Responses of Nontransformed Human Hepatocytes to Conditional Expression of Full-Length Hepatitis C Virus Open Reading Frame
2007; Elsevier BV; Volume: 171; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.070413
ISSN1525-2191
AutoresWeiliang Tang, Catherine A. Lázaro, Jean S. Campbell, W. Tony Parks, Michael G. Katze, Nelson Fausto,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoHepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. Hepatitis C virus (HCV) is a major cause of chronic hepatitis that can lead to cirrhosis and hepatocellular carcinoma. To study the effects of HCV protein expression on host cells, we established conditional expression of the full-length open reading frame (ORF) of an infectious cDNA clone of HCV (genotype 1a, H77 strain) in the nontransformed human hepatocyte line cell HH4 using the ecdysone receptor regulatory system. Treatment with the ecdysone analog ponasterone-A induced tightly regulated and dose-dependent full-length HCV ORF expression and properly processed HCV proteins. HCV Core, NS3, and NS5A colocalized in perinuclear regions and associated with the early endosomal protein EEA1. HCV ORF expression caused marked growth inhibition, increased intracellular reactive oxygen species, up-regulation of glutamate-l-cysteine ligase activity, increased glutathione level, and activation of nuclear factor κB. Although it was not directly cytotoxic, HCV ORF expression sensitized HH4 cells to Fas at certain concentrations but not to tumor necrosis factor-related apoptosis-inducing ligand. HCV ORF expression in HH4 cells up-regulated genes involved in innate immune response/inflammation and oxidative stress responses and down-regulated cell growth-related genes. Expression of HCV ORF in host cells may contribute to HCV pathogenesis by producing oxidative stress and increasing the expression of genes related to the innate immune response and inflammation. Hepatitis C virus (HCV) infects an estimated 4 million people in the United States and around 170 million worldwide.1Lauer GM Walker BD Hepatitis C virus infection.N Engl J Med. 2001; 345: 41-52Crossref PubMed Scopus (2505) Google Scholar For reasons that are still unclear, the majority of HCV-infected individuals are unable to clear the virus and become chronically infected. Chronic HCV infection causes liver injury, resulting in varying degrees of liver inflammation and associated fibrosis. In 10 to 30 years, chronically infected patients may develop liver cirrhosis and hepatocellular carcinoma. HCV infection is a serious public health problem, and HCV-induced liver disease is the leading indication for liver transplantation in the U.S.2Hoofnagle JH Hepatitis C: the clinical spectrum of disease.Hepatology. 1997; 26: 15S-20SCrossref PubMed Scopus (709) Google Scholar, 3Di Bisceglie AM Hepatitis C and hepatocellular carcinoma.Hepatology. 1997; 26: 34S-38SCrossref PubMed Scopus (432) Google Scholar, 4Alter MJ Kruszon-Moran D Nainan OV McQuillan GM Gao F Moyer LA Kaslow RA Margolis HS The prevalence of hepatitis C virus infection in the United States, 1988 through 1994.N Engl J Med. 1999; 341: 556-562Crossref PubMed Scopus (2441) Google Scholar A major obstacle toward understanding the mechanisms of HCV-induced liver disease has been the lack of suitable cell culture systems and small animal models for HCV replication. The development of HCV subgenomic and genomic replicon systems in 1998 greatly advanced studies on HCV replication.5Lohmann V Korner F Koch J Herian U Theilmann L Bartenschlager R Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line.Science. 1999; 285: 110-113Crossref PubMed Scopus (2497) Google Scholar A breakthrough in 2005 achieved the efficient propagation of HCV JFH-1 virus, an HCV genotype 2a, isolated from a Japanese patient with fulminant hepatitis, in the human hepatoma line Huh-7 cells.6Wakita T Pietschmann T Kato T Date T Miyamoto M Zhao Z Murthy K Habermann A Krausslich HG Mizokami M Bartenschlager R Liang TJ Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat Med. 2005; 11: 791-796Crossref PubMed Scopus (2417) Google Scholar, 7Lindenbach BD Evans MJ Syder AJ Wolk B Tellinghuisen TL Liu CC Maruyama T Hynes RO Burton DR McKeating JA Rice CM Complete replication of hepatitis C virus in cell culture.Science. 2005; 309: 623-626Crossref PubMed Scopus (1954) Google Scholar, 8Zhong J Gastaminza P Cheng G Kapadia S Kato T Burton DR Wieland SF Uprichard SL Wakita T Chisari FV Robust hepatitis C virus infection in vitro.Proc Natl Acad Sci USA. 2005; 102: 9294-9299Crossref PubMed Scopus (1518) Google Scholar This system has been widely used for HCV studies, particularly for those involving the virus life cycle and antiviral defense mechanisms. However, these studies use a transformed cell line as host cells and rely on the infection of an unusual HCV strain. Recently, our laboratory reported the development of a human fetal hepatocyte system in which HCV replication was achieved after transfection of unmodified virus of different genotypes and after infection by serum of patients with chronic HCV infection.9Lázaro CA Chang M Tang W Campbell J Sullivan DG Gretch DR Corey L Coombs RW Fausto N Hepatitis C virus replication in transfected and serum-infected cultured human fetal hepatocytes.Am J Pathol. 2007; 170: 478-489Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar To understand better the interactions between unmodified genotype 1a HCV, the most common type of HCV, and nontransformed human hepatocytes, we designed a system in which the expression of viral proteins would be controlled through a conditional mechanism. In this study, we report the development of conditional expression of the open reading frame (ORF) of an infectious cDNA clone of genotype 1a HCV (H77 strain) in HH4 cells, using the ecdysone-inducible system. HH4 cells, a nontransformed hepatocyte line derived from HPV E6/E7 immortalized adult human hepatocytes, express hepatocytic markers, such as albumin, α-antitrypsin, and transferrin and exhibit a nontransformed phenotype, as assessed by soft-agar assay and nude mouse transplantation. Our results demonstrate that HCV ORF expression in cultured human hepatocytes induced oxidative stress, activated nuclear factor κB (NF-κB), inhibited cell growth, sensitized cells to Fas-mediated apoptosis, and up-regulated genes involved in the innate immune response and inflammation. HH4 human hepatocyte cell line10Yan W Lee H Deutsch EW Lazaro CA Tang W Chen E Fausto N Katze MG Aebersold R A dataset of human liver proteins identified by protein profiling via isotope-coded affinity tag (ICAT) and tandem mass spectrometry.Mol Cell Proteomics. 2004; 3: 1039-1041Crossref PubMed Scopus (63) Google Scholar, 11Wang HC Chang WT Chang WW Wu HC Huang W Lei HY Lai MD Fausto N Su IJ Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes.Hepatology. 2005; 41: 761-770Crossref PubMed Scopus (111) Google Scholar (see description under Results) was cultivated in William's E medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), ITS premix (BD Biosciences, Bedford, MA), 50 μg/ml gentamicin, 0.2 mmol/L l-ascorbic acid-2-phosphate, 10 mmol/L nicotinamide, 2 mmol/L l-glutamine, 0.1 μmol/L dexamethasone, 20 mmol/L HEPES, 1 mmol/L sodium pyruvate, 17 mmol/L sodium carbonate, 14 mmol/L glucose, and 10 mg/L ciprofloxacin. For detection of hepatocytic markers, HH4 cells were cultivated in the above medium except that 10% fetal bovine serum was replaced by human epidermal growth factor (20 ng/ml, BD Biosciences). Phenix-Ampho (PNXA) cells (gift from Dr. Gary Nolan, Stanford University, Stanford, CA) were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, and penicillin (100IU/ml)/streptomycin (100 μg/ml). Zeocin was purchased from Clontech (Mountain View, CA), and hygromycin-B and ponasterone-A (ponA) were obtained from Stratagene (La Jolla, CA). Sources for antibodies used for immunoblot, immunohistochemistry (IHC), and immunofluorescence (IF) are listed in Table 1.Table 1Sources and Concentrations of Antibodies Used in This StudyAntigenSourceDilutionsHCV coreAffinity BioreagentsAIHC, 1:100; IF, 1:100; Immunoblot, 1:3000HCV E1BioDesign (Saco, ME)IF, 1:25; Immunoblot, 1:1000HCV E2BioDesignImmunoblot, 1:400HCV NS3Affinity BioreagentsIF, 1:50; Immunoblot, 1:8000HCV NS5AAustral Biologicals (San Ramon, CA)IF, 1:50HCV NS5ABioDesignImmunoblot, 1:3000AlbuminICN Pharmaceuticals (Aurora, OH)Immunoblot, 1:1000α1-AntitrypsinSigmaImmunoblot, 1:2000α1-FetoproteinDAKO Corp. (Carpinteria, CA)Immunoblot, 1:1000β-ActinSigmaImmunoblot, 1:10,000Caveolin2Santa Cruz BiotechnologyIF, 1:100EEA1Santa Cruz BiotechnologyIF, 1:100Golgi58KSigmaIF, 1:100P450 1A1Santa Cruz BiotechnologyImmunoblot, 1:500P450 3A4Santa Cruz BiotechnologyImmunoblot, 1:500Rab-8PharmingenIF, 1:125SARASanta Cruz BiotechnologyIF, 1:100TransferrinRockland (Gilbertsville, PA)Immunoblot, 1:1000 Open table in a new tab The 5.0-kb PstI/NotI fragment from plasmid pER3 (Stratagene) containing a CMV immediate early promoter driving the ecdysone receptor coding sequence was subcloned into PstI/NotI sites of plasmid pBSIIKS (Stratagene). The 5.0-kb EcoRI/NotI fragment from the resulting plasmid was then ligated into the EcoRI/NotI site of plasmid pLXSZ, derived from pLXSN (Clontech) by replacing the neomycin resistance gene with the zeocin selection gene, to generate the retroviral vector pLCERSZ. The ponA-inducible expression vector for the full-length HCV ORF, pE-HCV, was also constructed in multiple steps. The N-terminal portion of the HCV ORF was generated by PCR as a 259-bp fragment containing newly created XhoI and EcoRI sites at the 5′ end of the coding sequence for the first 80 amino acids of HCV core protein (nucleotides 342 to 580). The C-terminal part of the HCV ORF was a PCR-derived 174-bp fragment containing newly created BamHI sites at the 3′ end, and the coding sequence for the C terminus of NS5B protein (nucleotides 9220 to 9400). The 259-bp XhoI-EcoRI/KpnI fragment and 174-kb BamHI/NotI fragment were sequentially inserted into plasmid pCEP4 (Invitrogen) to make pCEP4-HCVΔ. The NotI site in the ecdysone-inducible expression vector pEGSH (Stratagene) was eliminated by NotI digestion followed by Klenow fill-in to make pEGSH2. The 0.43-kb EcoRI/BamHI fragment from pCEP4-HCVΔ was ligated into the MfeI/BamHI sites of pEGSH2 to make pE-HCVΔ. Finally, the 8.74-kb Acc65I/NotI fragment of p90/HCV-FLpU (a gift of Dr. Charles Rice, Rockefeller University, New York) was inserted into Acc65I/NotI sites of pE-HCVΔ to complete the construction of pE-HCV. The plasmid p90/HCV-FLpU contains an infectious HCV cDNA (genotype 1a; GenBank access no. AF009606), and it was used as template for generating PCR fragments for pE-HCV. All of the PCR-generated fragments were verified by sequencing. Control vectors pE-GFP and pE-Core allow ponA-inducible expression of green fluorescent protein (GFP) and HCV core protein, respectively. The pE-GFP was constructed by ligation of 0.7-kb Acc65I/XhoI fragment of pCEP4-GFP (in which a 0.7-kb NheI/XhoI fragment of pEGFP-C1 was inserted into the NheI/XhoI sites of pCEP4) into the Acc65I/XhoI sites of pEGSH2. pE-Core was made by ligation of a 0.6-kb EcoRI/SalI fragment containing the HCV core coding region into the MfeI/XhoI sites of pEGSH2. Retrovirus production and subsequent transduction of HH4 cells were performed according to a protocol developed by Gary Nolan (Stanford University). In brief, PNXA packaging cells were seeded at 5 × 106 in a 10-cm tissue culture dish and transfected with 25 μg of pLCERSZ plasmid DNA 16 to 24 hours later, using CalPhos Mammalian Transfection kit (Clontech). Cell culture medium was replaced 8 hours after transfection; culture supernatant containing retroviruses was collected 24 hours later and filtered through a syringe-top filter unit with 0.45-μm polyvinylidene difluoride membrane (Millipore, Bedford, MA). Retroviral transduction was performed by incubating recipient cells in culture medium containing 4 μg/ml Polybrene (Sigma, St. Louis, MO) and the retrovirus for 8 hours. Total cellular RNA was extracted from cell monolayers using acid-guanidinium thiocynanate-phenol-chloroform method as previously described.12Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar Probes used were antisense riboprobe (NS5B; nucleotides 8187 to 9220), for the detection of full-length HCV transcript, and a HCV core cDNA fragment (core; nucleotides 602 to 730), for the detection of truncated HCV transcript. The adequacy of RNA sample loading and efficiency of transfer were determined by ethidium bromide staining. Protein samples (20 μg) were resolved on a 12% SDS-polyacrylamide gel, transferred to Immuno-PVDF membrane (Millipore, Bedford, MA), blocked with 5% nonfat milk in 1× PBS plus 0.05% Tween 20, and probed with primary antibodies (Table 1) diluted in 1× PBS plus 0.05% Tween 20 containing 1% bovine serum albumin overnight at 4°C followed by incubation with secondary antibody conjugated with horseradish peroxidase (1:5000; Amersham, Piscataway, NJ) for 1 hour at room temperature. Membrane-bound antibodies were detected with the Pierce Chemiluminescent ECL Detection System (Pierce Biotechnology, Rockford, IL). Even sample loading and efficiency of transfers were routinely assessed via Ponceau-S staining and probing with β-actin antibody. Autoradiographies obtained from Northern blot or immunoblot analysis were scanned with a flatbed scanner and analyzed using NIH Image software (Wayne Rasdand; http://rsb.info.nih.gov/nih-image/Default.html). Blots exposed to phosphor screens were scanned using the Strom PhosphorImager System (Molecular Dynamics, Piscataway, NJ) and analyzed with the accompanied ImageQuant software. PonA-treated or untreated cells were fixed in cold methanol:acetone (1:1, v/v) for 25 minutes, washed in PBS, blocked in PBS containing 5% normal horse serum, and incubated with anti-core antibody (1:100; Affinity Bioreagents, Golden, CO) in blocking solution overnight at 4°C. The presence of anti-core primary antibody was detected using the Vectastain ABC System (Vector Laboratories, Burlingame, CA) in accordance with manufacturer's instructions. Cells were fixed in cold PBS containing 4% paraformaldehyde for 25 minutes, permeabilized in 0.1% Triton X-100 for 5 minutes, and rinsed in cold PBS containing 5% glycine for 15 minutes. Fixed cells were washed in PBS, blocked in PBS containing 10% horse serum for 1 hour at room temperature, and stained with primary antibody diluted in blocking solution overnight at 4°C. After three washes in PBS, cells were then incubated with Alexa Fluor488 or Alexa Fluor594-conjugated secondary antibodies (1:2000; Molecular Probes, Eugene, OR) for 1 hour at room temperature, washed in PBS, and mounted in Immunomount with 4,6-diamidino-2-phenylindole counterstaining (Vector Laboratories). Subcellular organelle markers used are Ds2Red-ER (Clontech) for endoplasmic reticulum, MitotrackerRed (Molecular Probes) for mitochondria, 4,6-diamidino-2-phenylindole (Vector Laboratories) for nucleus, Golgi58k (Sigma) for Golgi apparatus, Rab-8 (Pharmingen, San Diego, CA) for trans-Golgi apparatus/basolateral plasma membrane, SARA and caveolin2 (Santa Cruz Biotechnology, Santa Cruz, CA) for plasma membrane periphery, and Hsp27 (Stressgen, Victoria, BC, Canada) for microtubes. IF microscopy was performed using a Nikon Eclipse E600 fluorescence microscope with a QImaging Retigia EX CCD camera and a ×60 objective. Cell proliferation was measured by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay as previously described.13Kirillova I Chaisson M Fausto N Tumor necrosis factor induces DNA replication in hepatic cells through nuclear factor kappaB activation.Cell Growth Differ. 1999; 10: 819-828PubMed Google Scholar For cell growth rate assay, cell numbers were determined using a crystal violet assay as previously described.14Orsulic S Li Y Soslow RA Vitale-Cross LA Gutkind JS Varmus HE Induction of ovarian cancer by defined multiple genetic changes in a mouse model system.Cancer Cell. 2002; 1: 53-62Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay to measure apoptosis was performed using the In Situ Cell Death Detection kit (Roche Diagnosis, Indianapolis, IN) in accordance with manufacturer's instructions. After hematoxylin counterstaining, labeled nuclei were scored in 1000 cells on each slide. Cell viability was also assessed using trypan blue exclusion assay.15Freshney RI Culture of Animal Cells: A Manual of Basic Technique. Wiley Liss, New York1994Google Scholar The number of trypan blue-stained (dead) and unstained (live) cells were counted, and percent cell death was expressed as (number of blue cells × 100)/number of blue and unstained cells. Subconfluent HCV ORF- and GFP-inducible cells were treated with or without 10 μmol/L ponA for 24 hours followed by incubation with protein A-crosslinked APO-1-3 (Kamiya Biomedical, Seattle, WA), a Fas agonist, at various concentrations (0 to 250 ng/ml) for 36 hours and subjected to the trypan blue exclusion cell death assay. Similarly, a leucine zipper form of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that induces apoptosis in human hepatocytes16Walczak H Miller RE Ariail K Gliniak B Griffith TS Kubin M Chin W Jones J Woodward A Le T Smith C Smolak P Goodwin RG Rauch CT Schuh JC Lynch DH Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.Nat Med. 1999; 5: 157-163Crossref PubMed Scopus (2232) Google Scholar was added at various concentrations (0 to 200 ng/ml) for 8 hours to induce cell death in untreated and ponA-treated (10 μmol/L, 24 hours) HCV ORF and GFP cells. Untreated or ponA-treated (5 μmol/L, 2 or 6 days) subconfluent HCV ORF and GFP cells were stained with or without 2.5 μmol/L RedoxSensorRed for 10 minutes and analyzed immediately by flow cytometry using a FACScan (BD Biosciences). The difference in geometric means of the red fluorescence (600 nm wavelength) measurement in stained and unstained cells for each experimental condition was used as measurement of intracellular reactive oxygen species (ROS) level. Total intracellular glutathione level (GSH, reduced form, plus GSSG, oxidized form) was determined by a modification of the Tietze assay as described previously.17Franklin CC Krejsa CM Pierce RH White CC Fausto N Kavanagh TJ Caspase-3-dependent cleavage of the glutamate-L-cysteine ligase catalytic subunit during apoptotic cell death.Am J Pathol. 2002; 160: 1887-1894Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar For glutamate-l-cysteine ligase (GCL) activity measurements, cells were harvested, cell pellets were washed in PBS before lysis to remove any traces of cysteine remaining from the medium, and GCL-specific activity in the cell lysate was determined as described previously.18Thompson SA White CC Krejsa CM Diaz D Woods JS Eaton DL Kavanagh TJ Induction of glutamate-cysteine ligase (gamma-glutamylcysteine synthetase) in the brains of adult female mice subchronically exposed to methylmercury.Toxicol Lett. 1999; 110: 1-9Crossref PubMed Scopus (54) Google Scholar Binding of the transcription factors NF-κB to oligonucleotide probes containing consensus NF-κB sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′; Santa Cruz Biotechnology) was assessed by electrophoretic mobility shift assay (EMSA) as previously described,19Chaisson ML Brooling JT Ladiges W Tsai S Fausto N Hepatocyte-specific inhibition of NF-kappaB leads to apoptosis after TNF treatment, but not after partial hepatectomy.J Clin Invest. 2002; 110: 193-202Crossref PubMed Scopus (172) Google Scholar using 4 μg of nuclear extract prepared from HH4 cells inducible for HCV ORF or GFP. HCV ORF cells were cultured, in quadruplicate, in the presence or absence of 5 μmol/L ponA for 2 or 6 days with medium replacement every 2 days. One set of these cells was used to confirm ponA-inducible expression of HCV proteins by immunoblot. Total RNA samples were isolated from cultured cells using an acid-guanidinium thiocynanate-phenol-chloroform method as previously described.12Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar Microarray analysis of a single experiment comparing two samples using the Cy3/Cy5 dye reverse technique with an oligonucleotide microarray of approximately 20,000 unique probes was performed in triplicate for each time point as previously described.20Smith MW Yue ZN Korth MJ Do HA Boix L Fausto N Bruix J Carithers Jr, RL Katze MG Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers.Hepatology. 2003; 38: 1458-1467Crossref PubMed Google Scholar Mean ratios between the expression levels of each gene in the analyzed sample pair, SD, and P values were obtained and analyzed using Rosetta Resolver System 3.0 (Rosetta Biosoftware, Seattle, WA). Gene function information was obtained from Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) and GeneCards (http://thr.cit.nih.gov/cards/index.shtml). Differential gene expression detected by microarray was validated using quantitative real-time reverse transcription-PCR (QPCR). Total RNA samples that were used in the microarray analysis were treated with DNase using DNA-free DNase Treatment and Removal Reagents (Ambion, Inc., Austin, TX) to remove residual DNA contamination. Reverse transcription was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Primer and probe sets for each of selected five target sequences [interleukin 1-a (IL1A), CD14, toll-like receptor 2 (TLR2), colony stimulating factor 2 (CSF2), and E-cadherin (CDH1)] were chosen from the Applied Biosystems Assays-on-Demand product list. QPCR was performed on an ABI 7500 Real Time PCR System, using TaqMan chemistry (Applied Biosystems). Each target was run in triplicate, with 20-μl reaction volumes of TaqMan 2× PCR Universal Master Mix (Applied Biosystems). The quantification of each target was normalized to the 18S endogenous control. Values are expressed as mean ± SD. Group means calculated from 3 to 4 independent data sets were compared with the Student's two-tailed unpaired t-test using Prism software (version 4.0 for Macintosh; GraphPad Software, San Diego, CA). Differences with a P value less than 0.05 were considered significant. The HH4 cell line was created by the introduction of the HPV E6 and E7 genes into hepatocytes isolated from a normal adult liver, followed by derivation of a clonal, immortalized cell line. The transduction techniques used to create the HH4 line were similar to those reported for the development of immortalized cell lines from human prostate, pancreas, and melanocytes.21Furukawa T Duguid WP Rosenberg L Viallet J Galloway DA Tsao MS Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16.Am J Pathol. 1996; 148: 1763-1770PubMed Google Scholar, 22Le Poole IC van den Berg FM van den Wijngaard RM Galloway DA van Amstel PJ Buffing AA Smits HL Westerhof W Das PK Generation of a human melanocyte cell line by introduction of HPV16 E6 and E7 genes.In Vitro Cell Dev Biol Anim. 1997; 33: 42-49Crossref PubMed Scopus (64) Google Scholar, 23Schwab TS Stewart T Lehr J Pienta KJ Rhim JS Macoska JA Phenotypic characterization of immortalized normal and primary tumor-derived human prostate epithelial cell cultures.Prostate. 2000; 44: 164-171Crossref PubMed Scopus (37) Google Scholar Immunoblot analysis revealed that HH4 cells expressed hepatocytic markers such as albumin, α1-antitrypsin, and transferrin but not the fetal hepatocyte marker α-fetoprotein (Figure 1A). Exposure of HH4 cells to β-naphtoflavone or phenobarbital resulted in the induction of cytochorme P450 enzymes 1A1 and 3A4, respectively (Figure 1B). These results indicate that cultured HH4 cells retain the expression of adult hepatocytic markers. The growth properties of HH4 cells were analyzed by counting cell numbers of HH4 cultures over a period of 15 days. The average doubling time was 49 hours (58 hours for doubling at low cell density and 34 hours for doubling at high cell density). HH4 cells did not form anchorage-independent colonies in soft-agar assay (data not shown). Furthermore, whereas transplantation of HepG2 human hepatoma cells at 107 cells per mouse led to the formation of large tumors in nude mice by about 56 days, no tumors were detected for at least 111 days in mice transplanted with HH4 cells (at 107 per mouse). HH4 cells transduced with pLCERSZ-derived retrovirus were selected with zeocin for 10 to 14 days. About 20 of the resulting zeocin-resistant clones were each transfected with pEGSH-luc, and their luciferase activities in the presence or absence of ponA were measured. Among all, clone 4E2 exhibited the highest ponA inducibility (∼204-fold) and was chosen as the founder clone. The 4E2 cells were then transfected with pE-HCV, pE-Core, or pE-GFP and selected with hygromycin-B for 10 to 14 days. Stable hygromycin-resistant clones were screened for ponA-induced protein expression using various assays. The HCV ORF- and HCV C-inducible candidates were screened using IHC for HCV core protein, and clones that stained positive for HCV core were further tested for inducible expression of HCV proteins (C, E1, E2, NS3, and NS5A) by immunoblot analysis. For GFP-inducible clones, the resistant cells were cultured in the presence and absence of 10 μmol/L ponA for 24 hours and evaluated for the expression of GFP under a fluorescence microscope. Nearly one-half of 40 clones transfected with either pE-core or pE-GFP expressed these proteins on ponA induction, indicating that the system was fully functional. By contrast, we obtained only 2 full-length HCV ORF-inducible clones after screening 169 clones transfected with pE-HCV. These clones were designated as clones 16 and 50 and will be referred to as HCV-ORF. The low yield of full-length HCV ORF-inducible clones may have been due to the relatively large size of the HCV ORF sequence (approximately 9 kb) in the pE-HCV vector or perhaps because the HCV ORF is unstable in DNA form before integration into host chromosome. Interestingly, about 11% of pE-HCV-derived stable clones that expressed the core protein were inducible for core, E1, and E2 but not NS3 and NS5A proteins. These cells, which expressed part of the HCV ORF encoding the structural proteins, will be referred as HCVΔ cells. To verify that full-length HCV ORF transcript was induced, we performed Northern blot analysis on total RNA samples prepared from HCV ORF-inducible cells of clones 16 and 50. A single RNA transcript of 9.5 kb in length was detected in ponA-treated HCV-ORF cells when probed with 32P-labeled antisense riboprobe corresponding to the NS5B region of HCV ORF (Figure 2A), indicating the induction of full-length HCV ORF in the HCV-ORF cells. The HCV-specific RNA transcript was first detectable at 1 day, reached peak expression level at 2 days, and remained at maximal level for at least 4 days after 5 μmol/L ponA treatment. No RNA transcript was seen in HCVΔ cells when the NS5B riboprobe was used, demonstrating the lack of coding sequences for the C terminus of the HCV ORF. When probed with a 32P-labeled core probe (located at 5′-end of HCV ORF), two HCV-specific RNA transcripts (about 3.2 and 3.8 kb in length) were detected in the HCVΔ cells with induction kinetics similar to that of full-length HCV ORF transcript (Figure 2B). Based on sizes of the HCV transcripts detected in HCVΔ cells, it is likely that truncations of HCV ORF were located between NS2 and NS3 regions. To determine whether the HCV polyprotein was induced and faithfully processed, we investigated the expression of HCV proteins by immunoblot analysis in the two HCV ORF-inducible clones (16 and 50), using a panel of antibodies against HCV C, E1, E2, NS3, and NS5A proteins. We detected HCV proteins only in ponA-treated HCV- ORF cells (Figure 3A), confirming the high inducibility and low basal level of ponA-regulated HCV ORF expression in these cells. In the absence of SDS in the cell lysis buffer, HCV core, NS3, and NS5A proteins were
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