Journal of Medical Virology 77:374–381 (2005)

Hepatitis B Virus X Protein Upregulates SurvivinExpression in Hepatoma Tissues

Xiaodong Zhang,1* Nan Dong,1 Lihui Yin,1 Na Cai,1 Hongtao Ma,1 Jiacong You,2 Hang Zhang,1

Honghui Wang,2 Ran He,2 and Lihong Ye2**1Department of Cancer Research, Institute for Molecular Biology, Tianjin Key Laboratory of Microbial FunctionalGenomics, College of Life Sciences, Nankai University, Tianjin, P.R.China2Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin, P.R.China

The hepatitis B virus X protein (HBx) plays animportant role in the development of hepatocel-lular carcinoma (HCC). The relationship was ex-amined between HBV antigens and IAP (inhibitorof apoptosis) family in development of HCC.The expression levels of HBV antigens (HBsAg,HBcAg, and HBxAg) and members of the IAPfamily (survivin, XIAP, cIAP-1, and cIAP-2) weredetected immunohistochemically in tissues from34 cases of HCC and 30 cases of liver cirrhosis.The positive rate of survivin was higher thanthese three molecules in all three tissue types(P< 0.05). The positive rates of HBxAg and sur-vivin were high in HCC (76.5% and 88.2%),paratumor (85.3% and 91.2%), and liver cirrhosis(100% and 93.3%) tissues, with no significantdifferences between the survivin- and HBxAg-positive rates (each P> 0.05). To examine theeffect of HBx on survivin expression, plasmidpCMV-X (encoding the HBx gene) was trans-fected transiently with or without plasmidpcDNA3-sur (encoding the survivin gene) intoH7402 hepatoma cells and L-O2 human normalliver cells. Cells over-expressing HBx aloneshowed increased apoptosis along with a dose-dependent increase in survivin levels. However,co-expression of survivin inhibited the HBx-induced apoptosis. To examine the effect of HBxon survivin in hepatoma cells without apop-tosis, plasmid pCMV-X was transfected stablyinto human hepatomaH7402 cells and L-O2 cells.These H7402-X and L-O2-X cells showed high-level expressionof bothHBxand survivin, but didnot show apoptosis. The addition of pSilencer3.0-X, an RNAi vector targeting the HBx gene,reduced the expression levels of survivin proteinin H7402-X cells. Collectively, these data demon-strate that HBx upregulates survivin expressionin hepatoma tissues, suggesting that HBx andsurvivin may both be involved in carcinogenesisof HCC. J. Med. Virol. 77:374–381, 2005.� 2005 Wiley-Liss, Inc.

KEY WORDS: hepatoma; HBx; survivin; carci-nogenesis; normal liver cells

INTRODUCTION

The hepatitis B virus (HBV), a member of the familyhepadnaviridae, replicates its genome by reverse tran-scription and causes both acute and chronic infection ofthe liver [Beasley et al., 1981]. The HBV gene is highlyconserved among all mammalian hepadnaviruses andencodes a small peptide expressed at low levels duringthe natural course of HBV infection [Spandu and Lee,1988]. It has been reported that the HBV X protein(HBx) transactivates several cellular genes that mod-ulate cell growth and cell cycle checkpoints [Spandu andLee, 1988; Benn and Schneider, 1995]. Previous studieshave shown that the multifunctional HBx has a closerelationship with Bid and p53, and is capable of trans-activating many cellular factors, including c-Myc, c-fos,and c-jun [Chen and Lai, 2001; Huo and Wang, 2001;Nijhara et al., 2001; Su et al., 2001]. Notably, HBx canbind to HVDAC3, a human voltage-dependent anionchannel localized on mitochondria [Rahmani et al.,2000], inhibit caspase-3 [Gottlob, 1998], and induceexpression of FasL [Shin et al., 1999]. It seems thatthe fate of HBx-expressing infected cells is likely to bedetermined by the balance between apoptotic and anti-apoptotic signals of viral, cellular, and environmentalorigin [Gergametti et al., 1999; Diao et al., 2001]. It

Grant sponsor: Tianjin Natural Science Foundation, China;Grant number: 043113411.

*Correspondence to: Xiaodong Zhang, Department of CancerResearch, Institute for Molecular Biology, Tianjin Key Laboratoryof Microbial Functional Genomics.E-mail: zhangxd@nankai.edu.cn

**Correspondence to: Lihong Ye, Department of Biochemistry,College of Life Sciences, Nankai University, 94 Weijin Road,Tianjin, 300071, P.R.China. E-mail: yelihong@nankai.edu.cn

Accepted 14 June 2005

DOI 10.1002/jmv.20466

Published online in Wiley InterScience(www.interscience.wiley.com)

� 2005 WILEY-LISS, INC.

has been demonstrated that HBx upregulates the ex-pression and activity of human telomerase reversetranscriptase (hTERT) in hepatoma cells, suggestingthat hTERTmay be associatedwith tumor development[Zhang et al., 2005]. However, the actual impact of HBxin liver carcinogenesis and its precise mechanisms ofaction remain largely obscure.

The inhibitors of apoptosis (IAPs), which are crucialregulators in the molecular mechanisms of cancerand apoptosis [Nachmias et al., 2004], are characteriz-ed by functionally important RING finger and BIRdomains [Takahashi et al., 1998]. Seven IAPs have beenidentified to date, including survivin, XIAP, cIAP-1,cIAP-2, apollon,DIAP-1, andDIAP-2 [Chen et al., 1999].Gene (mRNA) expression of cIAP-1, cIAP-2, and XIAPhas been detected in most tissues. Moreover, studieshave shown that the expression of XIAP is a prognosticindex for lung cancer [Liston et al., 1996; Olie andSimoes-Wust, 2000]. Most of the IAPs are expressedbroadly in normal adult tissues, with the exception ofsurvivin, which is undetectable in normal adult tissues,but is expressed abundantly in transformed cells and avariety of human cancers [Ambrosini et al., 1997].Antisense knock-down of survivin expression and/orfunction was found to cause aberrant mitotic progres-sion and spontaneous apoptosis [Guo and Hay, 1999;Olie and Simoes-Wust, 2000]. In addition, survivinexpression in tumorshas beenassociatedwith increasedaggressiveness and decreased patient survival [Olie andSimoes-Wust, 2000; Trieb et al., 2003]. Inhibition ofapoptosis by survivin appears to occur via three path-ways. First, survivin suppresses apoptosis by inhibitingdirectly the activities of caspase 3 and caspase 7 [Sohnet al., 2003]. Second, survivin can bind with the cyclinkinases, cdk4 and p34cdc2, perhaps leading to abroga-tion of pro-apoptotic cell signal transduction. And third,survivin can suppress apoptosis by binding the smac/DIABLO complex [Song et al., 2003].

According to recent studies, survivin form complexeswith the hepatitis B X-interacting protein (HBXIP), acellular protein recognized originally for its associa-tion with HBx. The survivin-HBXIP complex, but notsurvivin or HBXIP alone, binds pro-caspase-9, prevent-ing its recruitment to Apaf1 and suppressing selectivelyapoptosis initiated via the mitochondria/cytochrome-cpathway. HBx also interacts with the survivin-HBXIPcomplex and suppresses caspase activation in a survi-vin-dependent manner. Thus, HBXIP functions as acofactor for survivin, forming a link between the cellularapoptotic machinery and a viral pathogen involved inhepatocellular carcinogenesis [Marusawa et al., 2003].However, the in vivo relationships in the hepatomatissue, hepatoma cell, and gene expression levels of HBxhave not been reported previously.

The relationships between HBx and the IAPs wasinvestigated by examining the expression patterns ofthree HBV antigens (HBsAg, HBcAg, and HBxAg) andfourmembers of the IAP family (survivin, XIAP, cIAP-1,and cIAP-2) in HBV-infected HCC and liver cirrhosistissues. The effect of HBx on survivin expression was

also studies by co-transfecting transiently plasmidspCMV-X (encoding the HBx gene) and/or pcDNA3-sur(encoding the survivin gene) into human hepatomaH7402 cells or L-O2 human normal liver cells, bytransfecting stablyHBx intoH7402 cells and L-O2 cells,and by expressing transiently an HBx-targeting RNAiin the stably transfected H7402 cells. The findingsindicate collectively that HBx induces upregulation ofsurvivin expression, suggesting that HBx and survivinmay be involved coordinately in carcinogenesis of hepa-tocellular Carcinoma.

MATERIALS AND METHODS

Immunohistochemical Detection of HBV X inHepatoma and Liver Cirrhosis Tissues

Samples were taken from 34 hepatocellular carci-noma (HCC) tissuesandadjacent tumor tissues,30casesof liver cirrhosis, and 6 normal livers without HBVinfection obtained at autopsy (Tianjin First CenterHospital, Tianjin, China). The mean age of patientswas 47.9� 9.4. All had undergone total or subtotalhepatectomies, and all patients had a history of HBV in-fection according to the hospital data. Five micrometer-thick sections of formalin-fixed, paraffin-embeddedsamples from each case were studied by immuno-histochemistry, using the Strept Avidin-BiotinComplexmethod (SABC Immunohistochemistry Kit, Boster,Wuhan, China). The primary antibodies includedmouseanti-HBsAg (SantaCruz,CA), rabbit anti-HBcAg(Santa Cruz, CA), rabbit anti-HBxAg (provided by Dr.Wang, Fourth Military Medical University,China,Wang et al., 1991; dilution �400), mouse anti-survivin(NeoMarkers, CA; dilution �400), rabbit anti-XIAP(R & D Systems, Inc., St. Paul, MN, dilution �1000),rabbit anti-cIAP-1 (R & D Systems, Inc., 1.0 mg/ml), andrabbit anti-cIAP-2 (R & D Systems, Inc., 1.5 mg/ml),respectively. Tissue sections were blocked and incu-bated with the appropriate primary antibodies over-night at 48C. The sectionswere thenwashed three timeswith PBS, incubated with a biotin-labeled secondaryantibody for 1 hr at 378C, washed three times with PBS,and incubated with the strept-avidin-labeled tertiaryantibody for 30 min at 378C. Finally, samples werewashed three times with PBS and positive staining wasdetected using the DAB substrate. For negative con-trols, PBS and normal rabbit IgG were used in place ofthe primary antibody. The normal liver tissues wereused as an additional negative control. The study wasapproved by the hospital’s Ethics Committee.

Positive signals in the cell nuclei or cytoplasm in theHCC and liver cirrhosis tissues were defined accordingto standard immunohistochemistry criteria [Marusawaet al., 2003]. The expression levels in the varioussamples were quantitated using a modification of themethod described previously for measuring survivinexpression [Marusawa et al., 2003]. Briefly, a meanpercentage of positive cells was determined at a mini-mum X400 magnification and assigned to one of thefollowing five categories: 0, <5%; 1, 5–25%; 2, 25–50%;

Survivin Expression in Hepatoma Tissues 375

3, 50–70%; and 4,>75%. The intensity of immunostain-ingwas scored as follows:weak, 0.5þ;moderate, 1þ; andintense, 2þ. For tissues that showed heterogeneousstaining, the predominant pattern was scored. Thepercentage of positive cells and the staining intensitywere multiplied to produce weighted scores for eachcase. Cases with weighted scores of less than 1 weredefined as negative, those with scores greater than 1were defined as positive.The w2 test was used to examine the relationships

between the immunohistochemical and histopathologi-cal findings.

Constructs and Transfections

A human survivin cDNA was obtained by reversetranscription-PCR (RT-PCR) of RNA derived fromH7402 cells, using primers based on the known survivinsequence (GenBank accession number U75285; for-ward, 50-GGGAATTCATGGGTGCCCCGACGTTGCC-30; reverse, 50-CTGGTACCTCAATCCATGGCAGCCA-GCT-30). The resulting PCR product was inserted intothe pUCm-T vector (Takara, Japan) to yield pUCm-T-sur. This construct was digested with EcoRI and XhoI,and the survivin gene fragment was ligated into thepcDNA3 vector (Invitrogen, CA) to generate pcDNA3-sur, which was confirmed by sequencing.The HBV-free H7402 human hepatoma cell line was

obtained from the People’s Hospital of Beijing Univer-sity [Liu et al., 2002]. In order to confirmthat the cell linewas hepatoma cells, RT-PCR was used to show that thecells were capable of expressing a-feto proteins (data notshown). Human normal liver cell line L-O2 [Li et al.,2003]was purchased fromNanjingKeyGenBiotechCo.,Ltd. (Nanjing, China) The H7402 and L-O2 cells weremaintained in RPMI Medium 1640 (Gibco, CA) supple-mented with heat inactivated 10% fetal calf serum(FCS), 100U/ml penicillin, and 100mg/ml streptomycinin a humidified atmosphere of 5% CO2 and 95% air.For transient transfections, H7402 cells were trans-

fected in 6-wells plates using a Lipofectamine (Gibco)with 3 mg of pCMV-X (encoding the HBx gene in thepcDNA3 vector; kindly provided by Dr. A. Graessmann,Freie University, Germany), 3 mg pcDNA3-sur, 3 mgpcDNA3 or with 3 mg each of pcDNA3-sur and pCMV-X. The transfection efficiency was measured by co-transfection with 0.1 mg of the GFP marker plasmid,pEGFP (Clontech). The cells were cultured for 36 hrpost-transfection, and then both floating and attach-ed cells were harvested and used for flow cytometryand Western blot analyses. For examining the dose-dependency of HBx-induced apoptosis in hepatomacells, 1, 2, and 3 mg of pCMV-X were transfected asabove. The transient transfection of 2 mg of pCMV-Xwasperformed in L-O2 cells as above.For stable transfection of theHBx gene, H7402 and L-

O2 cells were transfectedwith Lipofectamine, accordingto themanufacturer’s protocol (Invitrogen). Inbrief, 3mgof pCMV-X plasmid was mixed with 18 ml of Lipofect-amine in serum free medium and incubated at 378C

for 6 hr. The medium was then replaced with RPMI1640 containing 10% FCS. After 48 hr, the transfectedcells were incubated in selection medium containing600 mg/ml G418 (Genview, Carlsbad, CA), and selectionwas maintained for 3–4 weeks. For control purposes,empty pcDNA3 vector plasmids were transfected intoH7402 cells as above. The successful stable transfectionof theHBx gene was confirmed by RT-PCR andWesternblot analyses.

Using plasmid pSilencer 3.0 (Promega,Madison,WI),we used sequences 50GATCCCGGTCTTACATAAGA-GGACTTTCAAGAGAAGTCCTCTTATGTAAGACCTT-TTTTGGAAA-30 (sense) and 50AGCTTTTCCAAAAA-AGGTCTTACATAAGAGGACTTCTCTTGAAAGTCCT-CCTTATGTAAGACCGGG-30 (antisense) from the HBxgene to create theRNAi vector ofHBxgene [Shlomai andShaul, 2003].Thepurifiedvector ofpSilencer3.0-X (3mg)was transfected into H7402-X cells by Lipofectamine asdescribed above. Cells were harvested and lysed at 24,36, and 48 hr post-transfection, respectively, and theexpression levels of HBx and survivin proteins wereassessed by Western blot analysis.

Flow Cytometry

The medium containing detached cells was aspiratedand the attached cells were harvested using trypsin.The two fractions were pooled, washed with PBS, fixedin ice-cold 70% ethanol, and washed with Ca2þ/Mg2þ-free HBSS containing 1% BSA. The cells were thenincubated in 50 mg/ml propidium iodide containing 1mg/ml sodium citrate, 100 mg/ml RNase I, and 0.1% tritonX-100 for 30 min at 378C, and analyzed by flow cyto-metry in a fluorescence-activated cell sorter (EpicsXL.MCL,BeckmanCoulter) using theEXPO32 software.

Examination of HBV x mRNA and X Proteinand Survivin Protein

Total cellular RNA was isolated from H7402, H7402-X, L-O2, and L-O2-X cells using the Uniq-10 columntotalRNA isolationkit (Sangon,China), according to themanufacturer’s protocol. For RT-PCR analysis, 2 mgof total cellular RNA was reverse transcribed withSuperScriptTM III reverse transcriptase (Invitrogen),per themanufacturer’s instructions. The reactions werestopped by heat inactivation for 5 min at 908C. Thegenerated cDNA (2 ml) was then amplified in a DNAthermocycler (Geneamp PCR system, Perkin Elmer2400, USA) with 2U rTaq DNA polymerase (Takara,Japan) and 5 pM of each HBx gene primer (forward, 50-GGCTCGAGATGGCTG CTAGGCTGTGC-30, reverse,50-GGCGAATTCAGAAGTCGTCGTCGTCC-30; 510 bpamplified fragment) and each human GAPDH geneprimer (forward, 50-GGTCATCCCTGAGCTGAACG-30;reverse, 50-TCCGTTGTCATACCAGGAAAT-30; 298 bpamplified fragment) in a volume of 50 ml. The cyclingconditions consisted of 30 cycles of 948C for 30 sec, 608Cfor 30 sec, and 728C for 45 sec. The resultant PCRproduct was analyzed on a 1% TBE agarose gel.

376 Zhang et al.

For the examination of HBx and survivin, H7402 cellswith transient transfections of H7402-X cells wereharvested and lysed in lysis buffer (62.5 mM Tris-HCl,pH 6.8, 2% SDS, 5% 2mercaptoethanol, 10% glycerol).Equal amounts of protein (30 mg) were separated by 15%SDS–polyacrylamide gel electrophoresis (SDS–PAGE)and transferred onto a nitrocellulosemembrane for 1 hr.The membrane was blocked in blocking buffer (PBS, 5%skim milk, 0.1% Tween 20) at room temperature for2 hr, and then incubated with the appropriate primaryantibody (diluted in blocking buffer). The primaryantibodies included rabbit anti-HBx (1:50,000 dilution),mouse anti-survivin (1:1000 dilution), and mouse anti-b-tubulin (1:500 dilution, Sigma, MI). The membraneswere then washed three times in PBS with 0.1% Tween

20 and incubated for 1 hr in the secondary antibody(peroxidase-conjugated anti-rabbit or anti-mouse IgG).The membranes were then washed three times and thebands were visualized by ECL (Amersham, NJ). Survi-vin levels inH7402-XandL-O2-X cells expressing stablythe HBx gene were examined by Western blot analysisas above.

TABLE I. Expression of HBV Antigens and IAPs in HCC and Cirrhosis Tissues

HBV antigens Hepatoma (%) Paratumor (%) Cirrhosis (%)

HBsAg 20/34 (58.8) 22/34 (64.7%) 23/30 (76.7%)HBcAg 9/34 (26.5) 14/34 (41.2%) 20/30 (66.7%)HBxAg 26/34 (76.5)a 30/34 (85.3%)a 30/30 (100%)a

Survivin 30/34 (88.2)b 31/34 (91.2)b 28/30 (93.3)b

XIAP 19/34 (55.9) 18/34 (53.0) 18/30 (60.0)cIAP-1 9/34 (26.5) 4/34 (11.8) 2/30 (6.7)cIAP-2 10/34 (29.4) 8/34 (23.5) 0/30 (0)

w2 test.aP> 0.05 (vs. survivin).bP< 0.05 (vs. XIAP, cIAP-1, and cIAP-2, respectively).

Fig. 1. Immunohistochemical examination ofHBVantigens and IAPfamily proteins. HBxAg-positive signals were detected in tumor tissue(A) and paratumor tissue (B), whereas the negative control was notdetected in tumor tissue (C). Survivin-positive signals were detected intumor tissue (D), paratumor tissue (E), and cirrhosis tissue (F). XIAP-positive signals were detected in tumor tissue (G). cIAP-1-positivesignals (H) and cIAP-2 positive signals (I) were detected in tumortissue. SABC �100.

Fig. 2. Flow cytometric analysis of apoptosis in H7402 cellstransiently expressingHBX and survivin.A: Flow cytometric analysis.O, mock negative control; V, pcDNA3 empty vector; S, pcDNA3-sur; X,pCMV-X; X/S, both pCMV-X and pcDNA3-sur. B: The percentage ofapoptotic cells was 6.18% (O), 4.53% (V), 4.06% (S), 62.82% (X), and6.83% (X/S), respectively, in the transiently transfected H7402 cells.Each bar corresponds to the average�SD for at least three indepen-dent experiments w2 test **P< 0.01 (x/v vs. x).

Survivin Expression in Hepatoma Tissues 377

RESULTS

High-level Expression of HBxAg andSurvivin in Hepatoma Tissues

Immunohistochemistry showed that HBxAg and sur-vivin were highly expressed in hepatoma samples,corresponding paratumor and liver cirrhosis tissues(Table I). The positive rate of survivin was higher thanthese threemolecules in all three tissue types (P<0.05).The positive rates of HBxAg and survivin were high,with no significant differences between them (eachP>0.05) in HCC (76.5% and 88.2%), paratumor (85.3%and 91.2%), and liver cirrhosis (100% and 93.3%)tissues, respectively. The HBxAg- or survivin-positivecells were generally distributed in a diffuse pattern.Only a few survivin-positive signals were observed inthe nuclei of hepatoma cells; the majority of positivesignals were found in the cytoplasm (Fig. 1). Survivinexpression was noted in 58 out of 60 HBxAg-positivestaining hepatoma, paratumor, and liver cirrhosistissues. Signals were not detected in the negativecontrols.

Survivin Inhibits HBx-induced Apoptosis

The eukaryotic expression vector pcDNA3-sur wasconstructed and used to express survivin in cultur-ed hepatoma cells. Co-transfection of pCMV-X andpcDNA3-sur yielded transient expression of HBx andsurvivin in H7402 hepatoma cells. Flow cytometryrevealed varying percentages of apoptotic cells incultures transfected transiently with the various vec-tors (Fig. 2): 6.18% (mock, without transfection), 4.53%(empty pcDNA3 vector), 4.06% (pcDNA3-sur), 62.82%(pCMV-X), and 6.83% (pCMV-X/pcDNA3-sur). Thus,our results indicate that HBx expression inducedapoptosis in H7402 cells, while survivin expressioninhibited HBX-induced apoptosis.

HBx Protein Upregulates SurvivinExpression in H7402 and L-O2 cells

In transiently transfected cells expressing HBx, theendogenous expression level of survivin protein wasdose-dependent and increased in parallel with the levelofHBx (Fig. 3). StablyHBx-transfectedH7402 andL-O2cells (designated H7402-X and L-O2-X) were select-ed with G418 and confirmed by RT-PCR (Fig. 4) andWestern blot analysis (Fig. 5A,B). Control cells trans-fected stably with empty pcDNA3 vector were desig-nated H7402-P cells. Western blot analysis revealedthat H7402-X and L-O2-X cells showed increased sur-vivin expression (Fig. 5A,B). To investigate the effect ofHBx on survivin expression, HBx gene expression wasgenerated by HBx-specific siRNA in H7402-X cells.Transfection of the pSilencer 3.0-X into H7402-X cells,led to decreased HBx expression over time, as shown byimmunoblotting (Fig. 5C). Western blot analysis re-vealed further that the expression level of survivindecreased in parallel with that of HBx.

DISCUSSION

Hepatitis B virus infection is closely related to thedevelopment of HCC. However, no previous study hasexamined the relationship between HBV antigens andIAP family. In order to demonstrate the effect of HBVantigens on IAP family proteins, the expression levels

Fig. 4. RT-PCR analysis of HBxmRNA inH7402-X cells and L-O2-Xcells. Marker was DL 2000. H7402-X and L-O2-X cells were stabletransfected withHBx gene. H7402 and L-O2 cells were not transfectedwith HBx gene. GAPDH mRNA was a house-keeping gene, which wasused as internal reference.

Fig. 3. Western blot analysis for HBx and survivin protein ex-pression in H7402 cells. A: Western blot analysis. pCMV-X plasmid(1, 2, and 3 mg) was transiently transfected into H7402 cells to test thedose-response relationship between the expression levels of HBx andendogenous survivin. Transfection of pcDNA3 empty vector andMock (untransfected) cells were used as the negative controls. B: Thedensity of bands was analyzed with the Glyco BandScan software(PROZYME1, San Leandro, CA) for Western blot analysis. Each barcorresponds to the average�SD for at least three independentexperiments.

378 Zhang et al.

and distributions of HBV antigens (HBsAg, HBcAg, andHBxAg) and IAPmembers (survivin, XIAP, cIAP-1, andcIAP-2) were examined by immunohistochemistry. Itwas found that the positive rates of HBxAg and survivinwere the highest among the seven proteins in both HCCand liver cirrhosis samples (Table I). There was nosignificant difference between the two positive rates

(P> 0.05), suggesting that there is likely a close rela-tionship betweenHBxAgand survivin. Previously, sero-logical examination and immunohistochemical testsindicated that the existence and active replication ofHBV inHCC and cirrhosis tissues can cause liver tissueinjury [Nakamoto and Kaneko, 2003]. Our data suggestthat the abnormally high expression of survivin protein

Fig. 5. Examination of HBx and survivin proteins by Western blotanalysis. I: Western blot analysis of HBx and survivin protein levelsin HBx-expressing H7402-X and L-O2-X cells. A: Survivin proteinlevels were upregulated by HBx protein expression in H7402 cells.Lane 1: H7402-X, H7402 cells stably transfected with the HBx gene;(Lane2)H7402-P,H7402 cells stably transfectedwith empty pcDNA3;(Lane 3) H7402, mock. B: Survivin protein levels were upregulatedby HBx protein expression in L-O2 cells. Lane 1: L-O2-X, L-O2 cellsstably transfected with the HBx gene; (Lane 2) L-O2 cells transiently

transfected with HBx gene; (Lane 3) II-O2 cells, mock. C: Survivinprotein levels were downregulated by the pSilencer 3.0-X HBx-targeting RNAi vector. Lane 1: H7402-X without treatment;(Lane 2) 24 hr after addition of RNAi; (Lane 3) 36 hr after additionof RNAi; (Lane 4) 48 hr after addition of RNAi. II: The bands wereanalyzed byGlycoBandScan software (PROZYME1, SanLeandro,CA)for Western blot. (A) refers to I A, (B) to I B and (C) to I C. Each barcorresponds to the average�SD for at least three independentexperiments.

Survivin Expression in Hepatoma Tissues 379

in these tissues may act to suppress the HBV-inducedapoptosis of liver cells, leading to retention of injuredcells and subsequent malignant transformations. Thus,the observed high-level expression of HBxAg and sur-vivin in the examined liver tissuesmay play a role in thedevelopment of HCC.We examined further the relationship between HBx

and survivin at the molecular level by transientand stable transfection experiments in hepatoma andnormal human liver cells. Flow cytometric measure-ment of apoptotic cells revealed that the apoptotic rate incells expressing transiently HBx was much higher thanthat in cells expressing both HBx and survivin (Fig. 2),as detected by Western blot analysis (Fig. 3). Theseresults suggest thatHBx induced apoptosis in the testedHCC cells, and that this induction was suppressed bysurvivin. In addition, we observed that endogenous sur-vivin levels increased in a dose-dependent manner inH7402 cells transiently expressing HBx alone (Fig. 3),although the expression levels of survivin proteindecreased over time due to apoptosis of the expres-sing cells. The results in stably HBx-expressing cellssuggest that HBx promotes survivin expression in non-apoptotic hepatoma and normal liver cells (Fig. 5A,B).Furthermore, the experimentswithHBx-targetedRNAidemonstrated that survivin expression decreased instably HBx-expressing cells in which HBx expressionwas depressed (Fig. 5C), suggesting that expression ofHBx protein promotes that of survivin.A previous report showed that survivin-transfected

293 cells grew larger, with more numerous colonieswhen compared with control cells, indicating that theanti-apoptotic gene, survivin, could promote cell trans-formation [Zhu et al., 2002]. Survivin form complexeswith HBXIP, which is able to bind with HBx. Survivin-HBXIP complexes, but not survivin or HBXIP alone,bind pro-caspase-9 and prevent its recruitment to Apaf-1, thereby suppressing selectively apoptosis initiatedvia the mitochondria/cytochrome c pathway. The viralHBX protein also interacts with the Survivin-HBXIPcomplex and suppresses caspase activation in a survi-vin-dependent manner [Gottlob et al., 1998], thusblocking additionally apoptosis initiated via the mito-chondria/cytochrome-c pathway. Our findings are con-sistent with these results.In summary, it was shown that HBx promotes the

upregulation of survivin expression in hepatoma andnormal liver cells, regardless of apoptosis. These find-ings suggest that survivin andHBxmay play importantroles in carcinogenesis of hepatocellular carcinoma.

ACKNOWLEDGMENTS

This work was supported by a grant from the TianjinNatural Scientific Foundation (No. 043113411). Wethank Dr. A Graessmann from Freie University,Germany, forproviding thepCMV-Xplasmid containingthe HBx gene. We further thank Dr. RZ QI from HongKong University of Science and Technology for discuss-ing the manuscript.

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