1. Volume 127, Issue 3, Pages 845-858 (September 2004)
Helicobacter pylori and H2O2 increase AP endonuclease-1/redox factor-1 expression in human gastric epithelial cells 
Song-Ze Ding, Ann M. O’Hara, Tim L. Denning, Bernadette Dirden-Kramer, Randy C. Mifflin, Victor E. Reyes, Kieran A. Ryan, Susan N. Elliott, Tadahide Izumi, Istvan Boldogh, Sankar Mitra, Peter B. Ernst, Sheila E. Crowe 
Gastroenterology 
Volume 127, Issue 3, Pages (September 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
APE-1/Ref-1 protein expression is increased in gastric epithelial cells from H. pylori-infected human subjects. Epithelial cell preparations from gastric antral biopsy samples from H. pylori-infected (n = 3) and -uninfected (n = 3) human subjects were examined for APE-1/Ref-1 expression by Western blotting. A representative Western blot showing APE-1/Ref-1, a 37-kilodalton protein, is shown. The graph depicts the band density of samples from each subject group as mean ± SEM. APE-1/Ref-1 protein is expressed constitutively by human gastric epithelial cells and is increased by chronic H. pylori infection in vivo (*P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 2
APE-1/Ref-1 is expressed in situ by native human gastric epithelial cells. Gastric epithelial cells were specifically excised from an H. pylori-negative human gastric antral mucosal biopsy specimen by using the laser capture microdissection technique. (A) Section of human gastric mucosa from an H. pylori-negative subject before laser capture microdissection. (B) Microdissected epithelial cells captured onto CapSure HS LCM transfer film caps. (C) Section of remaining mucosa. (D) Agarose gel electrophoresis of PCR product from complementary DNA from the microdissected sample with APE-1/Ref-1-specific primers. Lane 1, 50-base pair (bp) molecular weight ladder; lane 2, PCR product prepared from a complementary DNA sample with reverse transcriptase (APE-1/Ref-1 + reverse transcriptase); lane 3, PCR product from complementary DNA sample prepared without reverse transcriptase (no-reverse transcriptase control). A 169-bp APE-1/Ref-1 PCR product was successfully amplified from the captured epithelial cells, but not from the no-reverse transcriptase control, indicating that the APE-1/Ref-1 gene is expressed in normal human gastric epithelium.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 3
H. pylori and H2O2 increase constitutive APE-1/Ref-1 protein expression in gastric epithelial cells. Freshly isolated gastric epithelial cells from uninfected human subjects (A) or Kato III cells (B) were stimulated with 300:1 H. pylori or 200 μmol/L H2O2 for 3 hours, and APE-1/Ref-1 expression was assayed by Western blotting. Untreated cells served as controls. (A) Increased APE-1/Ref-1 expression was observed in native gastric epithelial cells after infection with H. pylori or stimulation with H2O2 compared with untreated control cells (*P < 0.05). The graph depicts the densitometric analysis values of immunoblots from 3 separate experiments expressed as percentage increase over untreated control cells, shown as mean ± SEM. Similar results were observed with Kato III cells (B) and AGS and N87 cells (data not shown). A representative Western blot with Kato III cells is shown, and the graph depicts the densitometric analysis values of immunoblots from 3 separate experiments, expressed as percentage increase over control cells, shown as mean ± SEM. Infection with H. pylori or stimulation with H2O2 significantly enhanced the expression of APE-1/Ref-1 in Kato III cells by 2–3-fold (*P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 4
C. jejuni does not increase constitutive APE-1/Ref-1 protein expression in gastric epithelial cells. AGS cells were stimulated with 300:1 C. jejuni for 3 hours, and APE-1/Ref-1 expression was assayed by Western blotting. Untreated cells served as controls. No increase of APE-1/Ref-1 expression was observed in AGS cells after infection with C. jejuni compared with untreated control cells. The graph depicts the densitometric analysis values of immunoblots from 4 separate experiments, expressed as percentage increase over untreated control cells, shown as mean ± SEM.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 5
New protein synthesis contributes to H. pylori- and H2O2-induced APE-1/Ref-1. Immunoprecipitated nuclear extracts from N87 cells stimulated with H. pylori or H2O2 for 3 hours during a pulse-chase experiment were analyzed by SDS-polyacrylamide gel electrophoresis. A representative autoradiograph is shown, and the graph depicts the density of the [35S]-labeled APE-1/Ref-1 immunoprecipitated bands, expressed as percentage increase over untreated N87 cells (mean ± SEM of 4 separate experiments). Stimulation with H. pylori or H2O2 significantly increased newly synthesized APE-1/Ref-1 protein compared with untreated cells (*P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 6
H. pylori (Hp)-induced APE-1/Ref-1 up-regulation is independent of the cag PAI. A representative Western blot is shown of N87 cells infected for 3 hours with 300:1 Hp (cag PAI positive), Hp 8-1 (isogenic mutant; cag PAI negative), Hp LC-11 (cag PAI positive), or Hp AH 244 (#cagA and cagE deficient but full cag PAI status not established). All 4 strains of Hp increased APE-1/Ref-1 expression in N87 cells and in AGS cells (data not shown), indicating that the cag PAI is not required for the Hp-induced increase of APE-1/Ref-1 protein expression in gastric epithelial cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 7
H. pylori and ROS mediate an accumulation of APE-1/Ref-1 in the nucleus. Kato III cells were stimulated with 300:1 H. pylori or 200 μmol/L H2O2 for 15, 30, or 60 minutes. Nuclear proteins were extracted and assayed for APE-1/Ref-1 expression by Western blot. Low-level APE-1/Ref-1 expression was detected in the nucleus of untreated cells. (A) Infection with H. pylori resulted in a time-dependent accumulation of APE-1/Ref-1 in the nucleus of the cells, with maximum levels detected 1 hour after infection. (B) Treatment with H2O2 induced a rapid nuclear accumulation of APE-1/Ref-1, with maximum levels detected within 15 minutes. Representative Western blots are depicted.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

9. Figure 8
Cellular distribution of APE-1/Ref-1. (A) Immunofluorescence staining of untreated AGS cells or AGS cells infected with 300:1 Helicobacter pylori or 200 μmol/L H2O2 for 1 hour. Under basal conditions, APE-1/Ref-1 was distributed in both the nucleus and the cytoplasm of AGS cells. After stimulation with H. pylori or H2O2, APE-1/Ref-1 showed a more predominant nuclear distribution. (B) Representative confocal microscopy images of untreated AGS cells or AGS cells infected with 300:1 H. pylori or 200 μmol/L H2O2 for 15 minutes. Under basal conditions, APE-1/Ref-1 (green) was predominantly located in the cytoplasm of AGS cells, with the nucleus stained red. After treatment with H. pylori or H2O2, APE-1/Ref-1 was rapidly translocated to the nucleus, where it appears yellow.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

10. Figure 9
NAC inhibits Helicobacter pylori- and ROS-induced APE-1/Ref-1 expression in cultured and freshly isolated human gastric epithelial cells. N87 cells (A) or freshly isolated gastric epithelial cells from uninfected subjects (B) were pretreated with 10 mmol/L NAC for 30 minutes before stimulation with 300:1 H. pylori or 200 μmol/L H2O2 for 3 hours. Whole cell lysates were extracted and assayed for APE-1/Ref-1 expression by Western blot. In both N87 (A) and native gastric epithelial cells (B), infection with H. pylori or stimulation with H2O2 significantly enhanced APE-1/Ref-1 expression compared with untreated control cells (*P < 0.05). Pretreatment with NAC before stimulation significantly decreased H. pylori- or H2O2-induced APE-1/Ref-1 up-regulation almost to basal levels. The graphs depict the densitometric analysis values of immunoblots from 4 (A) or 3 (B) separate experiments, expressed as percentage increase over untreated control cells, shown as mean ± SEM. NAC had no effect on basal levels of APE-1/Ref-1 in either N87 or native gastric epithelial cells. Results similar to those in N87 cells were shown in AGS and Kato III cells.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

11. Figure 10
APE-1/Ref-1 overexpression increases AP-1 activity. AGS cells were transfected with 50 ng of (AP-1)3p54IL-8Luc, with or without co-transfection of 50 ng of pFLAG-APE-1 cDNA3.1. Subsequently, cells were infected with Helicobacter pylori at a 1:300 ratio of cells to bacteria, challenged with 200 μmol/L of H2O2, or left untreated (control) before harvesting 8 hours later. Luciferase activity was determined and is expressed as light units per milligram of protein for each group of cells (mean ± SEM; n = 3). For all treatment groups, AP-1 luciferase activity was significantly increased in cells in which APE-1/Ref-1 was overexpressed compared with cells with constitutive levels of APE-1/Ref-1 (*P < 0.05).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions