagA− at 14 weeks post-inoculation. In the 7.13 group, 5 gerbils had invasive carcinoma and 5 had dysplasia, while in the 7.13 cagA− group all had chronic gastritis only. (A) Left and middle panels: levels of SMO protein and 8-oxoguanosine, respectively, measured by flow cytometry; right panel: correlation (Pearson coefficient) between SMO and 8-oxoguanosine levels. (B) Representative dot plots. (C) Percentage of cells active caspase-3high (left panel); 8-oxoguanosinehigh (middle panel); active caspase-3low,8-oxoguanosinehigh (right panel). (D) Representative dot plots. In panels A and C, each circle is a different gerbil; **P < .01, ***P < .001 vs uninfected control; §§P < .01, §§§P < .001 vs gerbils infected with 7.13. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 SMO and DNA damage levels in gastric epithelial cells in mice. (A) C57BL/6 mice were infected with PMSS1 or its cagE isogenic mutant, and gastric epithelial cells were isolated at 2 months post-inoculation. Left and middle panels: summary data for levels of SMO protein and 8-oxoguanosine, respectively, measured by flow cytometry. Right panel: correlation (Spearman coefficient) between levels of SMO protein and 8-oxogaunosine. (B–D) FVB/N INS-GAS mice infected with PMSS1 for 4 months. (B) H&E staining showing dysplastic and dilated glands in infected and uninfected mice, and invasive carcinoma in H pylori–infected mice. (C) Representative dot plots of SMO and 8-oxoguanosine levels in gastric epithelial cells. (D) Representative dot plots of active caspase-3 and 8-oxoguanosine levels. In panel A, n = 5 per group; In panels B–D, n = 3 uninfected and 6 infected mice. For panel A, **P < .01 vs control, §§P < .01 vs PMSS1. Gastroenterology 2011 141, 1696-1708.e2DOI: (10.1053/j.gastro.2011.07.045) Copyright © 2011 AGA Institute Terms and Conditions