Nitric Oxide and Acid Induce Double-Strand DNA Breaks in Barrett’s Esophagus Carcinogenesis via Distinct Mechanisms  Nicholas J. Clemons, Kenneth E.L. McColl, Rebecca C. Fitzgerald  Gastroenterology  Volume 133, Issue 4, Pages 1198-1209 (October 2007) DOI: 10.1053/j.gastro.2007.06.061 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Acid induces DNA DSBs in Barrett’s esophagus nondysplastic, high-grade dysplasia, and adenocarcinoma cell lines. Acid-induced DSB formation was assessed by immunostaining for γH2AX followed by FACS or confocal microscopy. Examples are shown from FACS (A; black, isotype control; red, γH2AX) and confocal microscopy (B; blue, DAPI; pink, γH2AX) of SEG cells incubated in pH 3.5 medium for the indicated times. (C) Quantitative FACS results from all cell lines showing the mean fold increase in fluorescence (compared with control) ± SEM. Statistical analysis by Student t test, *P < .05 compared with corresponding control treatment. Cell viability (D) and survival (E) following incubation at pH 3.5 for the indicated time periods was assessed by trypan blue exclusion after 24 hours or by clonogenic assay immediately after incubation in acid, respectively. (F) Annexin V-FITC labelling of SEG cells treated at pH 3.5 for 25 minutes followed by recovery at pH 7.4 for 20 minutes or 6 hours. Cells treated at pH 7.4 for 25 minutes or 100 μmol/L staurosporine are negative and positive controls, respectively. The percentage of positive cells ± SD from 3 independent experiments is shown. Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Acid induces intracellular production of ROS. SEG cells were preincubated with the ROS reactive fluorescent dye CM-H2DCFDA followed by incubation at pH 3.5 for the indicated periods and analyzed by FACS (A) or confocal microscopy (B; blue, DAPI; green, CM-H2DCFDA fluorescence). Quantitative FACS results for acid-induced ROS production following incubation at pH 3.5 for 10, 15, 20, or 25 minutes for all cell lines are shown (C). Data are presented as the mean fold increase in fluorescence compared with control ± SEM. Statistical analysis by ANOVA, *P < .01. FACS histograms (D) and quantitative results showing mean fold increase in fluorescence compared with control ± SEM (E) of γH2AX labelling in SEG cells pretreated with or without the antioxidant NAC prior to incubation at pH 3.5 for 15 minutes. Statistical analysis by Student t test, #P = .01 vs –NAC. Mitochondrial membrane polarization was assessed in SEG cells treated with acid (pH 3.5) for 10 or 20 minutes (F). Cells were stained with tetramethylrhodamine ethyl ester and analyzed by FACS. Positive control cells were treated with CCCP to depolarize the mitochondria. Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 In vitro NO production from nitrite. Production of NO from sodium nitrite was measured with an NO probe when 100, 300, or 600 μmol/L sodium nitrite was added to acidified medium at pH 3.5, 3.0, or 2.5 containing 2 mmol/L AA (A) or 600 μmol/L of sodium nitrite was added to acidified medium containing 0.5, 1.0, or 2.0 mmol/L AA (B). The results show mean concentration ± SEM. Statistical analysis by ANOVA, *P < .0002. FLO cells preincubated with DAF-FM diacetate were incubated with 100 μmol/L MAHMA NONOate for the indicated times and analyzed by FACS (C). Quantitative FACS results for FLO and QhTERT cells treated with the indicated doses of MAHMA NONOate for 30 minutes (D). The results show the mean fold increase ± SEM of the geometric mean of DAF-FM diacetate fluorescence histograms. Statistical analysis by Student t test, *P < .05 vs control cells. Confocal images of DAF-FM diacetate fluorescence in FLO cells treated with 100 μmol/L MAHMA NONOate for 20 minutes (E). DAPI staining of DNA is shown in blue and DAF-FM diacetate fluorescence in green. Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Physiologic concentrations of NO cause DSBs in Barrett’s esophagus nondysplastic, high-grade dysplasia, and adenocarcinoma cell lines. NO-induced DSB formation was assessed by staining for γH2AX followed by FACS or confocal microscopy. Example histograms from FACS (A; black, isotype control; red, γH2AX) and images from confocal microscopy (B; blue, DAPI; green, γH2AX) of SEG cells treated with 100 μmol/L MAHMA NONOate for 45 minutes are shown. Quantitative FACS data for all of the cell lines giving mean percentage of positive cells ± SEM (C, nondysplastic and high-grade dysplasia cells) and (D, adenocarcinoma cells). Statistical analysis by ANOVA, *P = .003, P = .0002, P = .0005 for QhTERT, ChTERT, and GihTERT, respectively, **P = 2 × 10−17, P = 2 × 10−12, and P = 9 × 10−13 for BIC, FLO, and SEG, respectively. NO-induced DSBs in SEG cells were confirmed by neutral comet assay (E). Confocal images of γH2AX labelling in primary Barrett’s esophagus cells treated with 100 μmol/L MAHMA NONOate (F; blue, DAPI; green, γH2AX). Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 NO-mediated DSBs occur preferentially in S-phase cells. SEG cells treated with the indicated doses of MAHMA NONOate for 45 minutes were analyzed by FACS for DNA content (DAPI) and γH2AX expression to examine production of DSBs in relation to cell cycle (A). Long-term survival of Barrett’s esophagus nondysplastic, high-grade dysplasia, and adenocarcinoma cell lines following incubation with MAHMA NONOate for 45 minutes was assessed by clonogenic assay (B). The kinetics of NO-induced γH2AX was assessed in SEG cells (C). Expression of γH2AX was examined by Western blotting in SEG cells treated with 100 μmol/L NO for the indicated times (D). Lysates from cells treated with 10 Gy ionizing radiation or 100 J/m2 UV radiation followed by recovery for 30 minutes were used as positive controls. Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Nitrite-induced DSBs in Barrett’s esophagus nondysplastic, high-grade dysplasia, and adenocarcinoma cells. SEG cells were treated with 600 μmol/L nitrate or nitrite in the presence of 2 mmol/L ascorbate at pH 3.5 or 7.4 with or without PTIO, and γH2AX was detected by FACS (A). Data show mean percentage of positive cells ± SEM. Statistical analysis by Student t test, *P = 7 × 10−6, #P = 3 × 10−4, **P = .003. (B) 2-D FACS plots of γH2AX and DAPI-labelled SEG cells treated with 600 μmol/L nitrate or nitrite at pH 3.5. Gastroenterology 2007 133, 1198-1209DOI: (10.1053/j.gastro.2007.06.061) Copyright © 2007 AGA Institute Terms and Conditions