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GASTROENTEROLOGY 2004;127:845–858

elicobacter pylori and H2O2 Increase AP Endonuclease-1/edox Factor-1 Expression in Human Gastric Epithelial Cells

ONG–ZE DING,* ANN M. O’HARA,‡ TIM L. DENNING,§,� BERNADETTE DIRDEN–KRAMER,*ANDY C. MIFFLIN,* VICTOR E. REYES,§,� KIERAN A. RYAN,‡ SUSAN N. ELLIOTT,‡

ADAHIDE IZUMI,¶ ISTVAN BOLDOGH,¶ SANKAR MITRA,¶ PETER B. ERNST,‡ andHEILA E. CROWE‡

Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas; ‡Department of Internal Medicine, Digestive Healthenter of Excellence, University of Virginia, Charlottesville, Virginia; and Departments of §Microbiology and Immunology, �Pediatrics, andHuman Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas

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ackground & Aims: Helicobacter pylori infection causesnflammation, accumulation of reactive oxygen species,nd oxidative DNA damage in the gastric mucosa.purinic/apyrimidinic endonuclease-1 (APE-1)/redox fac-or-1 (Ref-1) repairs damaged DNA and reductively acti-ates transcription factors, including activator protein-1.onsidering that H. pylori generate reactive oxygen speciesnd that reactive oxygen species modulate APE-1/Ref-1 inther cell types, we examined the effect of H. pylori, oxi-ative stress, and antioxidants on APE-1/Ref-1 expressionn human gastric epithelial cells. Methods: Human gastricpithelial cell lines or cells isolated from mucosal biopsyamples were stimulated with H. pylori, Campylobacterejuni, and/or H2O2 in the presence or absence of antioxi-ants. APE-1/Ref-1 expression was assayed by Westernlot or reverse-transcription polymerase chain reaction,nd its cellular distribution was determined by using indi-ect conventional and confocal immunofluorescence. Newrotein synthesis was detected by [S35]methionine label-ng. APE-1/Ref-1 function was assessed by using a lucif-rase-linked reporter construct containing 3 activator pro-ein 1 binding sites. Results: APE-1/Ref-1 protein andessenger RNA were detected in resting gastric epithelialells. APE-1/Ref-1 protein expression was increased aftertimulation with H2O2 or live cag pathogenicity island–earingH. pylori, but not cag pathogenicity island–negative. pylori or C. jejuni. H. pylori– or reactive oxygen species–ediated increases in APE-1/Ref-1 expression involved deovo protein synthesis that was inhibited by antioxidants.. pylori or H2O2 also induced nuclear accumulation ofPE-1/Ref-1, and overexpression of APE-1/Ref-1 in-reased activator protein 1 binding activity. Conclusions:he data show that H. pylori or reactive oxygen speciesnhance APE-1/Ref-1 protein synthesis and nuclear accu-ulation in human gastric epithelial cells and implicatePE-1/Ref-1 in the modulation of the pathogenesis of H.ylori infection.

he gram-negative flagellated bacterium Helicobacterpylori colonizes the human gastric mucosa. Infection

ith H. pylori leads to diverse clinical and pathologicutcomes, including chronic superficial gastritis, duode-al or gastric ulceration, and gastric adenocarcinoma.1–3

ore severe disease manifestations are attributed to in-ection with H. pylori strains that encode a cluster of cagenes within a region referred to as the cag pathogenicitysland (PAI).2 Studies show that H. pylori infection in-erferes with the equilibrium between proliferation andpoptosis in the gastric epithelium and that the numberf apoptotic epithelial cells increases during infection.4–7

nfection is characterized by an infiltration of polymor-honuclear and mononuclear cells within the gastricucosa.8,9 These immune cells produce reactive oxygen

pecies (ROS), and H. pylori itself also generates ROS10;his leads to an accumulation of ROS in gastric epithelialells in vitro.11,12 Together, these events result in thencreased levels of ROS that have been measured in theastric mucosa of H. pylori–infected patients.13,14

An excess of ROS in vivo can alter intracellular reduc-ion/oxidation (redox) homeostasis. ROS can react ad-ersely with biological components of the adjacent gas-ric epithelial cells and induce oxidative DNA damage inhe gastric mucosa.15,16 Oxidative DNA damage mayesult in apurinic/apyrimidinic lesions of DNA that canlock DNA replication and that are cytotoxic and mu-

Abbreviations used in this paper: AP, activator protein; APE-1/Ref-1,purinic/apyrimidinic endonuclease-1/redox factor-1; BHA, butylatedydroxyanisole; DTT, dithiothreitol; FCS, fetal calf serum; GSH, gluta-hione; HBSS, Hank’s balanced salt solution; NAC, N-acetylcysteine;F, nuclear factor; PAI, pathogenicity island; PCR, polymerase chaineaction; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate.

© 2004 by the American Gastroenterological Association0016-5085/04/$30.00

doi:10.1053/j.gastro.2004.06.017

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846 DING ET AL. GASTROENTEROLOGY Vol. 127, No. 3

agenic.17 Apurinic/apyrimidinic endonuclease-1/redoxactor-1 (APE-1/Ref-1) is a ubiquitous multifunctionalrotein involved in the base excision repair of oxidativeNA damage.18 In addition, APE-1/Ref-1 has been im-licated in the redox regulation of transcription factorNA binding activity via the reduction of a conserved

ysteine residue in the DNA binding domain of severalranscription factors. These include the activator proteinAP)-1 proteins, Fos and Jun, nuclear factor (NF)-�B,arly growth response-1, Myb, p53, and others (reviewedy Evans et al.19). Furthermore, APE-1/Ref-1 acts as aegative regulator of its own gene.20 Homozygous dele-ion of the APE-1/Ref-1 gene is embryonically lethal,21

hus highlighting the essential role of APE-1/Ref-1 inellular homeostasis. APE-1/Ref-1 shows a complex andeterogeneous localization pattern that differs among cellypes.22 Physically separate domains of the protein arenvolved in its DNA repair and redox regulatory activi-ies.23 The dual activities of APE-1/Ref-1 suggest thathe repair of ROS-induced DNA lesions and the regula-ion of a transcriptional response to an oxidizing envi-onment are tightly coupled.

Several studies have shown that ROS, including H2O2,ediate and enhance APE-1/Ref-1 expression and activity

n fibroblasts, HeLa, macrophages, B cells, and other cellypes.24–28 Furthermore, increased APE-1/Ref-1 activityorrelates with decreased DNA damage induced by oxi-ants or other DNA-damaging agents,29–31 and APE-1/ef-1 expression levels are inversely related to apoptosis inany cell types.32,33 However, the expression of APE-1/ef-1 in gastric epithelial cells has not previously been

tudied. Although studies show that H. pylori infectionctivates transcription factors NF-�B and AP-1,34,35 whichre known to be regulated by APE-1/Ref-1, and that oxi-ants also activate NF-�B,36 the effect of H. pylori or anyther infection on APE-1/Ref-1 expression has not beeneported. In this study, we investigated whether H. pylori orOS affect APE-1/Ref-1 expression in human gastric epi-

helial cells. Our data indicate that APE-1/Ref-1 is consti-utively expressed by human gastric epithelial cells, withnhanced expression and nuclear translocation after infec-ion with H. pylori or stimulation with ROS, and that thisffect is inhibited by antioxidants. Moreover, our resultshow that APE-1/Ref-1 plays a functional role in humanastric epithelial cells by enhancing AP-1–mediated tran-cription.

Materials and Methods

Gastric Epithelial Cell Lines

The human gastric epithelial cell lines Kato III, NCI-87 (N87), and AGS were obtained from the American Type

ulture Collection (Rockville, MD). Kato III and N87 cellines were cultured in RPMI 1640 (GIBCO-BRL, Grand Is-and, NY) supplemented with 10% heat-inactivated fetal calferum (FCS; GIBCO-BRL), whereas the AGS cells were grownn Hams F12 (GIBCO-BRL) supplemented with 10% FCS.ll cells were grown in a monolayer in 25- or 75-cm2 tissue

ulture flasks at 37°C in a humidified, 5% carbon dioxidencubator until they approached approximately 80%–90%onfluency.

Human Gastric Epithelial Cell IsolationFrom Mucosal Biopsy Samples

Gastric biopsy specimens were collected from consent-ng adults undergoing gastroesophageal duodenoscopy for var-ous clinical indications, according to a University of Texas

edical Branch Institutional Review Board–approved proto-ol. Patients were considered infected if H. pylori were detectedy rapid urease testing or by histopathology of biopsy samples.iopsy specimens (4–6 pinches) from the antral gastric mu-osa of infected or uninfected individuals were collected in coldalcium- and magnesium-free Hank’s balanced salt solutionHBSS; GIBCO-BRL) supplemented with 5% FCS, 100 U/mLenicillin (Sigma Chemical Company, St. Louis, MO), and 100g/mL streptomycin (Sigma). Epithelial cells were isolated

rom the specimens as described previously.7,37,38 Briefly,insed tissue was agitated at 37°C in HBSS containing 0.1mol/L dithiothreitol (DTT; Sigma) and 0.1 mmol/L ethyl-

nediaminetetraacetic acid (EDTA; Sigma) for 15 minutes.he supernatant was decanted, replaced with fresh HBSS/DTA/DTT, and incubated for another 15 minutes. Subse-uently, the biopsy tissue was placed in a 2.4 U/mL dispaseolution (Boehringer Mannheim, Mannheim, Germany), agi-ated at 37°C for 15 minutes, and then centrifuged at 200g for

minutes. Fresh dispase solution was added, and the proce-ure was repeated. The isolated cells were confirmed to be90% epithelial cells according to morphology with May–runwald–Giemsa (Sigma) staining and immunofluorescence

taining of epithelial anticytokeratin antigen, as describedreviously.38 The viability of the isolated cells was determinedy trypan blue exclusion, and cells were not used unlessiability exceeded 90%. The freshly isolated gastric epithelialells were maintained in RPMI 1640 containing 10% FCS andere used immediately in assays as described below.

Bacteria

H. pylori 26695, a cag PAI–bearing strain,39 and thesogenic cag PAI–deficient knockout strain, H. pylori 8-1, wererovided by D. Berg (Washington University School of Med-cine, St. Louis, MO). H. pylori LC-11, a cag PAI–bearingtrain isolated from a clinical specimen,40 was provided by P.herman (University of Toronto, Toronto, Ontario, Canada).. pylori AH 244, a naturally occurring cagA- and cagE-eficient strain, was obtained from R. Alms (AstraZeneca,oston, MA). A nongastric mucosal pathogen, Campylobacter

ejuni, was provided by R. Guerrant (University of Virginia,harlottesville, VA). All strains of bacteria were maintained on

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September 2004 H. PYLORI AND ROS INCREASE APE–1/REF–1 EXPRESSION 847

lood agar plates (Becton Dickinson, Cockeysville, MD). Be-ore use in experiments, the bacteria were cultured overnight at7°C in 25 mL of brucella broth (GIBCO-BRL) supplementedith 10% FCS under microaerophilic conditions. Subse-uently, the bacteria were harvested, and the bacterial numberas determined as described previously.7 Before use, bacterialobility was confirmed by phase-contrast microscopy.

Stimulation of Gastric Epithelial Cells

For all assays, gastric epithelial cell viability was de-ermined by trypan blue exclusion, and a known number ofGS (5 � 105), N87 (1 � 106), or Kato III (5 � 105) cellsere seeded into 9.5-cm2 6-well plates. Freshly isolated gastric

pithelial cells (1–2 � 105) were seeded into 1.9-cm2 24-welllates. The gastric epithelial cell lines were incubated for 24ours before stimulation, whereas the native gastric epithelialells were used immediately after isolation. To study thenduction of APE-1/Ref-1 expression, dose–response studiesere performed to determine an optimal bacterial/epithelial

ell ratio and the optimal concentrations of H2O2 (Sigma) tose for stimulation of gastric epithelial cells. Dose–responsetudies showed that infection with H. pylori 26695 at ratios of0:1 to 1000:1 or stimulation with 200–400 �mol/L H2O2

ignificantly enhanced APE-1/Ref-1 expression in gastric epi-helial cell lines compared with control cells. Increased levelsf APE-1/Ref-1 were significantly and consistently detectedith 200 �mol/L H2O2 and a bacterial/epithelial cell ratio of00:1, a dose we have used in previous studies.7 Stimulationith H. pylori or H2O2 at these doses resulted in a time-ependent up-regulation of APE-1/Ref-1 expression, withaximal levels detected after 3–6 hours. Consequently, in

ubsequent assays, epithelial cells were stimulated for 3 hoursith 300:1 H. pylori or 200 �mol/L H2O2 to observe their

ffect on APE-1/Ref-1 expression. In some assays, epithelialells were co-stimulated with 300:1 H. pylori and 200 �mol/L

2O2 to determine whether H. pylori and H2O2 together exertsynergistic effect on APE-1/Ref-1 expression. The effect of

ntioxidants on H. pylori– or ROS-induced APE-1/Ref-1 ex-ression was examined by preincubation of the epithelial cellsith 10 mmol/L N-acetylcysteine (NAC; Sigma), 10 mmol/Llutathione (GSH; Sigma), or 100 �mol/L butylated hydroxy-nisole (BHA) for 30 minutes before stimulation. These con-entrations were based on our observations in dose–responsetudies, and similar concentrations have been used in othertudies of oxidative injury.41,42 To evaluate whether live H.ylori were necessary to up-regulate APE-1/Ref-1 expression,ormalin-killed H. pylori, prepared as previously described,43

ere used to infect gastric epithelial cells at a ratio of 300:1.o compare the effect of another human gut mucosal patho-en, C. jejuni, a nongastric gram-negative pathogen was usedo infect gastric epithelial cells, also at a ratio of 300:1.

Extraction of Whole Cell Lysates andNuclear Protein

To examine APE-1/Ref-1 expression, whole cell lysatesf untreated or stimulated gastric epithelial cells were prepared

s previously described.44 Briefly, cells were lysed in cold lysisuffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% Non-det P-40 [Sigma], 50 mmol/L NaF, 1 mmol/L NaVO3, 1mol/L phenylmethylsulfonyl fluoride, 25 �g/mL aprotinin,

5 �g/mL pepstatin A, and 25 �g/mL leupeptin) for 5 min-tes on ice and centrifuged at 14,000 � g for 5 minutes at°C. The protein concentration of the resulting lysates wasetermined by Bradford assay (Bio-Rad, Hercules, CA), andhe samples were stored at �80°C.

To determine whether APE-1/Ref-1 was expressed in celluclei, nuclear proteins were extracted from H. pylori– or

2O2-treated N87 (1 � 107), Kato III (5 � 106), or AGS (5106) cells. At different time points, the cells were harvested

nd were washed once with cold phosphate-buffered salinePBS). Subsequently, nuclear pellets were prepared by resus-ending cells in 400 �L of hypotonic lysis buffer [10 mmol/LEPES, 10 mmol/L KCl, 1 mmol/L DTT, 0.1 mmol/L EDTA,

.1 mmol/L ethylene glycol-bis(�-aminoethyl ether)-N,N,=,N=-tetraacetic acid, and 1% protein inhibitor cocktail;

igma] at 4°C for 30 minutes. Then 25 �L of 10% Nonidet-40 was added, and the suspension was mixed vigorously.fter a 30-second centrifugation (14,000 � g; 4°C), theelleted nuclei were resuspended in 100 �L of extractionuffer [20 mmol/L HEPES, 0.4 mol/L KCl, 1 mmol/L DTT,.1 mmol/L EDTA, 0.1 mmol/L ethylene glycol-bis(�-amino-thyl ether)-N,N, N=,N=-tetraacetic acid, and 1% protein in-ibitor cocktail] and incubated on ice for 20 minutes. Therotein concentration of the extracts was determined by Brad-ord assay, and the nuclear extracts were stored at �80°C untilssayed.

Western Blot Analysis

Immunoblotting was performed on whole cell lysatesr nuclear proteins as previously described.44 Protein samples10 �g) were fractionated by sodium dodecyl sulfate (SDS)-olyacrylamide gel electrophoresis (12%) in a discontinuousuffer system. After electrophoresis, the separated proteinsere transferred onto nitrocellulose membranes with therans-blot cell (Bio-Rad). Immunodetection analysis was per-

ormed by incubating the membrane with a specific rabbitnti-human APE-1/Ref-1 polyclonal antibody24 at a 1:2000ilution, followed by goat anti-rabbit immunoglobulin Gorseradish peroxidase conjugate (Santa Cruz Biotechnology,anta Cruz, CA) as a second antibody. Immunoreactions wereisualized by enhanced chemiluminescence (Amersham Bio-ciences, Buckinghamshire, UK) and autoradiographed. Quan-itative changes were determined by densitometric analysis ofhe immunoreactive band by using an Alphaimager image-nalysis system (Alpha Innotech, San Leandro, CA). Proteinoading was normalized by stripping the membrane withtripping buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS,nd 100 mmol/L �-mercaptoethanol) for 30 minutes at 56°C,ollowed by reprobing with mouse anti-human �-actin anti-ody (Sigma) and goat anti-mouse immunoglobulin G horse-adish peroxidase conjugate (Cell Signaling Technology, Bev-rly, MA), as described previously.

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848 DING ET AL. GASTROENTEROLOGY Vol. 127, No. 3

Isolation of Native Gastric Epithelial Cellsby Using Laser Capture Microdissection

To determine whether APE-1/Ref-1 is expressed initu in the human gastric mucosa, gastric biopsy samplesere taken from an H. pylori–negative subject undergoingedically indicated endoscopy at the Digestive Healthenter of Excellence, University of Virginia, in accordanceith an institutional review board–approved protocol. Tis-

ue was snap-frozen in OCT compound (Sakura Finetechni-al Co., Ltd., Tokyo, Japan) and stored at �80°C untilrocessing. Sections (10 �m) were prepared with a cryostatnd mounted on a clean plain uncoated slide, counterstainedith H&E (Fisher Scientific, Pittsburgh, PA), dried, and

tored under desiccation and vacuum. Epithelial cells wereicrodissected onto CapSure HS LCM transfer film caps

Arcturus Engineering Inc., Mountain View, CA) by usinghe PixCell Laser capture microdissection apparatus (Arc-urus Engineering Inc.). RNA was extracted from the cellsaptured on the transfer film caps by using the PicoPureNA Isolation kit (Arcturus Engineering Inc.), according

o the manufacturer’s instructions. Subsequently, comple-entary DNA was prepared by using the SuperScript First-

trand synthesis system protocol for reverse transcriptionInvitrogen, Carlsbad, CA). To test for DNA contamina-ion, a negative control was prepared for each sample by themission of reverse transcriptase from the reaction mixtureno–reverse transcriptase control). Polymerase chain reac-ion (PCR) for human APE-1/Ref-1 was performed in aNA Engine thermocycler (MJ Research, Watertown, MA)

y using an APE-1/Ref-1 primer set designed in our laboratory:=-TGGATTGTGGATGGG-CTTCGAGCC-3= (forward) and=-AAGGAGCTGAC-CAGTATTGATGA-3= (reverse). TheCR reaction mixture comprised 0.3 �mol/L of each primer, 3mol/L MgCl2, 1� PCR reaction buffer, 200 nmol/L PCR

ucleotide mix, and 0.75 U of Platinum Taq DNA polymeraseInvitrogen). PCR cycling conditions consisted of 95°C for 5inutes, followed by 40 cycles of 95°C for 1 minute, 60°C for 1inute, and 72°C for 1 minute. Subsequently, the PCR productsere analyzed by 2% agarose gel electrophoresis.

Metabolic Pulse-Chase Labeling,Immunoprecipitation, and Electrophoresis

N87 cells (1 � 107) were incubated in methionine-nd cysteine-free RPMI 1640 medium for 1 hour and thenere metabolically radiolabeled with [35S]methionine (ICN,

rvine, CA) and cysteine (0.18 mCi/107 cells; ICN). Subse-uently, cells were stimulated with 300:1 H. pylori or 200mol/L H2O2 for 3 hours. In some experiments, 10 mmol/LAC was added 30 minutes before infection with H. pylori or

2O2. After stimulation, the cells were washed and lysed asreviously described.38 Solubilized membrane samples wererecleared with normal rabbit serum and formalinized Staph-lococcus aureus Cowen strain A (Chemicon, El Segundo, CA).mmunoadsorbents were subsequently prepared as describedreviously.38 After immunoprecipitation, the eluted samples

ere analyzed on 12% polyacrylamide gels by 1-dimensionalDS-polyacrylamide gel electrophoresis. Then, the separatedamples were transferred onto nitrocellulose membranes asescribed previously and were autoradiographed.

Immunofluorescence Staining

AGS cells (0.5 � 104) were stimulated with H. pylori6695 or 200 �mol/L H2O2 for 1 hour. Subsequently, the cellsere fixed onto ethanol-cleaned slides with cold methanol for 5inutes at �20°C, air-dried for 5 minutes, and washed 3 timesith 5% FCS/PBS solution. After washing, the samples were

ncubated with a 1:100 dilution of rabbit anti-human APE-1/ef-1 polyclonal antibody24 for 1 hour at room temperature in aoist chamber, washed thrice with 5% FCS/PBS, and incubated

or a further 45 minutes with a 1:100 dilution of fluoresceinsothiocyanate–conjugated goat anti-rabbit antibody (Santa Cruziotechnology) as outlined previously.45 As a negative control foronspecific staining, some samples were incubated with an equalmount of normal rabbit serum in the absence of primary anti-ody. Immunofluorescence-labeled samples were visualized byV microscopy (Nikon, Melville, NY).For confocal microscopy studies, AGS cells (1 � 105) were

eeded on glass coverslips, treated, fixed, and stained withnti–APE-1/Ref-1 polyclonal antibody as described previ-usly. Subsequently, the cells were incubated with a 1:400ilution of Alexa Fluor 594– conjugated goat anti-rabbit an-ibody (Molecular Probes, Eugene, OR) for 2 hours at roomemperature in a moist chamber. Nuclei were counterstainedith 5 mmol/L SYTOX green (Molecular Probes) for 10inutes. After washing with PBS, slides were mounted withector-H1000 media (Vector Laboratories, Burlingame, CA).he fluorescence was studied with a Noran OZ Intervisionideo-rate laser scanning confocal microscope (Noran Instru-ents, Middleton, WI) equipped with an argon laser and aikon 60� oil immersion lens. The excitation wavelengths

sed were 594 nm for Alexa Fluor and 488 nm for SYTOXreen. Colocalization analysis was performed with Metamorph4.67 software (Universal Imaging Corporation, Downing-

own, PA) by using recorded digital data of 2-color fluores-ence from red and green. The red and green images wereigitally reversed; thus, stained APE-1/Ref-1 appeared green,nd stained nuclei were visualized as red. Subsequently, themages were merged, and colocalization appears as yellowecause of the mixture of green and red.

Overexpression of APE-1/Ref-1 in AGS Cells

For APE-1/Ref-1 overexpression, an APE-1/Ref-1 expres-ion vector, FLAG-APE-1 cDNA3.1, was prepared by insertingn EcoRI/BamHI fragment containing the human APE-1/Ref-1pen reading frame with an N-terminal FLAG epitope tag intohe EcoRI/BamHI site of pcDNA3.1Zeo(�) (Invitrogen), thuslacing APE-1/Ref-1 under the control of the cytomegalovirusmmediate early promoter. The AP-1 reporter plasmid used,AP-1)3p54IL-8Luc, contains 3 copies of a consensus AP-1 bind-ng site linked to the basal promoter elements (�54 nucleotides)

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f the human interleukin-8 gene driving expression of a fireflyuciferase reporter gene, as described by Casola et al.46

For transient transfections, AGS cells (1 � 105) were seeded in2-well plates and were incubated in Hams F12 medium with0% fetal bovine serum for 24 hours. Cells were transientlyransfected with 50 ng of the luciferase reporter plasmid, with orithout co-transfection of the APE-1/Ref-1 expression plasmid

50 ng), by using the heat-inactivated, psoralen cross-linked ad-noviral delivery system described by Allgood et al.47 (purchasedrom the Department of Cell Biology, Baylor College of Medicine,ouston, TX) with a virus/cell ratio of 250:1. A promoterlessL3-Basic plasmid (Promega, Madison, WI) was also used toetermine background expression levels. After 24 hours, theransfection reagent was removed, and cells were incubated for aurther 48 hours in 2 mL of Hams F12 with 0.2% heat-inacti-ated fetal bovine serum to minimize the AP-1 activity. Subse-uently, the cells were either stimulated with H. pylori 26695 at1:300 cell/bacteria ratio or challenged with 200 �mol/L of

2O2 in serum-free media for 8 hours and then were lysed bysing Luciferase Reporter Lysis buffer (Promega) according to theanufacturer’s instructions. Luciferase activity in the extracts was

etermined by using a commercially available luciferase assay kitPromega) in a Monolight luminometer 2010 (Analytical Lumi-escence Laboratory, San Diego, CA). The total protein concen-ration in each extract was determined by using the Bradford assays described previously. Luciferase activity was normalized to therotein level and expressed as luciferase activity per milligram ofrotein.

Statistical Analysis

All quantitative data are expressed as mean � SEM. Dataere compared by using paired or unpaired Student t tests, andifferences were considered significant if P values were �0.05.

Results

APE-1/Ref-1 Is Expressed by Native HumanGastric Epithelial Cells and Increased byH. pylori Infection In Vivo

To examine the effect of chronic H. pylori infec-ion on in vivo expression of APE-1/Ref-1 in the humanastric mucosa, epithelial cell preparations from gastricucosal biopsy samples from H. pylori–infected anduninfected subjects were examined for APE-1/Ref-1xpression by Western blotting. As shown in Figure 1,PE-1/Ref-1, a 37-kilodalton protein, is constitutively

xpressed by gastric epithelial cells from H. pylori–nega-ive human subjects (n 3). However, the expression ofPE-1/Ref-1 in epithelial cells from H. pylori–infected

ubjects (n 3) was 4–5-fold higher than in cells fromninfected individuals (P � 0.05; Figure 1).To confirm the constitutive expression of APE-1/Ref-1

n the normal gastric epithelium, epithelial cells werexcised from an H. pylori–negative human gastric muco-

al biopsy specimen by using the laser capture microdis-ection technique (Figure 2A–C). After RNA extractionnd reverse transcription, a 169–base pair APE-1/Ref-1CR product was successfully amplified from the cap-ured epithelial cells (Figure 2D). This shows for the firstime that the APE-1/Ref-1 gene is expressed in situ inhe normal gastric epithelium.

H. pylori or Reactive Oxygen SpeciesIncrease Constitutive APE-1/Ref-1Expression in Freshly Isolated and CulturedGastric Epithelial Cells In Vitro

Western blot analysis of whole cell lysates ofpithelial cells freshly isolated from uninfected humanubjects showed basal expression of APE-1/Ref-1 proteinhat was increased 2–3-fold (P � 0.05 compared withasal) after 3 hours of treatment with 300:1 H. pylori6695 or 200 �mol/L H2O2. Figure 3A depicts densi-ometric analysis of immunoblots from 3 separate exper-ments. The 3 gastric epithelial cell lines used in thistudy also expressed APE-1/Ref-1 constitutively. Infec-

igure 1. APE-1/Ref-1 protein expression is increased in gastric ep-thelial cells from H. pylori–infected human subjects. Epithelial cellreparations from gastric antral biopsy samples from H. pylori–in-ected (n 3) and –uninfected (n 3) human subjects were exam-ned for APE-1/Ref-1 expression by Western blotting. A representativeestern blot showing APE-1/Ref-1, a 37-kilodalton protein, is shown.

he graph depicts the band density of samples from each subjectroup as mean � SEM. APE-1/Ref-1 protein is expressed constitu-ively by human gastric epithelial cells and is increased by chronic H.ylori infection in vivo (*P � 0.05).

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850 DING ET AL. GASTROENTEROLOGY Vol. 127, No. 3

ion with 300:1 H. pylori 26695 or stimulation with 200mol/L H2O2 for 3 hours significantly (2–3-fold) en-anced the expression of APE-1/Ref-1 in Kato III, AGS,nd N87 cells compared with untreated control cells (P

0.05). A representative Western blot of Kato III cellss shown in Figure 3B, and the graph depicts densito-

etric analysis of immunoblots from 3 separate experi-ents. The similar results in both native epithelium and

ultured gastric epithelial cell lines suggest that thebserved findings are not an artifact of using cancer-erived cell lines. Treatment of gastric epithelial cellsith both H. pylori and H2O2 did not result in augmen-

ation of APE-1/Ref-1 protein expression over levelsnduced by either stimulus alone (n 5; data nothown). The nongastric pathogen C. jejuni did not in-rease APE-1/Ref-1 protein expression over levels inninfected control cells (Figure 4).

New Protein Synthesis Contributes toH. pylori and H2O2-Induced APE-1/Ref-1

By using pulse-chase experiments, [35S]methi-nine-labeled N87 cells treated with H. pylori or H O

igure 2. APE-1/Ref-1 is expressed in situ by native human gastric eylori–negative human gastric antral mucosal biopsy specimen by uastric mucosa from an H. pylori–negative subject before laser capapSure HS LCM transfer film caps. (C) Section of remaining mucosa.

rom the microdissected sample with APE-1/Ref-1–specific primers.repared from a complementary DNA sample with reverse transcripomplementary DNA sample prepared without reverse transcriptase (uccessfully amplified from the captured epithelial cells, but not froms expressed in normal human gastric epithelium.

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or 3 hours were used to determine whether the observedncreases in APE-1/Ref-1 expression after stimulationere due to new protein synthesis. Nuclear extracts were

mmunoprecipitated and analyzed by Western blotting.s shown in Figure 5, stimulation with H. pylori 26695 or2O2 increased APE-1/Ref-1 expression in N87 cells by

-fold compared with untreated N87 cells (P � 0.05). Thishows that H. pylori– or ROS-mediated increases in APE-/Ref-1 expression involve de novo protein synthesis.

H. pylori–Induced APE-1/Ref-1Up-regulation Requires Live Bacteriabut Is Independent of the cagPathogenicity Island

Considering that infection with cag PAI–bearing. pylori, particularly cagA-positive strains, is associatedith more severe disease manifestations,2 we sought toetermine whether H. pylori–induced up-regulation ofPE-1/Ref-1 was dependent on the presence of the cagAI. AGS and N87 gastric epithelial cells were stimu-

ated with different strains of H. pylori for 3 hours, andhe lysates were examined for APE-1/Ref-1 expression by

lial cells. Gastric epithelial cells were specifically excised from an H.the laser capture microdissection technique. (A) Section of humanmicrodissection. (B) Microdissected epithelial cells captured ontogarose gel electrophoresis of PCR product from complementary DNA1, 50–base pair (bp) molecular weight ladder; lane 2, PCR product(APE-1/Ref-1 reverse transcriptase); lane 3, PCR product from

verse transcriptase control). A 169-bp APE-1/Ref-1 PCR product waso–reverse transcriptase control, indicating that the APE-1/Ref-1 gene

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estern blot. H. pylori 26695 and H. pylori LC-11 are cagAI–bearing strains, whereas H. pylori 8-1 is a cag PAI–eficient isogenic mutant of strain 26695, and H. pyloriH 244 is a cagA- and cagE-deficient strain. The repre-

igure 3. H. pylori and H2O2 increase constitutive APE-1/Ref-1 pro-ein expression in gastric epithelial cells. Freshly isolated gastricpithelial cells from uninfected human subjects (A) or Kato III cells (B)ere stimulated with 300:1 H. pylori 26695 or 200 �mol/L H2O2 forhours, and APE-1/Ref-1 expression was assayed by Western blot-

ing. Untreated cells served as controls. (A) Increased APE-1/Ref-1xpression was observed in native gastric epithelial cells after infec-ion with H. pylori or stimulation with H2O2 compared with untreatedontrol cells (*P � 0.05). The graph depicts the densitometric anal-sis values of immunoblots from 3 separate experiments expresseds percentage increase over untreated control cells, shown as meanSEM. Similar results were observed with Kato III cells (B) and AGS

nd N87 cells (data not shown). A representative Western blot withato III cells is shown, and the graph depicts the densitometricnalysis values of immunoblots from 3 separate experiments, ex-ressed as percentage increase over control cells, shown as mean �EM. Infection with H. pylori 26695 or stimulation with H2O2 signifi-antly enhanced the expression of APE-1/Ref-1 in Kato III cells by–3-fold (*P � 0.05).

entative Western blot in Figure 6 shows that all 4 H.ylori strains tested increased the expression of APE-1/ef-1 in N87 cells, and similar results were obtainedith the AGS cell line (data not shown), indicating that. pylori–induced up-regulation of APE-1/Ref-1 expres-

ion is cag PAI independent. Infection of AGS cells withormalin-killed H. pylori did not significantly augmentPE-1/Ref-1 protein expression above control levels

data not shown), suggesting that this process requiresive bacteria.

H. pylori and Reactive Oxygen SpeciesMediate an Accumulation of APE-1/Ref-1in the Nucleus

Nuclear proteins extracted from gastric epithelialells stimulated with H. pylori or H2O2 for varying timesere assayed for APE-1/Ref-1 expression by Westernlot. In all 3 gastric epithelial cell lines tested, APE-1/ef-1 protein was detected in nuclear extracts underasal conditions. Infection with H. pylori 26695 or stim-lation with H2O2 resulted in a time-dependent accu-ulation of APE-1/Ref-1 in the nucleus of Kato III cells

Figure 7). Increased nuclear APE-1/Ref-1 was detectedithin 15 minutes of H. pylori infection, with maximum

evels detected 1 hour after infection (Figure 7A). Stim-lation with H2O2 rapidly induced APE-1/Ref-1 nuclear

igure 4. C. jejuni does not increase constitutive APE-1/Ref-1 proteinxpression in gastric epithelial cells. AGS cells were stimulated with00:1 C. jejuni for 3 hours, and APE-1/Ref-1 expression was assayedy Western blotting. Untreated cells served as controls. No increasef APE-1/Ref-1 expression was observed in AGS cells after infectionith C. jejuni compared with untreated control cells. The graph depicts

he densitometric analysis values of immunoblots from 4 separatexperiments, expressed as percentage increase over untreated con-rol cells, shown as mean � SEM.

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852 DING ET AL. GASTROENTEROLOGY Vol. 127, No. 3

xpression within 15 minutes, but the levels detected atater time points were not significantly different fromhose 15 minutes after stimulation (Figure 7B). Similaresults were obtained with nuclear extracts from N87nd AGS cell lines, and in data not shown, increaseduclear APE-1/Ref-1 expression was still evident 24ours after stimulation. The results indicate that H.ylori or ROS induce a rapid nuclear accumulation ofPE-1/Ref-1 in gastric epithelial cells.

Cellular Distribution of APE-1/Ref-1

Immunofluorescence studies showed that APE-1/ef-1 is distributed in both the nucleus and cytoplasm ofGS gastric epithelial cells (Figure 8A). In addition,

hese studies confirmed that infection with H. pylori ortimulation with H2O2 resulted in a time-dependentccumulation of APE-1/Ref-1 protein in the nuclei ofells. Figure 8A depicts H. pylori– or H2O2-induceduclear localization of APE-1/Ref-1 in AGS cells 1 hourfter stimulation. Confocal microscopy studies (FigureB) showed a predominant cytoplasmic distribution of

igure 5. New protein synthesis contributes to H. pylori– and H2O2-nduced APE-1/Ref-1. Immunoprecipitated nuclear extracts from N87ells stimulated with H. pylori 26695 or H2O2 for 3 hours during aulse-chase experiment were analyzed by SDS-polyacrylamide gellectrophoresis. A representative autoradiograph is shown, and theraph depicts the density of the [35S]-labeled APE-1/Ref-1 immuno-recipitated bands, expressed as percentage increase over untreated87 cells (mean � SEM of 4 separate experiments). Stimulation with. pylori or H2O2 significantly increased newly synthesized APE-1/ef-1 protein compared with untreated cells (*P � 0.05).

PE-1/Ref-1 (depicted as green) in untreated AGS cells.fter stimulation with H. pylori or H2O2 for 15 minutes,PE-1/Ref-1 was less evident in the cytoplasm, and

ncreased APE-1/Ref-1 was detected in the nucleusshown as yellow color; Figure 8B). Thus, an early con-equence of H. pylori infection or H2O2 treatment isranslocation of APE-1/Ref-1 from the cytoplasm to theucleus.

H. pylori– or H2O2-Induced APE-1/Ref-1Expression Is Inhibited by Antioxidants

NAC, GSH, and BHA, commonly used antioxi-ants, were used to assess the effect of antioxidants on H.

igure 6. H. pylori (Hp)-induced APE-1/Ref-1 up-regulation is indepen-ent of the cag PAI. A representative Western blot is shown of N87ells infected for 3 hours with 300:1 Hp 26695 (cag PAI positive), Hp-1 (isogenic mutant; cag PAI negative), Hp LC-11 (cag PAI positive),r Hp AH 244 (#cagA and cagE deficient but full cag PAI status notstablished). All 4 strains of Hp increased APE-1/Ref-1 expression in87 cells and in AGS cells (data not shown), indicating that the cagAI is not required for the Hp-induced increase of APE-1/Ref-1 proteinxpression in gastric epithelial cells.

igure 7. H. pylori and ROS mediate an accumulation of APE-1/Ref-1n the nucleus. Kato III cells were stimulated with 300:1 H. pylori6695 or 200 �mol/L H2O2 for 15, 30, or 60 minutes. Nuclearroteins were extracted and assayed for APE-1/Ref-1 expression byestern blot. Low-level APE-1/Ref-1 expression was detected in theucleus of untreated cells. (A) Infection with H. pylori 26695 resultedn a time-dependent accumulation of APE-1/Ref-1 in the nucleus ofhe cells, with maximum levels detected 1 hour after infection. (B)reatment with H2O2 induced a rapid nuclear accumulation of APE-1/ef-1, with maximum levels detected within 15 minutes. Representa-ive Western blots are depicted.

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September 2004 H. PYLORI AND ROS INCREASE APE–1/REF–1 EXPRESSION 853

ylori– or H2O2-induced APE-1/Ref-1 expression. Gas-ric epithelial cell lines were pretreated with 10 mmol/LAC, 10 mmol/L GSH, or 100 �mol/L BHA for 30inutes before stimulation with H. pylori or ROS. As

hown in Figure 9A, H. pylori– or H2O2-induced up-egulation of APE-1/Ref-1 in N87 cells was decreasedlmost to basal levels by pretreatment with NAC. Sim-lar results were detected in N87 cells after pretreatmentith GSH or BHA and also in AGS and Kato III cellsretreated with any of the 3 antioxidants. However,reatment of resting cells with these antioxidants aloneid not affect constitutive APE-1/Ref-1 expression (dataot shown).To ensure that the antioxidant effect of NAC was

ot restricted to immortal cell lines, NAC was alsoested for its effect on H. pylori– or H2O2-inducedp-regulation of APE-1/Ref-1 in native gastric epi-helial cells. Freshly isolated human gastric epithelialells from uninfected biopsy specimens were pre-reated with NAC for 30 minutes and then stimulatedith H. pylori or H2O2 for 3 hours. Western blot

nalysis indicated that infection with H. pylori 26695r H2O2 significantly enhanced APE-1/Ref-1 expres-ion by 2–3-fold compared with untreated native hu-

igure 8. Cellular distribution of APE-1/Ref-1. (A) Immunofluores-ence staining of untreated AGS cells or AGS cells infected with00:1 Helicobacter pylori 26695 or 200 �mol/L H2O2 for 1 hour.nder basal conditions, APE-1/Ref-1 was distributed in both theucleus and the cytoplasm of AGS cells. After stimulation with H.ylori or H2O2, APE-1/Ref-1 showed a more predominant nuclearistribution. (B) Representative confocal microscopy images of un-reated AGS cells or AGS cells infected with 300:1 H. pylori 26695 or00 �mol/L H2O2 for 15 minutes. Under basal conditions, APE-1/ef-1 (green) was predominantly located in the cytoplasm of AGSells, with the nucleus stained red. After treatment with H. pylori or2O2, APE-1/Ref-1 was rapidly translocated to the nucleus, where itppears yellow.

an gastric epithelial cells. However, pretreatmentith NAC reduced APE-1/Ref-1 expression to nearlyasal levels (Figure 9B). NAC was also shown tonhibit newly synthesized APE-1/Ref-1 protein in-uced by ROS or infection with H. pylori (data nothown). Together, these results show that NAC andther antioxidants inhibit H. pylori– or ROS-enhancedPE-1/Ref-1 protein expression in both native and

ultured human gastric epithelial cells.

igure 9. NAC inhibits Helicobacter pylori– and ROS-induced APE-1/ef-1 expression in cultured and freshly isolated human gastric epi-helial cells. N87 cells (A) or freshly isolated gastric epithelial cellsrom uninfected subjects (B) were pretreated with 10 mmol/L NAC for0 minutes before stimulation with 300:1 H. pylori 26695 or 200mol/L H2O2 for 3 hours. Whole cell lysates were extracted andssayed for APE-1/Ref-1 expression by Western blot. In both N87 (A)nd native gastric epithelial cells (B), infection with H. pylori or stim-lation with H2O2 significantly enhanced APE-1/Ref-1 expression com-ared with untreated control cells (*P � 0.05). Pretreatment with NACefore stimulation significantly decreased H. pylori– or H2O2-inducedPE-1/Ref-1 up-regulation almost to basal levels. The graphs depicthe densitometric analysis values of immunoblots from 4 (A) or 3 (B)eparate experiments, expressed as percentage increase over un-reated control cells, shown as mean � SEM. NAC had no effect onasal levels of APE-1/Ref-1 in either N87 or native gastric epithelialells. Results similar to those in N87 cells were shown in AGS andato III cells.

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854 DING ET AL. GASTROENTEROLOGY Vol. 127, No. 3

APE-1/Ref-1 Increases Transcription FactorActivity

The overexpression of APE-1/Ref-1 in AGS cellsransiently transfected with pFLAG-APE-1 cDNA3.1ompared with cells transfected with a promoterless con-rol vector was confirmed by Western blot (data nothown). In control AGS cells transfected with the pro-oterless GL3 vector, luciferase activity was negligible

n all samples, irrespective of co-transfection withFLAG-APE-1 cDNA3.1 or posttransfection treatmentdata not shown). In AGS cells with basal levels ofPE-1/Ref-1 transfected with the (AP-1)3p54IL-8Luclasmid, exposure to H2O2, but not H. pylori 26695,oubled the luciferase activity of the AP-1 multimerompared with unstimulated control cells (P � 0.05). Ashown in Figure 10, in cells co-transfected with pFLAG-PE-1 cDNA3.1, the overexpression of APE-1/Ref-1

ncreased luciferase activity 2–3-fold in all treatmentroups compared with cells with constitutive levels ofPE-1/Ref-1 (P � 0.05). These results show that inuman gastric epithelial cells, APE-1/Ref-1 plays a func-ional role in the activation of the transcription factor

igure 10. APE-1/Ref-1 overexpression increases AP-1 activity. AGSells were transfected with 50 ng of (AP-1)3p54IL-8Luc, with or withouto-transfection of 50 ng of pFLAG-APE-1 cDNA3.1. Subsequently, cellsere infected with Helicobacter pylori 26695 at a 1:300 ratio of cells

o bacteria, challenged with 200 �mol/L of H2O2, or left untreatedcontrol) before harvesting 8 hours later. Luciferase activity was de-ermined and is expressed as light units per milligram of protein forach group of cells (mean � SEM; n 3). For all treatment groups,P-1 luciferase activity was significantly increased in cells in whichPE-1/Ref-1 was overexpressed compared with cells with constitutive

evels of APE-1/Ref-1 (*P � 0.05).

P-1 at baseline and during H. pylori infection or oxi-ative stress.

Discussion

This study shows that APE-1/Ref-1 is expressedn situ in the normal human gastric epithelium and thatpithelial cells isolated from individuals chronically in-ected with H. pylori express higher levels of APE-1/ef-1 protein. We show that APE-1/Ref-1 is constitu-

ively expressed by human gastric epithelial cells isolatedrom H. pylori–negative biopsy specimens and by cul-ured human gastric epithelial cell lines and that thendogenous expression of APE-1/Ref-1 is enhanced aftern vitro infection with H. pylori or stimulation with

2O2. Moreover, H. pylori– or ROS-mediated increasesn APE-1/Ref-1 expression involve de novo protein syn-hesis and a rapid accumulation of APE-1/Ref-1 in celluclei. H. pylori–induced up-regulation of APE-1/Ref-1xpression requires live bacteria but is not restricted toag PAI–bearing strains. To our knowledge, this is therst report of a human pathogen affecting the expressionf the essential, multifunctional APE-1/Ref-1. The anti-xidants NAC, BHA, and GSH inhibited the increase ofotal APE-1/Ref-1 protein, and NAC also inhibited thencrease of newly synthesized APE-1/Ref-1 protein thatccurred in response to infection or ROS. Our resultsmplicate a redox-sensitive regulation of APE-1/Ref-1xpression and a functional role of this enzyme in thectivation of AP-1 in human gastric epithelial cells,henomena previously shown only in cells of nongastro-ntestinal origin.

APE-1/Ref-1 expression is ubiquitous, but it shows aomplex and heterogeneous staining pattern that differsmong tissue types and even between neighboringells.19 This study is the first to provide evidence thatPE-1/Ref-1 is expressed constitutively in situ in theormal human gastric epithelium. Gastric mucosal in-egrity is maintained by a balance between the rates ofell loss and epithelial cell regeneration, and the highasal expression of APE-1/Ref-1 in gastric epithelial cellsetected in the study may reflect the essential role ofPE-1/Ref-1 in DNA repair and cellular homeosta-

is.21,24,26,27 Our data indicate that like other cell types,19

astric epithelial cells show both cytoplasmic and nuclearocalization of APE-1/Ref-1, although under basal con-itions, its distribution is predominantly cytoplasmic.Our finding that H2O2 enhances APE-1/Ref-1 expres-

ion in gastric epithelial cells is similar to observations inther cell types (fibroblasts, macrophages, HeLa, B cells,nd Chinese hamster ovarian cells) in which ROS andxidizing agents increase APE-1/Ref-1 expression.24

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September 2004 H. PYLORI AND ROS INCREASE APE–1/REF–1 EXPRESSION 855

owever, to our knowledge, this study is the first tomplicate a microbial pathogen in the modulation ofPE-1/Ref-1 expression. The data indicate that the in-uction of APE-1/Ref-1 by H. pylori requires live bacte-ia, but not the cag PAI. H. pylori regulate host intracel-ular signal transduction by injecting CagA protein, aroduct of the cag PAI, into the host cell cytosol.48–50

owever, because cag-deficient H. pylori enhanced APE-/Ref-1 expression as much as cag PAI–bearing strains,agA injection and subsequent tyrosine phosphoryla-

ion48–50 do not seem to be the mechanism responsibleor the increased expression of APE-1/Ref-1. Anotherram-negative human pathogen, C. jejuni, had no effectn the expression of APE-1/Ref-1 in gastric epithelialells, suggesting that the effect is restricted to gastricathogens.ROS have been implicated in H. pylori–related gastri-

is, and increased levels of ROS have been found in theastric mucosa of H. pylori–infected patients.13,14

hether H. pylori–induced up-regulation of APE-1/ef-1 expression in gastric epithelial cells is due to the

bility of H. pylori itself to generate ROS10 with anccumulation of ROS in epithelial cells11,12 or to otherechanisms resulting from the direct interaction be-

ween H. pylori and the gastric mucosa is unclear. How-ver, our results show that treatment of human gastricpithelial cells with different types of antioxidants in-ibited H. pylori–induced APE-1/Ref-1 protein expres-ion. This implies a redox-sensitive regulation of APE-/Ref-1 expression in H. pylori–infected gastric epithelialells. In agreement with our findings that NAC inhib-ted H2O2-induced APE-1/Ref-1 expression in gastricpithelial cells, NAC has been shown to abrogate ROS-nduced APE-1/Ref-1 expression in HeLa cells.24

Oxidative DNA damage has been found in the gastricucosa of H. pylori–infected subjects15,16 and is thought

o play a role in the development of gastric cancer inome infected individuals.51 It is well established thatOS can induce oxidative DNA damage, which may

ontribute to cell death and malignant transforma-ion.17,52 However, several studies have shown “adaptiveesponses” and have shown that increased APE-1/Ref-1ctivity protects cells from the genotoxicity of DNA-amaging agents, including oxidants.24,30,31 Conversely,isruption of APE-1/Ref-1 by using antisense techniquesensitizes cells to DNA-damaging agents.29,53 It is un-lear whether enhanced cellular sensitivity to such agentsesults from a loss of the DNA-repair function of APE-/Ref-1 or its redox activity. However, APE-1/Ref-1 ishe rate-limiting enzyme in the repair of ROS-inducedNA damage,24 and thus, ROS-mediated induction ofPE-1/Ref-1 may be a compensatory mechanism. Alter-

atively, APE-1/Ref-1 may regulate gene expression asart of the cellular response to oxidative stress. In addi-ion to transcriptional regulation by ROS, APE-1/Ref-1s regulated at the posttranslational level by phosphory-ation54 and redox modification by thioredoxin,55 and itay autoregulate by binding to its own promoter,

hereby inhibiting transcription.20 Nonetheless, the ac-ivities of APE-1/Ref-1 are tightly coupled, and APE-1/ef-1 represents a unique link between DNA base exci-

ion repair, transcription factor regulation, and oxidativeignaling.

This study suggests that the process of APE-1/Ref-1nduction in gastric epithelial cells by H. pylori or ROSnvolves new protein synthesis and nuclear accumulation.ur results showing de novo APE-1/Ref-1 protein syn-

hesis after H. pylori or H2O2 treatment is similar totudies in other cell types. It has been shown in HeLa and

cells that oxidative stress promotes APE-1/Ref-1 pro-ein synthesis.24,45 Our findings of APE-1/Ref-1 translo-ation from the cytoplasm to the nucleus within 15inutes of H. pylori infection or stimulation with H2O2

re consistent with findings in B lymphocytes and thy-oid cells. In those studies, a similar rapid response toOS with nuclear translocation of APE-1/Ref-1 was

hown.45,56 However, in another study with HeLa cells,he activation of APE-1/Ref-1 after ROS was delayed.24

t is known that H. pylori infection and H2O2 activate thePE-1/Ref-1–regulated transcription factors NF-�B andP-1.34–36 Regulation of transcription factor activationccurs mostly in the nucleus, and the DNA bindingctivity of several transcription factors is redox-regulatedy APE-1/Ref-1.57 APE-1/Ref-1–mediated reduction ofconserved cysteine residue in the DNA binding domainf transcription factors promotes binding to DNA bind-ng sites upstream of genes involved in various cellularesponses.19 Therefore, the rapid induction and nuclearranslocation of APE-1/Ref-1 implicates it as an impor-ant signaling molecule in gastric epithelial cell re-ponses to H. pylori infection or oxidative stress. Ourndings that APE-1/Ref-1 is functionally involved in thectivation of AP-1 in gastric epithelial cells furthertrengthen this proposal. In the nucleus, APE-1/Ref-1erves to regulate gene expression, with the potential toodulate the pathogenesis of H. pylori infection.Several studies have shown increased numbers of ap-

ptotic epithelial cells during H. pylori infection,4–7 andn other cell types, APE-1/Ref-1 levels were linkedlosely to apoptosis.32,33 Whether the APE-1/Ref-1 in-uced by H. pylori functions to repair the DNA damagessociated with infection or regulates gene expression andignaling responses presumably depends on the extent ofnfection. Nevertheless, the rapid nuclear translocation of

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PE-1/Ref-1 in gastric epithelial cells suggests that itsnitial induction may serve to reductively activate earlyesponse transcription factors such as AP-1 and NF-�B,hereby coordinating cellular responses to infection. Al-ered levels or cellular location of APE-1/Ref-1 have beenound in some cancers, including ovarian, cervical, androstate,58–60 and an APE-1/Ref-1–like gene was re-ently shown to be associated with gastric cancer.61

urthermore, APE-1/Ref-1 is reported to mediate telo-erase activity.62 Considering that H. pylori has been

mplicated in gastric carcinogenesis,2,3 our finding that. pylori infection augments APE-1/Ref-1 expression inastric epithelial cells implicates APE-1/Ref-1 in thenitiation or development of gastric cancer.

In conclusion, our data show that H. pylori or ROSnhance the expression of APE-1/Ref-1 in human gastricpithelial cells. H. pylori– or ROS-mediated increases inPE-1/Ref-1 expression involve new protein synthesis

nd result in a rapid accumulation of APE-1/Ref-1 in theucleus of cells. Our results also indicate that APE-1/ef-1 is involved in the activation of the transcription

actor AP-1 in gastric epithelial cells. Collectively, theata implicate APE-1/Ref-1 in the modulation of theathogenesis of H. pylori infection, and future studiesill focus on determining the role that APE-1/Ref-1 maylay in coordinating gastric epithelial cellular responseso H. pylori infection or oxidative stress.

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Received October 30, 2003. Accepted June 10, 2004.Address requests for reprints to: Sheila E. Crowe, M.D., Departmentf Internal Medicine, University of Virginia, P.O. Box 800709, Char-ottesville, Virginia 22908-0708. e-mail: scrowe@virginia.edu.The authors thank Drs. Gang Ye, Jide Wang, and Carlos Barrera for

echnical assistance.Supported by grants RO1 DK61769 (to S.E.C.) and RO1 ES08457 (to.M.), the UTMB President’s Cabinet Award (to S.E.C.), the McLaughlinostdoctoral Fellowship Award (to S.-Z.D.), and a John Sealy Memorial

ndowment Fund Development Grant (to P.B.E.).