Human Hepatitis B Virus-X Protein Alters Mitochondrial Function and Physiology in Human Liver Cells

The hepatitis B virus-X protein (HBx) regulates fundamental aspects of mitochondrial physiology. We show that HBx down-regulates mitochondrial enzymes involved in electron transport in oxidative phosphorylation (complexes I, III, IV, and V) and sensitizes the mitochondrial membrane potential in a hepatoma cell line. HBx also increases the level of mitochondrial reactive oxygen species and lipid peroxide production. HBx does not activate apoptotic signaling, although it sensitizes hepatoma cells to apoptotic signaling, which is dependent on reactive oxygen species. Increased intrahepatic lipid peroxidation in HBx transgenic mice demonstrated that oxidative injury occurs as a direct result of HBx expression. Therefore, we conclude that mitochondrial dysfunction is a crucial pathophysiological factor in HBx-expressing hepatoma cells and provides an experimental rationale in the investigation of mitochondrial function in rapidly renewed tissues, as in hepatocellular carcinomas. The hepatitis B virus-X protein (HBx) regulates fundamental aspects of mitochondrial physiology. We show that HBx down-regulates mitochondrial enzymes involved in electron transport in oxidative phosphorylation (complexes I, III, IV, and V) and sensitizes the mitochondrial membrane potential in a hepatoma cell line. HBx also increases the level of mitochondrial reactive oxygen species and lipid peroxide production. HBx does not activate apoptotic signaling, although it sensitizes hepatoma cells to apoptotic signaling, which is dependent on reactive oxygen species. Increased intrahepatic lipid peroxidation in HBx transgenic mice demonstrated that oxidative injury occurs as a direct result of HBx expression. Therefore, we conclude that mitochondrial dysfunction is a crucial pathophysiological factor in HBx-expressing hepatoma cells and provides an experimental rationale in the investigation of mitochondrial function in rapidly renewed tissues, as in hepatocellular carcinomas. Hepatitis B virus-X protein (HBx), 1The abbreviations used are: HBx, hepatitis B virus-X; Ab, antibody; CCCP, carbonyl cyanide p-chlorophenylhydrazone; COX, cyclooxygenase; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; DPI, diphenyliodonium; Δψm, mitochondrial membrane potential; HBV, hepatitis B virus; HE, hydroethidine; MDA, malonyl dialdehyde; MPT, mitochondrial permeability transition; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SMP, submitochondrial particle(s); TMRM, tetramethylrhodamine methyl ester; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP end labeling; VDAC, voltage-dependent anion channel; VK3, vitamin K3. 1The abbreviations used are: HBx, hepatitis B virus-X; Ab, antibody; CCCP, carbonyl cyanide p-chlorophenylhydrazone; COX, cyclooxygenase; DCF, dichlorofluorescein; DCFH-DA, dichlorofluorescein diacetate; DPI, diphenyliodonium; Δψm, mitochondrial membrane potential; HBV, hepatitis B virus; HE, hydroethidine; MDA, malonyl dialdehyde; MPT, mitochondrial permeability transition; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; ROS, reactive oxygen species; SMP, submitochondrial particle(s); TMRM, tetramethylrhodamine methyl ester; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP end labeling; VDAC, voltage-dependent anion channel; VK3, vitamin K3. known as a multifunctional protein, is involved in the activation of a wide variety of different enhancer/promoter functions by direct or indirect interactions with transcription factors (1Lara-Pazzi E. Armesilla A.L. Majano P.L. Redondo J.M. Lopez-Cabrerea M. EMBO J. 1998; 17: 7066-7077Crossref PubMed Scopus (88) Google Scholar), activation of signal transduction pathways (2Klein N.P. Schneider R.J. Mol. Cell. Biol. 1997; 17: 6427-6436Crossref PubMed Scopus (218) Google Scholar), sensitization of cells to apoptosis (3Su F. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8744-8749Crossref PubMed Scopus (287) Google Scholar), loss of cell cycle checkpoints (4Benn F. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11215-11219Crossref PubMed Scopus (265) Google Scholar), induction cell growth arrest (5Park U.S. Park S.K. Lee Y.I. Park J.G. Lee Y.I. Oncogene. 2000; 19: 3384-3394Crossref PubMed Scopus (84) Google Scholar), and modulation of proteolytic degradation pathways in cells (6Hu Z. Zhang Z. Doo E. Coux O. Goldberg A.L. Liang T.J. J. Virol. 1999; 73: 7231-7240Crossref PubMed Google Scholar). Circumstantial evidence also suggests that it may play roles directly or indirectly in the genesis of hepatocellular carcinoma (7Kim C.M. Koike K. Saito T. Miyamura T. Gillbert J. Nature. 1991; 353: 317-320Crossref Scopus (1044) Google Scholar, 8Feitelson M.A. Duan L.X. Am. J. Pathol. 1997; 150: 1141-1157PubMed Google Scholar). HBx is therefore an essential viral protein with pleiotropic activity that might act directly or indirectly in the development of hepatocellular carcinoma during chronic hepadnavirus infection. Mitochondria are cellular organelles where vital functions such as electron transfer and oxidative phosphorylation reside. In addition to their role as the supplier of ATP in cells, mitochondria also function as major players in the regulation of cell death (9Zamzami N. Susin S.A. Marchetti P. Hirsh H. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar). There are five enzyme complexes (complexes I-V) involved in oxidative phosphorylation in mitochondria (10Sheffler I.E. Mitochondria. John Wiley & Sons, Inc., New York1999Crossref Google Scholar). Electrons are passed along these respiratory enzymes located in the inner mitochondrial membrane. The energy released by this electron transfer is used to pump protons across the inner mitochondrial membrane, resulting in the establishment of an electrochemical gradient, the mitochondrial membrane potential (Δψm). The Δψm is a component of the overall proton motive force that drives ATP production in mitochondria. Its decrease has been shown to have implications for a variety of pathophysiological conditions, in particular for apoptosis. Cells undergo a decrease in the Δψm caused by the opening of mitochondrial permeability transition (MPT) pores before they exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation) (9Zamzami N. Susin S.A. Marchetti P. Hirsh H. Gomez-Monterrey I. Castedo M. Kroemer G. J. Exp. Med. 1996; 183: 1533-1544Crossref PubMed Scopus (1262) Google Scholar). MPT, which determines the Δψm of mitochondria, is composed of the voltage-dependent anion channel (VDAC) located in the outer mitochondrial membrane and adenine nucleotide translocase in the inner membrane. It has been reported that proapoptotic proteins, Bak and Bax, of the Bcl-2 family, can bind to VDAC and stimulate its opening and decrease of Δψm, leading to cytochrome c release (11Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1909) Google Scholar). A decrease in the Δψm has also been observed in apoptosis triggered by growth factor withdrawal, and Bcl-XL has been shown to prevent the decrease in the Δψm, thereby inhibiting apoptosis (12Heiden M.G. Chandel M.S. Willianmson E.K. Schumacker P.T. Thompson C.B. Cell. 1997; 91: 627-637Abstract Full Text Full Text PDF PubMed Scopus (1233) Google Scholar). A recent report by Shirakata and Koike (13Shirakata Y. Koike K. J. Biol. Chem. 2003; 278: 22071-22078Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) also showed that HBx induces cell death by down-regulating Δψm. Based on these experimental results it is recognized that cells with a low Δψm are committed to undergo apoptosis, whereas those with a high Δψm are capable of exiting the apoptotic pathway (14Heiden M.G. Chandel M.S. Schumacker P.T. Thompson C.B. Mol. Cell. 1999; 3: 159-167Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). Recent reports on HBx interaction with the outer mitochondrial VDAC, VDAC3 (15Rahmani Z. Huh K.W. Siddiqui A. J. Virol. 2000; 71: 2840-2846Crossref Scopus (265) Google Scholar), suggested a key role of HBx in affecting mitochondrial physiology, metabolism, and other relevant functions. HBx association with mitochondria induces oxidative stress, which leads to the activation of a series of transcription factors, nuclear factor-κB and signal transducers and activators of transcription-3 (16Waris G. Huh K.W. Siddiqui A. Mol. Cell. Biol. 2001; 21: 7721-7730Crossref PubMed Scopus (280) Google Scholar). Oxidative stress, imposed either directly by the virus or by the host immune response, has been suggested as a potentially important pathological mechanism in chronic liver diseases (17Kageyama F. Kobayashi Y. Kawasaki T. Toyokuni S. Uchida K. Nakamura H. Am. J. Gastroenterol. 2000; 95: 1041-1050Crossref PubMed Google Scholar). A link between oxidative stress and pathogenesis is supported by a pilot study of antioxidant therapy which suggested improvement in liver injury in chronic hepatitis C (18Okuda M. Li K. Beard M.A. Showalter L.A. Scholle F. Lemon S.M. Weinman S.A. Gastroenterology. 2000; 122: 366-375Abstract Full Text Full Text PDF Scopus (798) Google Scholar). Little is understood about the mechanism that produces oxidative stress and its effect on cells after HBV infection. In this report, we show that HBx alters mitochondrial functions; we also demonstrate down-regulation of enzymes in electron transport in oxidative phosphorylation, sensitization of the Δψm to inhibitors of electron transport, and sensitization of hepatoma cells to apoptotic stimuli. These results together with the observed increase in the cellular abundance of ROS with a consequential increase in cellular lipid peroxidation shed new light on the physiological significance of the HBx effect on mitochondria which can contribute to liver disease associated with HBV infection.EXPERIMENTAL PROCEDURESCell Lines and Materials—The open reading frame of HBx of pHBV-D plasmid (19Kim Y.S. Kang H.S. Kor. Biochem. J. 1984; 17: 70-79Google Scholar) was cloned into the pMAMneo expression vector (Clontech) and named pMAMneo-X. Hepatoma cells and HBx-transfected hepatoma cells (20Lee Y.I. Bong Y.S. Poo H.R. Lee Y.I. Park J.G. Oh S.O. Sohn M.J. Lee S. Park U.S. Kim N.S. Hyun S.W. Gene (Amst.). 1998; 207: 111-118Crossref PubMed Scopus (15) Google Scholar) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (JBI, Daegu, Korea), 2 mm glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. The in situ cell death detection kit for the TUNEL assay was from Roche Applied Science. The anti-cytochrome c monoclonal antibody was from Pharmingen (San Diego). The monoclonal antibody against the DNA repair enzyme, poly(ADP-ribose) polymerase (PARP), was from Enzyme System Products (Dublin, CA). Tetramethylrhodamine methyl ester (TMRM), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and hydroethidine (HE) were from Molecular Probes, Inc. (Eugene, OR). Fluorescein isothiocyanate-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch Laboratory (West Grove, PA). Carbonyl cyanide p-chlorophenylhydrazone (CCCP), glutathione, Hoechst 33258, propidium iodide, staurosporine, and vitamin K3 (VK3) were from Sigma.Differential Display-PCR Analysis—Differential display-PCR analysis for the detection of differentially expressed genes in HepG2 and HBx-transfected HepG2-3X cells was performed essentially by the method of Liang and Pardee (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4687) Google Scholar) using the RNAmap kit according to the manufacturer's instructions as described previously (22Oh S. Kim N.S. Lee Y.I. Mol. Cells. 1998; 8: 212-218PubMed Google Scholar).Submitochondrial Particle Preparation and Immunoblotting—HepG2 and HepG2-1X, 3X, and 4X cell mitochondria were prepared according to Bhat et al. (23Bhat N.K. Niranjan B.G. Avadhani N.G. Biochemistry. 1982; 21: 2452-2460Crossref PubMed Scopus (43) Google Scholar) and stored in 250 mm sucrose suspension at -20 °C. Submitochondrial particles (SMP) were prepared essentially as described by Pedersen et al. (24Pedersen P.C. Greenawalt J.W. Reynafarje B. Hullihen J. Decker G.I. Soper J.W. Bustamente E. Methods Cell Biol. 1978; 20: 411-481Crossref PubMed Scopus (380) Google Scholar), except that EDTA was omitted from the sonication medium. Briefly, frozen mitochondrial suspension was thawed and diluted with 250 mm sucrose to a concentration of about 20∼30 mg/ml. The mitochondria were then subjected to sonication for 2 min at the maximal output with a Branson sonifier in an ice bath under a nitrogen stream. The suspension was decanted and centrifuged at 105,000 × g for 50 min. The resulting pellet, consisting of SMP, was washed and suspended in 250 mm sucrose. Proteins were determined by the usual biuret method using bovine serum albumin as standard. For immunoblot analysis, proteins were run on 12% SDS-urea gel and transferred to nitrocellulose membrane. Transferred proteins were probed with cytochrome oxidase I, II, III, IV, and VI-b mouse monoclonal antibodies (Molecular Probes). Reactive bands were detected using the enhanced chemiluminescence (ECL) Plus™ detection reagent (Amersham Biosciences).Measurement of Enzyme Activities—NADH-CoQ oxidoreductase (complex I) activity in SMP from HepG2 and HBx-expressing HepG2 cell lines was assayed by measuring the NADH disappearance at A340 nm as described previously (25Estornell E. Fato R. Pallotti F. Lenaz G. FEBS Lett. 1993; 332: 127-131Crossref PubMed Scopus (191) Google Scholar). Briefly, 10∼15 μg of SMP was diluted in an assay medium (50 mm KCl, 10 mm Tris-HCl, 7.4, 1 mm EDTA, 2 mm KCN, pH 7.4), and 75 μm NADH and electron acceptor 40 μm decylubiquinone were added for enzyme activity assay. Oxidation of NADH (NADH disappearance) was followed at A340 nm after adding 40 μm decylubiquinone, and the values were expressed as nmol/min/mg of SMP. Succinate:ubiquinone oxidoreductase (complex II) activity was measured by following the reduction of ubiquinone observed at 278 nm using an extinction coefficient of 14.7 cm-1 mm as described (26Kita K. Vibat C.R. Meinhardt S. Guest J.R. Gennis R.B. J. Biol. Chem. 1989; 264: 2672-2677Abstract Full Text PDF PubMed Google Scholar). 30 μm decylubiquinone was used as the electron acceptor. Ubiquinone-cytochrome c oxidoreductase (complex III) activity was measured using diode array spectrophotometer (Agilent 8453, Palo Alto, CA) by following the increase in reduced cytochrome c absorbance at 540 nm. The SMP sample (10 μg) was added to 3 ml of the assay mixture (50 μm ferricytochrome c in 50 mm phosphate buffer, pH 7.2), and the reaction was started by the addition of 30 μm decylubiquinone. The activity was calculated using an extinction coefficient of 19.1/cm/mm for reduced cytochrome c. The specific activity of the enzyme is expressed as nmol of cytochrome c reduced/min/mg of SMP. Cytochrome c oxidoreductase (complex IV) activity in SMP was assayed by measuring the rate of oxidation of ferrocytochrome c at 550 nm. The reaction medium contained 50 mm sodium phosphate, pH 7.0, 1% sodium cholate, 80 μm ferrocytochrome c, 1 mm EDTA, and 1∼2 μg of SMP protein in a total volume of 1 ml. Ferrocytochrome c concentrations were determined using an extinction coefficient (of 21.1/cm/mm at 550 nm) and the values expressed as nmol/min/mg of SMP. ATP synthase (complex V) activity in SMP was assayed by measuring ATP formation as described previously (27Das A.M. Mol. Genet. Metab. 2003; 79: 71-82Crossref PubMed Scopus (93) Google Scholar).Flow Cytometric Analysis of the Mitochondrial Membrane Potential—HepG2 and HBx-transfected HepG2 cells were plated on 100-mm Petri dishes. After a 48-h incubation with complete medium, the cells were treated by FACScan flow cytometer according to the instructions of the manufacturer. Then, cells were trypsinized, transferred to 15-ml Falcon tubes, and centrifuged at 1,200 rpm for 3 min. The supernatant was removed, and the pellet was washed with 5 ml of PBS. The cells were resuspended in complete medium containing the fluorochrome TMRM at a final concentration of 150 nm. After incubation for 30 min at 37 °C, the emission fluorescence was measured using the FL2 channel (57,526 nm) of a FACScan flow cytometer at 549 nm (excitation) and 573 nm (emission). Values for mitochondria with depleted Δψm were determined by simultaneous treatment of cells with the protonophore CCCP (final concentration, 100 nm) and TMRM.Flow Cytometric Analysis of the Reactive Oxygen Species—HE and DCFH-DA were used to measure ROS. HE and DCFH-DA were used to measure the production of superoxide and hydrogen peroxide, respectively. Superoxide oxidized HE to yield ethidium bromide, and hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF). HepG2 or HepG2-3X cells were pretreated with 5 μm DCFH-DA for 1 h at 37 °C. For the measurement of the superoxide anion, 10 μm HE was treated for 15 min at 37 °C. Intracellular superoxide anion levels were assessed by ethidium bromide fluorescence using a FACScan flow cytometer with excitation and emission settings of 488 nm and 600 nm, respectively.Measurement of Lipid Peroxidation Products—Appropriate cell culture (5 × 106 cells) lysates were prepared by sonication and stored at -70 °C after the addition of 5 mmol/liter butylated hydroxytoluene (Sigma). 4-Hydroxyalkenals and malondialdehyde were measured in these homogenates using a commercial assay kit (LPO-586; OXIS International, Inc., Portland, OR). The protein concentration was determined by a Coomassie assay (Pierce).Detection of DNA Fragmentation, TUNEL Assay, and Hoechst Staining—DNA fragmentation was evaluated qualitatively by agarose gel electrophoresis as described previously (28Lee Y.I. Park S.M. Lee Y.I. J. Biol. Chem. 2001; 276: 16969-16977Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). To confirm the effect of VK3 and the antioxidant pyruvate or glutathione on HBx-sensitized HepG2 cells, 1 × 105-4 × 105 cells were plated on coverslips in 6-well plates and grown to 60-80% confluence. Cells were washed with PBS and 100 μl paraformaldehyde (4% in PBS, pH 7.4) for 30 min at room temperature. After washing once with PBS, cells were permeabilized with 100 μl of permeabilization buffer (0.1% Triton X-100, 0.1% sodium citrate) for 2 min at 4 °C. The TUNEL reaction was carried out by incubating cells in a moisture chamber for 1 h at 37 °C with TUNEL reaction mixture (Roche Applied Science) in a total reaction volume of 50 μl. After washing three times, samples were analyzed under a fluorescence microscope. Hoechst staining was done according to the manufacturer's instructions as described previously (28Lee Y.I. Park S.M. Lee Y.I. J. Biol. Chem. 2001; 276: 16969-16977Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar).Detection of Bax Content—For the detection of Bax, mitochondrial and cytosolic fractions (15 μg of protein) were analyzed by SDS-PAGE in a 15% gel with equal amounts of protein loaded into each well as determined by the Bradford assay (Bio-Rad). Kaleidoscope prestained standards (Bio-Rad) were used to determine the molecular mass. The gels were then electroblotted onto polyvinylidene difluoride transfer membranes (Amersham Biosciences). For the detection of Bax, anti-mouse IgG against Bax (Santa Cruz Biotechnology, Santa Cruz, CA) was used as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody. ECL was used for detection of Bax expression.Analysis of PARP Cleavage and Immunoblotting—For Western blot analysis of PARP cleavage, both floating and attached cells were rinsed in cold PBS, pH 7.4, and then collected with 2 ml of lysis buffer (62.5 mm Tris, pH 6.8, 8 m deionized urea, 10% glycerol, 2% SDS, and protease inhibitors). The cells were then sonicated on ice for 20 s. After the addition of loading buffer, the samples were incubated at 65 °C for 15 min, and equal amounts of protein were resolved on a 7.5% SDS-polyacrylamide gel. Immunoblotting for PARP cleavage was performed using a monoclonal antibody that specifically detects human PARP at a 1:2,000 dilution. Visualization of the signal was by ECL.Production of HBx Transgenic Mice—The HBx transgenic mice generated using a pHEX-1 expression vector were described previously (29Yu D.Y. Moon H.B. Son J.K. Jeong S. Yu S.L. Yoon H. Han Y.M. Lee C.S. Park J.S. Lee C.H. Hyun B.H. Hyun B.H. Murakami S. Lee K.K. J. Hepatol. 1999; 31: 123-132Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar).Immunohistochemical Staining Analysis—The immunohistochemical staining for lipid peroxidation products and HBx protein was basically followed as described previously (29Yu D.Y. Moon H.B. Son J.K. Jeong S. Yu S.L. Yoon H. Han Y.M. Lee C.S. Park J.S. Lee C.H. Hyun B.H. Hyun B.H. Murakami S. Lee K.K. J. Hepatol. 1999; 31: 123-132Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). For the primary antibodies, a HBx antibody (30Cheong J.H. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 142-150Crossref Scopus (239) Google Scholar) and a malonyl dialdehyde-specific antibody (MDA-Ab) (31Montine T.J. Amaranath V. Martin M.E Strittman H.J. Graham D.G. Am. J. Pathol. 1996; 48: 89-95Google Scholar) for lipid peroxidation products were used. For the secondary antibody, a universal secondary antibody (DACO, Foster City, CA) was used. Sections were labeled with peroxide-conjugated streptavidin (DACO) for 10 min, incubated in diaminobenzidine, and washed in Immuno/DNA buffer solution (Research Genetics, Huntsville, AL). Finally, the slides were counterstained with Mayers hematoxylin, washed in distilled water, and mounted with universal mount (Research Genetics). The specificity of immunohistochemical staining was verified using PBS in place of primary antibodies. Negative controls always gave negative staining of the tissues.RESULTSThe HBx Protein Down-regulates Mitochondrial Enzymes Involved in Oxidative Phosphorylation—Human hepatoma cell lines expressing the HBx protein in HepG2 (1X, 3X, and 4X) and HepG3B (6X and 8X) cells were established and characterized (20Lee Y.I. Bong Y.S. Poo H.R. Lee Y.I. Park J.G. Oh S.O. Sohn M.J. Lee S. Park U.S. Kim N.S. Hyun S.W. Gene (Amst.). 1998; 207: 111-118Crossref PubMed Scopus (15) Google Scholar). By using these cell lines we screened the genes that are expressed differentially by HBx. We employed the differential display-PCR technique, using the RNAmap kit (GenHunter Corp, Brookline, MA) according to the manufacturer's instructions. Total RNA from HepG2 and HepG2-3X cell lines was reverse transcribed into cDNA with three anchored oligo(dT) primers. Selected portions were amplified by PCR with the first strand cDNA primers and with eight different kinds of 5′-arbitrary primers as described in the RNAmap kit. The PCR products were separated on a 6% denaturing polyacrylamide gel. We repeated the same experiment three times with the same primer set and obtained similar results (data not shown). The PCR products that showed differences in band intensity in HBx-transfected and nontransfected cells were cloned into the PTZ18R vector (Amersham Biosciences). These cDNAs were sequenced by automatic sequencing methods and compared with the GenBank, EMBL, and SWISS-PROT data bases. Among the isolated cDNAs, three cDNAs showed more than 99% identity to mitochondrially encoded subunit I of cytochrome c oxidase, ATP synthase subunit 6, and NADH-CoQ oxidoreductase. Three different cDNA fragments (NADH-CoQ oxidoreductase, cytochrome c oxidase I, and ATP synthase subunit 6) as mentioned above were used as probes for Northern blot analysis containing total RNA of the HepG2, Hep3B, and HBx-transfected HepG2, Hep3B cells (HepG2-1X, 3X, and 4X; Hep3B-6X and 8X). As shown in Fig. 1A-I, mRNAs of the expected size of each product were detected. The intensity of the signals of each mRNA was 50-80% lower in HBx-transfected cells than in the control (Fig. 1A-I). Among the five enzyme complexes involved in steps of oxidative phosphorylation in mitochondria, we isolated three enzymes involved in electron transport. Because there are two more complexes involved in oxidative phosphorylation (complex III, ubiquinone-cytochrome c oxidoreductase; complex II, succinate:ubiquinone oxidoreductase), we did Northern blot analysis using respective gene probes as shown in Fig. 1A-II. The intensity of expression of ubiquinone-cytochrome c oxidoreductase (complex III) in each HBx-transformed cell line was lower, whereas the succinate:ubiquinone oxidoreductase (complex II) signal was unchanged compared with control HepG2 cells (Fig. 1A-II). The pYKM ND-5 vector, which expresses the 70-amino acid N-terminal deleted mutant form of HBx and showed defects in transactivating various promoters (32Kim Y.H. Kang S.K. Lee Y.I. Biochem. Biophys. Res. Commun. 1993; 197: 894-903Crossref PubMed Scopus (23) Google Scholar), was used for transformation of the HepG2 cell line. The resulting cell line HepG2-ND5X was used as a control. The enzyme activities of complexes I-V were measured on SMP from HepG2, HepG2-1X, 3X, and 4X cell lines (Table I). Enzyme activity analysis also showed that HBx down-regulates the enzyme activities involved in oxidative phosphorylations (complexes I, III, IV, and V) (Table I). These results further confirmed that HBx down-regulates the enzymes that are specifically involved in electron transport and proton translocations (complexes I, III, IV, and V), whereas it does not down-regulate complex II, succinate:ubiquinone oxidoreductase, which is involved in electron transport but not in proton translocations in mitochondria. The research on the molecular mechanism of HBx down-regulation of complexes I, III, IV, and V but not complex II has progressed in this laboratory. To verify the specificities of the enzyme activities we treated inhibitors of electron transport or uncoupling of oxidative phosphorylations in each complexes and found that inhibitors specifically abolish the enzyme activities as shown in Table I. Next we questioned whether the same reduction in expression was observed in cytochrome c oxidase subunits other than subunit I, so the expression of four subunits (subunits II, III, IV, and VI-b) in HepG2, Hep3B, and HBx-transfected cells was analyzed. Similar to Fig. 1A, mitochondrial encoded cyclooxygenase (COX) subunits (COX I, II, and III) showed consistent underexpression in HBx-transfected cells (Fig. 1B-I), whereas the levels of nuclear encoded subunits IV and IV-b, among 10 nuclear encoded subunits, did not show differences in the levels of the control (HepG2 and Hep3B) (Fig. 1B-II). These results also revealed another interesting result showing that only the mitochondrially encoded subunit genes are down-regulated by HBx, whereas nuclear encoded subunit genes are not affected by HBx. We did the Western blotting analysis using COX I, II, III, IV, and VI-b antibodies to confirm the Northern results as shown in Fig. 1C. The Western analysis results also showed that mitochondrially encoded subunit genes are down-regulated by HBx, whereas nuclear encoded subunit genes are not affected by it. Immunohistochemical analysis of COX I (encoded by mitochondrial DNA) and COX IV (encoded by nuclear DNA) showed that both COX I and COX IV were expressed in HepG2 cells, whereas COX I expression was decreased dramatically in HepG2-3X; this further confirmed that HBx down-regulates mitochondrial encoded COX subunits at the protein level (Fig. 1C, I and II).Table IComplex I, II, III, and IV enzyme activities of SMP in HepG2, -1X, -3X, and -4X cell lines and inhibitors effect on each enzymesActivitiesHepG21X3X4XHepG2Rotenone (1.25 μm)TTFA (12.5 μm)Antimycin A (12.5 μm)Azide (500 μm)Oligomycin B (6.25 μm)nmol·min−1mg−1 SMPComplex I (NADH-CoQ oxidoreductase)82729231327547Complex II (succinate:ubiquinone oxidoreductase)1,8621,7361,8291,832262Complex III (ubiquinone-cytochrome c oxidoreductase)1,726632718536136Complex IV (cytochrome c oxidoreductase)2,376897976732128Complex V (ATP synthase)3,8241,5231,2311,421432 Open table in a new tab HBx Expression Increases Cellular ROS Productions in Hepatoma Cells—Because we demonstrated that HBx down-regulates genes involved in oxidative phosphorylation at mitochondria, a major ROS generator, we questioned whether HBx induces ROS production in HBx-transfected cells. We detected intracellular ROS production by staining with the hydrogen peroxide-sensitive fluorescent dye DCFH-DA, and the H2O2 levels were measured using a FACScan flow cytometer with excitation and emission settings of 488 and 530 nm, respectively, and with a laser scanning confocal microscopy (Fig. 2, A and B). As shown in Fig. 2, A and B, the DCF fluorescence displayed an increase in HBx-transfected HepG2 cells, whereas GSH or pyruvate treatment showed a decline in the fluorescence to the basal level (Fig. 2B). We next quantified the total cellular lipid peroxidation products in extracts from these cells (Fig. 2C). This information provides a measure of the consequences of oxidative stress because peroxidation of lipids is a result of elevated cellular ROS. The HBx expression resulted in a significant increase in the abundance of lipid peroxide products in both HepG2-3X and Hep3B-6X cells (Fig. 2C). This result confirms that HBx protein expression induces oxidative injury in these cells. The Δψm in HepG2 and HBx-transfected HepG2 cells was measured using the fluorochrome TMRM. For the positive control, 50 μm CCCP was added. Cells were analyzed with a FACScan flow cytometer. As shown in Fig. 2D, there was no difference in the Δψm between HepG2 and HBx-expressi