Hepatitis B Virus X Protein Sensitizes Primary Mouse Hepatocytes to Ethanol- and TNF-α-Induced Apopt

Cellular Molecular ImmunologyArticleHepatitis b virus X Protein Sensitizes Primary Mouse Hepatocytesto Ethanol- and TNF-a-Induced apoptosis by a Caspase-3-DependentMechanismWon-Ho Kim,4, Feng Hong, Barbara Jaruga!, Zhengsheng Zhang, Saijun Fan, T. Jake Liang.and Bin gaoIt is well-documented that alcohol drinking together with hepatitis viral infection accelerates liver injury; howeverthe underlying mechanisms remain unknown. In this paper, we demonstrated that primary hepatocytes fromtransgenic mice overexpressing hepatitis B virus X protein(HBX)were more susceptible to ethanol-and TNF-a-induced apoptotic killing. Compared to normal control mouse hepatocytes, ethanol and/or TNF-a treatment led toa significant increase in reactive oxygen species, mitochondrial permeability transition, cytochrome C release,caspase-3 activity, and poly(ADP-ribose) polymerase degradation in hepatocytes from HBX transgenic mice.Blocking caspase-3 activity antagonized ethanol- and TNF-a-induced apoptosis in primary hepatocytes from HBXtransgenic mice. Taken together, our findings suggest that HBX sensitizes primary mouse hepatocytes to ethanol-and TNF-a-induced apoptosis by a caspase-3-dependent mechanism, which may partly explain the synergisticeffects of alcohol consumption and hepatitis B virus infection on liver injury. Cellular Molecular Immunology2005;2(1):40-48Key Words: HBV X protein, ethanol, apoptosis, TNF-a, caspase-3Introductioninvolved in the regulation of hepatocyte apoptosis. Amongthem, the effects of hepatitis C virus core protein(HCV core)e two major causes of liver disease worldwide, hepatand hepatitis B virus X protein(HBX)on cell apoptosis havently coexist in been extensively investigated in transformed hepatoma orpatients with chronic liver disease. Together, they nonhepatic cell lines (5-ID), but discordant findings havecirrhosis, and hepatocellular carcinoma(1-4). However, the through several mechanisms, including a p53-indepenBir nsynergistically promote the development of liver damage, been reported. HBX has been shown to promote apoptomolecular and cellular mechanisms underlying the mechanism(12), prolonged stimulation of the N-Myc andsynergistic effect of alcohol consumption and hepatitis viral stress-mediated mitogen-activated-protein kinase kinaseinfection on liver disease remain obscure (1-4). Recent (MEKKD)pathway (13), alteration of mitochondrial membranestudies have shown that several hepatitis viral proteins are and activation of caspase 3(14), interaction with c-FLIP andenhancement of death-inducing signal(15),and othermechanisms(16, 17). Conversely, HBX has also been shownto inhibit apoptosis through activating protein kinase B(18,19), c-Jun N-terminal kinase(20), inhibiting caspase-3(21),and Alcoholism, National Institutes of Health,Bethesda, MD 20892, USAor through binding p53(22). Here, we examined ethanol-andtumor necrosis factor a(TNF-a-mediated apoptosis inLIver Diseases Branch, National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.primary hepatocytes isolated from HBX transgenicDepartment of Oncology. Lombardi Cancer Center, Georgetown University3970 Reservoir Road, Nw, Washington DC 20057, USAReceived Feb 7, 2005. Accepted Feb 17, 2005Corresponding to: Dr. Won-Ho Kim, Division of Metabolic Disease.National Institutes of Health. #5 Nokbun-Dong, Eunpyung-gu, Seoul 122. Abbrevrotein: HCv core701, South Korea. E-mail: jhkwh@hanmailnetYH中国煤化工factor-KB: TNF-a.a P tumorCNMHyl transferase-mediatedCopyright o 2005 by The Chinese Society of ImmunologyduTP nick-eMPT, mitochondnial permeability transition.Volume 2 Number 1 February 2005Cellular Molecular Immunologywild-type control mice. Our data showed that primary Flow cytometric analysis of apoptosishepatocytes from HBX transgenic mice were more After trypsinization, approximately 10 cells were collectedusceptible to TNF-a- and ethanol-induced apoptotic cell by centrifugation at 1,000 g for 5 min. Cells were thendeath by a caspase-3-dependent mechanismwashed in phosphate-buffered saline(PBS)followed byre-suspension and fixation in 70% ethanol for approximatelyMaterials and Methods2 h. Next, cells were washed with PBS and re-suspended in500 HL BS containing 100 ug RNase, followed by a 30-minAnimalsincubation at 37C. Cellular DNA was then stained by theHBX transgenic mice were originally provided by Dr. addition of 50 ug propidium iodide, and cells were analyzedFrancis Chisari(The Scripps Research Institute, La Jolla, CA) on FACScan utilizing Cellquest software( Becton Dickinsonand bred at the niddK animal facility. details of theFranklin Lakes, NJ)generation of this mouse strain were described previously(23). The HBX gene is controlled by the developmentally Cultured hepatocytes were washed twice with PBS and lysedregulated liver-specific mouse major urinary protein(MUP) in buffer (100 mM NaCl, 10 mM Tris-HCL, pH 8.0, 25 mMlivers of HBX transgenic mice were confirmed by reversEDTA, 0.5% SDS, and 0. 1 mg/mL proteinase K)at 37C fortranscriptase-polymerase chain reaction. Male C57BL/6 18 h. DNA was extracted with an equal volume of phenol/background control mice were purchased from the Jacksonchloroform(1: 1)and precipitated at- 70C. DNA pellets wereLaboratory(Bar Harbor, Maine)re-suspended in 10 ul of 10 mM Tris(pH 7.8), 1 mM EDTAfor I h at 37'C with 1 ug/ml RNasMaterials( Roche Molecular Biochemicals, Indianapolis, IN)to removeCaspase-3 inhibitor I(DEVD-CHO)was purchased from RNA DNA pellets were electrophoresed for 2-3 h at 90VCalbiochem(San Diego, CA). Murine TNF-a was purchased1.8% agarose gels. The gel was stained with ethidiumfrom Bioscience Intemational(Camarillo, CA, USA). Ethanolbromide and the dna fragments were visualized underwas obtained from Sigma Chemicals(St Louis, MO, USA).ultraviolet lightTerminal deoxynucleotidyl transferase-mediated duTp nickIsolation and culture of primary mouse hepatocytesMale C57BL/6 background HBX transgenic mice and controlend labeling(TUNel)C57BL6 mice weighing 30-35 g were anesthetized with Apop Tag(Oncor, Gaitherburg, MD), an in situ apoptosissodium pentobarbital (30 mg/kg intrperitoneally), and theportal vein was cannulated under aseptic conditions. The terminal transferase and avidine- labelled-dUTP, followed byliver was perfused with EGTA solution (5.4 mM KCI, 0. 44 further incubation with FITC-labeled anti-avidine antibodymM KH2PO4, 140 mM NaCl, 0. 34 mM NayHPO4, 0.5 mM The slides were then examined under fluorescence micro-GTA, 25 mM Tricine, pH 7. 2)and Dulbeccos ModifiedEagle Medium(GIBCO BRL, Gaithersburg, MD), anddigested with 0.075% collagenase solution. Isolated mouse Measurement of reactive oxygen species(ROS)hepatocytes were then cultured in Hepato-ZYME-SFM Intracellular ROS generation was quantified using flowmedia(GIBCO BRL)in rat-tail collagen coated plates for 24 cytometric measurement of the metabolite fluoresceneh, followed by drug treatment.2 7-dichlorofluorescein (Molecular Probes, Inc, Eugene,OR). ROS produced by cells oxidizes dCFH-Da into theWestern blottinghighly fluorescent green compound (27'-dichlorofluorescein)Liver tissues were lysed in lysis buffer(30 mM Tris, PH 7.5, whose fluorescence intensity is directly proportional to ROS150 mM sodium chloride, I mM phenylmethylsulfonyl production(24). Cultured hepatocytes or freshly isolatedfluoride, I mM sodium orthovanadate, 1% Nonidet P-40, hepatocytes(2 x 10,)were loaded for I h with CM-DCF at a9% glycerol) for 15 min at 4"C, vortexed and centrifuged at final concentration of 10 Hmol/L. Fluorescene 27'-dichlo-16,000 rpm at 4 C for 10 min. The supernatants were mixed rofluorescein was measured by flow cytometry. Results werein Laemmli running buffer, boiled for 4 min, and then normalized to control cells and expressed as relative fluo-subjected to SDS-PAGE. After electrophoresis, proteins were rescence intensityransferred onto nitrocellulose membranes and blotted againstprimary antibodies for 16 h. Membranes were washed with Measurement of mitochondrial permeability membraneTPBS(0.05% [vol/vol] Tween 20 in phosphate-buffered transition (MPT)saline [pH 7.4 D and incubated with a 1: 4,000 dilution of MPT was measured using DiOC6(Molecular Probes Inc.horseradish peroxidase-conjugated secondary antibodies for Eugene, OR), which is electrophoretically re-distributed45 min. Protein bandsacrosschemiluminescence reaction(Amersham Pharmacia Biotech, memt中国煤化工 according to thetes(2×10)werePiscataway, NJ)and quantified using a Phospholmager incubatCN MH Glium containing 100(Amersham Pharmacia Biotech).nM DiOc6. Cells were then washed, re-suspended in DMEMVolume 2 Number 1 February 2005HBX Sensitizes Hepatocytes to Ethanol- and TNF-a-Induced ApoptosisCtrl (4h)ETOH (24h)rNF-(4h)CtrlI HBXmFigure 1. HBx transgenic mouse hepatocytes are moresusceptible to ethanol- and TNF-a-induced DNA fragmentation.trol(Ctrl) and HBXtransgenic mice were cultured in serum-free media in the absence orpresence of various concentrations of ethanol (E)or TNF-a, as Figure 2. TUNEL assay of ethanol. and TNF-aindicated, for 24 h Apoptosis was measured by DNA fragmentation apoptosis in wild-type control(CtrD)and HBX hepatocas described under "Materials and Methods. "A representative Mouse hepatocytes from control( Ctrl)and HBX transgenicexample of 3 independent experiments is shownwere cultured in serum-free media in the absence or presence of 100mM ethanol or 20 ng/ml of TNF-a for 24 h. Apoptosis wasexamined by TUNEL staining(panel A)as described under"Materials and Methods. Apoptotic hepatocytes(means t SEM)medium. The MPT was analyzed using a FACScan flow were counted from 3 independent experiments and depicted in panelcytometer( Becton Dickinson)equipped with a 488 nm argon B. .p< 0.01 in comparison with corresponding controls, whichlaser. Each sample containing a minimum of l0 cells was were assigned as 100%analyzed through the FLl-H channel. Data were acquired inlist mode and analyzed using the CellQuest softwareCleavage of the substrate was monitored at 405 nm and thePrimary hepatocytes isolated from wild-type mice and HBx specific activity was expressed in pmol of the producttransgenic mice were plated and treated with ethanol orTNF-a for various time points. Following treatments, cellsStatistical analysiswere re-suspended and homogenized in isolation buffer(20 For comparing values obtained in three or more groups,mmol/L Hepes, pH7. 4, 10 mmol/L KCl, 1.5 mmol/L MgCII mmol/L sodium-EDTA, I mmol/L dithiothreitol, 10 mmolL one-factor analysis of variance (ANOVA)was used,phenylmethylsulfonylfluoride, 10 Hg/ml leupeptin, and 10 followed by Tukey's post hoc test; p 0.05 was taken toue/ml aprotinin in 250 mmol/L sucrose). Unbroken cells andnuclei were removed by two rounds of centrifugation at2, 500 g at 4C. The mitochondria were then pelleted by Resultscentrifugation at 9,000 g at 4C for 30 min to obtainThe supematant from the first 9,000 g centrifugation was to ethanol- and TNF-a-mediated apoptotic kill usceptibleheavy membrane(HM)fraction enriched with mitochondria. Hepatocytes from HBX transgenic mice are moresubsequently centrifuged at 100,000 g to obtain the cytosolic To understand the synergistic effect of alcohol drinking andsupernatant and light membrane (LM) fractionsepatitis virus infection on liver injury, ethanol- andMitochondria and cytosolic fractions(60 Hg)were separated TNF-a-mediated apoptosis were examined in hepatocyteson 15% SDS-paGe gels and immunoblotted with the isolated from HBX transgenic mice and wild-type controlantibody against cytochrome C (Santa Cruz, San Diego, CA)mice. As shown in Figure l, treatment of primary hepatocytesfrom wild-type C57BL/6 mice with 25-50 mM ethanol orEnzymatic assay of caspase-3INF中国煤化工 significantly induceCleavage of the caspase-3 substrate 1(Ac-DEVD-PNA) DNAh 100 mM ethanol or(Calbiochem, San Diego, CA)was used as a measure of ethaCNMHGinduced DNA fragcaspase-3 activity. The p-Nitroaniline was used as the standard. mentation. In contrast, treatment of HBX transgenic mouse2 Number 1 February 2005Cellular Molecular Immunology12h24hM=1m=1M=MCtrlIce如ror%oateMarker GateEtOH33M231M217CtrlHBX性mIceM2EtOHM-712100010000Figure 3. Flow cytometric analysis of ethanol-mediated apoptosis in wild-type control( Ctrl)and HBX hepatocytes. Mouse hepatocytesfrom wild-type control (Ctrl)and HBX transgenic mice were cultured in serum-free media in the absence or presence of 100 mM ethanol forvarious time points. Apoptosis was examined by propidium iodide staining as described under"Materials and Methods. MI phase, sub-Glpeak, represents the percentage of apoptotic cells. Representative histograms of propidium iodide staining from 4 independent experiments areshown. Similar results were obtained for each of the 4 independent experimentsocytes with 50-100 mM ethanol alone, TNF-a alone, or ethanol-treated wild-type hepatocytes vs. 33.31% in ethanolnol plus TNF-a for 24 h caused significant DNA treated HBX hepatocytes at 24 h)(Figure 3). Collectively,mentationthese findings suggest that HBX sensitizes primary mouseThe TUNEL assay and flow cytometric analyses were hepatocytes to ethanol- and TNF-a-mediated apoptotic cellIso used to confirm ethanol-and TNF-a-mediated apoptosis deathin HBX transgenic mouse hepatocytes. As shown in Figure 2,a slight increase(about 1.5-2.0 fold)in apoptotic cells was Hepatocytes from HBX transgenic mice are more susceptibleobserved in hBX transgenic mouse hepatocytes compared to to ethanol-or TNF-a-mediated induction of ROS and MPTnormal mouse hepatocytes. Ethanol and TNF-a treatment for To understand the underlying mechanism by which HBX24 h caused, respectively, a 1.8+ 0.2 and 1.6+ 0.5 fold sensitizes hepatocytes to ethanol- and TNF-a-induced apop-increase in TUNEL-positive cells in normal primary mouse tosis, we examined ethanol- and TNF-a-mediated inductionhepatocytes, whereas the same treatments, respectively, of ROS and MPT in normal mouse hepatocytes and HBXcaused a 5.6+ 0.6 and 4.7+ 0.6 fold increase in TUNEL- transgenic mouse hepatocytes. As shown in Figure 4,positive cells in HBX transgenic mouse hepatocytes. Flow exposure to ethanol or TNF-a alone did not cause significantcytometric analyses showed that treatment with 100 mM ROSethanol for 24 h slightly induced apoptosis in normal primary but中国煤化工 pe C57BL6mice,oduction in HBXhepatocytes but markedly induced apoptosis in HBX transgeC N MH Gk shifted to the right)transgenic mouse hepatocytes(Ml, sub-Gl phase, 13.04% in Treatment with ethanol and INF-a together caused aVolume 2 Number I February 2005HBX Sensitizes Hepatocytes to Ethanol-and TNF-a-Induced ApoptosisHBXbut mPT induction in HBX mouse hepatocytes was muchControlhigher than that in normal mouse hepatocytes(M1 96.58%inM1:53.62%M:5150%HBX hepatocytes vs. 80.65% in normal hepatocytes). Tofurther examine the effect of ethanol and TNF-a onmitochondrial injury, expression of cytochromeexamined in cytosolic and mitochondrial fractions. Asin Figure 5B, treatment of HBX transgenic causcytochrome C release from the mitochondria into the cytosolM1:5563%M1:66.93%at 12 h and 24 h, whereas the same treatment did not causecytochrome C release in wild-type control hepatocytTNF-aFurthermore, both ethanol and TNF-a treatment enhancedexpression of proapoptotic Bax protein in HBx-transgenicT1‘80"12"t14mice compared to normal mice( Figure 5C)Ml:6L37%BX transgenic miM1:82.07%to ethanol- or TNF-a-mediated activation of caspase-3The effects of ethanol and TNF-a on the activation ofcaspase-3 were examined in normal and HBX transgenicmousehepatocytes.As shown in Figure 6A, treatment ofnormal mouse hepatocytes with 50 mM ethanol or TNF-adid not significantly induce caspase-3 activation, whereas theM1:83.38%Ml:9444%same treatments caused a 5-fold(ethanol) and 16-foldTNF-C(TNF-a)increase in caspase 3 activity in HBX transgenicmouse hepatocytes. Moreover, 100 mM ethanol treatmentEtoHinduced a 3-fold activation of caspase-3 in normal mousehepatocytes, but induced a 9-fold activation in HBXTo determine whether ethanol-activated3 isDCF fluorescencefunctionally active in HBX transgenic mouse hepatocytes,Figure 4. HBX mouse hepatocytes are more susceptible to ethanol. cleavage of poly (ADP-ribose) polymerase(PARP)wasand TNF-a-induced ROS Hepatocytes isolated from male C57BL6 examined by Westem blotting analysis. As shown in Figurebackground HBX transgenic mice and wild-type C57BL/6 mice 6B, treatment of cells with ethanol rapidly caused PARPwere incubated with ethanol (100 mM)and/or TNF-a(20 ng/ml) cleavage in HBX transgenic mouse hepatocytes, but not infor 24 h ROS was measured by flow cytometry as described under normal hepatocytes. PARP cleavage in HBX transgenicMaterials and Methods. "MI represents the relative ROS activity. hepatocytes was blocked by pretreatment with the caspase-3Representative histograms of ROS from 3 independent experimentsare shown. Similar results were obtained for each of the 3 inhibitor DEVD-CHOHBX sensitizes primary mouse hepatocytes to ethanolapoptotic killing by a caspase-3-dependent mechanismTo further confirm the involvement of caspase-3 in ethanol-significant increase in ROS production in normal mouse and TNF-a-mediated apoptosis, the caspase-3 inhibitorhepatocytes, which was further enhanced in HBX transgenic DEVD-CHO was used. As shown in Figure 7A, treatment ofnouse hepatocytes(MI 94. 44% in HBX hepatocytes vs. HBX transgenic mouse hepatocytes with ethanol caused83.38% in normal hepatocytes).significant DNA fragmentation, which was markedlyThe effect of ethanol and TNF-a on MPT induction was suppressed by pretreatment with DEVD-CHO. Flowalso examined by flow cytometry using DiOC(Figure 5A). cytometric analyses showed that treatment of HBXExposure to TNF-a alone did not induce mPt in normal transgenic mouse hepatocytes with ethanol for 24 h inducedmouse hepatocytes, but significantly induced MPT in HBX apoptosis, which was markedly attenuated by DEVD-CHOtransgenic mouse hepatocytes(MPT peak shifted to the left, (33.31% MI sub-G1 peak in ethanol-treated group vsindicating that fewer cells retained DiOC in their 12. 68% DEVD-CHO- plus ethanol-treated group)(Figuremitochondria). Exposure to ethanol for 24 h markedly 7B). Similarly, TNF-a-mediated induction of apoptosis ininduced MPT in HBX transgenic mouse hepatocytes, HBX transgenic mouse hepatocytes was also blocked by thewhereas the same treatment only slightly caused MPT caspaa not shown). Theseinduction in normal mouse hepatocytes. Co-treatment with findi中国煤化zes pnmary mouseethanol and TNF-a induced significant MPT in both normal hepatCNMHby a caspase-3mouse hepatocytes and HBX transgenic mouse hepatocytes, depenucrVolume 2 Number I February 2005Cellular Molecular ImmunologyControlHBXM:5535%M1:65.%8%Figure 5. HBX mouse hepatocytes arCvtochrome Cmore susceptible to ethanol and TNF-aCytosolMitochondria induction of MPT.(A)HepatocytesETod6122406124lated from male c57bl/6 backgoundHBx transgenic mice and wild-type會"m°"n)""w"tM1:4293%ng/ml) for 24 h MPT was measuredM1:84.21%Cytosol Mitochondria flow cytometry as described underTNEa3导8TNF-a(h12240 12 24"Materials and Methods. "MI representsthe relative MPT. (B)and (C) culturedwild-type mouse and HBX transgenicHBX103104 100house hepatocytes were treated with00 mM)or TNF-a(2M1:6876%1M:9047%vanous time points. Cytmitochondrial proteins were isolated andEtoHanti-cytochrome C antibody(panel B)oranti-Bax antibody(panel C). A repreCsentative example of 3 independentNormalHBx experiments is shown in panels A-c.TNF-a!M1:80.65%IM:9%589 ETOH(h Lo61220612282TNFa堡。距EToH%103104DiOC6 fluorescenceDiscussioncell types, and apoptotic stimuli used. For example, inweakly expressing cells, HBX is exclusively or predomiThe combined effects of alcohol drinking and hepatitis viral nantly localized in the nuclei, but in highly expressing cells itinfection on liver injury are well documented, but the accumulates in the cytoplasm(37). Also, HBX is typicallymechanisms by which ethanol accelerates progression of expressed in distinctive granular patterns in human hepatoma,liver disease in patients with hepatitis viral infection are notHuh-7 cells, but in squamous carcinoma A431 cells, HBX isfully understood (1-4). Several mechanisms have been expressed in dispersed, non-granular patterns(37).Cellconsidered(1), including ethanol suppression of interferon specific HBX expression pattems may be responsible forantiviral immune response (25-27), promotion of viraopposing effects on cell survival. Thus, the pro-apoptoticreplication(28, 29), inhibition of the immune system(30), effect of HBx demonstrated in primary mouse hepatocytes inattenuation of liver regeneration(31), and potentiation of this paper is likely more physiological than studieshepatitis viral protein-mediated activation of inflammatory previously conducted in transformed hepatoma and non-signals(32). Here we demonstrate for the first time that HBx hepatic cell lines(12, 18-22, 33-36)sensitizes primary hepatocytes to ethanol- and TNF-a-Several mechanisms may be involved in the sensitizationinduced ROS, MPT, and apoptosis, which may contribute to of HBX hepatocytes to apoptosis induced by ethanol andthe synergistic effects of alcohol drinking and viral hepatitis TNF-a. First, ethanol and TNF-a treatment inducedinfection on liver injury. Furthermore, we demonstrate that a significant caspase-3 activation in HBX transgenic mousecaspase-3-dependent mechanism may be involved in HBx hepatocytes, but not in normal control primary hepatocytessensitization of hepatocytes to ethanol- and TNF-a-induced Moreover, blocking caspase-3 activation with a specificapoptosIinhibitor abolished ethanol- and TNF-a-induced apoptosis inAlthough the effects of HBX on cell apoptosis have been HBX transgenic mouse hepatocytes. These findings suggestextensively investigated(12, 18-22, 33-36), contradictory that a caspase-3-dependent mechanism is involved infindings have been reported. It has been shown that HBX can ethaneither promote or inhibit apoptosis. The opposing effects by mous中国煤菜化in HBX transgeHBX in these studies could be due to differences in HBx cytesCNMHGand TNF-a-inducedexpression levels, transient and stable expression of HBX, ROSction of KUS ana MPt has been shownVolume 2 Number 1 February 2005HBX Sensitizes Hepatocytes to Ethanol- and TNF-a-Induced ApoptosisFigure 6. Ethanol and TNF-a activate caspase-3 in HBXFL24transgenic mouse hepatocytes. (A)Hepatocytes isolated fromwild-type mice and HBX transgene mice were incubated with ,gure 7. HBX sensitizes primary mouse hepatocytes to ethanol.induced apoptosis by a easpase3-dependent mechanism HBX50-100 mM ethanol or 20 ng/ml of TNF-a for 24 h, and then transgenic mouse hepatocytes were incubated in the absence orcaspase-3 activity was measured. Values shown are means t SEMfrom 3 independent experiments. p<0.05, **p<0.01, significant presence of the caspase-3 inhibitor DEVD-CHO with ethanol(100(panel A)or propidium iodide staining(Ml, sub-Gl peak represents(B)Hepatocytes isolated from normal control (Ctrl) and HBX apoptosis)(panel B)as described under"Materials and Methods. "Aansgenic mice were incubated with 100 mM ethanol in thabsence or presence of the caspase-3 inhibitor DEVD-cHo panels A and Brepresentative example of 3 independent experiments is shown inabbreviation: D) for various time points as indicated. Cell extractswere then prepared and subjected to Western blotting using annti-PARP antibody. The 86 kd band represents the degradedPARPalcoholics with HBV infection(45, 46)Referencesto play an important role in hepatocyte apoptosis(38-40)Thus, higher induction of ROs and MPt may partl1. Gao B. Alcohol and hepatitis virus interactions in liverontribute to ethanol- and tNF-a-induced cell death in HBXpathology. In: Preedy eds Comprehensive Handbook of Alcoholtransgenic mouse hepatocytes. Currently, the underlyingRelated Pathology. New York, Elsevier Ltd; 2005: 819-832.mechanism by which HBX sensitizes primary mouse hepato-rya R, Shuhart MC. Hepatitis Ccytes to ethanol and TNF-a induction of ROS and MPt isractions, outcomes, and implications. J Clin Gastroenterolnot clear. Co-localization and interaction of HBX protein2003:36:242-252with several mitochondrial proteins may play an important3. Schiff ER. Hepatitis C and alcohol. Hepatology, 1997: 26: 39S-role in HBX sensitization of he4. Safdar K, Schiff ER. Alcohol and hepatitis C. Semin Liver DisTNF-a-induced ROS and MPT (37, 41, 42).200424:305-315In summary, this paper demonstrates that HBX sensitizes 5. Yang SH, Lee CG Lee CW, et al. 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