Cell Culture and Reagents
Huh-7 and Huh-7.5 cells were provided by Apath (Brooklyn, NY). Antibodies specific for IKK, phospho-IKK, phospho-IκB, JNK, phospho-JNK, X-linked inhibitor of apoptosis protein (XIAP), cellular-FLICE inhibitory protein (c-FLIP), and FLAG were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, p65, and horseradish-peroxidase–conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Human recombinant TNF-α was acquired from R&D Systems (Minneapolis, MN). The NF-κB inhibitor, SN50, was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). The JNK inhibitor, SP600125, was purchased from Calbiochem (La Jolla, CA). Recombinant HCV protein core, NS3, NS4, and NS5B were obtained from ViroGen (Watertown, MA). The caspase-3 substrate, Ac-DEVD-AMC, was purchased from Calbiochem.
HCV Preparation and Infection.
The JFH-1 strain (genotype 2a) of HCV was produced by transfecting Huh-7.5 cells with linearized RNA from a plasmid encoding the full genome of JFH-1 HCV (provided by Apath). Huh-7.5 cells were transfected with DMRIE-C reagent (Invitrogen, Carlsbad, CA) using in vitro–transcribed JFH-1. After RNA transfection, cell-culture supernatants at the peak of HCV production were used to infect naïve Huh-7.5 cells. HCV-infected Huh-7.5 cells were passaged, and cell-culture supernatants with the highest HCV production were selected as described previously.39 The selected HCV supernatants were filtered (0.45 μm) and frozen at −70°C until use. Naïve Huh-7 and Huh-7.5 cells were infected with HCV supernatants at a multiplicity of infection (MOI) of 0.01. Cells were subcultured every 3.5 days. At the time of subculture, a portion of the cells was permeabilized and immunostained with an anti-HCV core antibody (Affinity BioReagents, Golden, CO) and FITC-anti-mouse immunoglobulin (Ig) (BD Biosciences, San Jose, CA) to determine the percentage of HCV-infected cells. When >80% of cells were infected, cells were used for TNF-α treatment and further analyses.
The HCV RNA Replicon System.
Huh-7.5 cells carrying the full-length H77 (genotype 1a) replicon were maintained in complete Dulbecco's modified Eagle's medium (DMEM), supplemented with 1 g/L of G418 (A.G. Scientific, San Diego, CA). For elimination of HCV RNA, cells were maintained in complete DMEM, supplemented with 10 μg/L of interferon-beta (IFN-β) instead of G418. After HCV became undetectable, HCV-cured cells were maintained in complete DMEM without IFN-β and G418.
Cell-Death and Viability Assays.
After treatment with TNF-α for 24 hours, cells were washed twice with phosphate-buffered saline (PBS), trypsinized, suspended in binding buffer, and stained with propidium iodide (PI) and Annexin V/allophycocyanin (Pharmingen, San Diego, CA). The stained cells were analyzed on an LSR II flow cytometer (BD Biosciences). A water-soluble tetrazolium (WST)-1 assay was also performed to measure cell viability and cell death. Huh-7 and Huh-7.5 cells were seeded in 24-well plates, and WST-1 reagent (Nalgene, Rochester, NY) was added to each well. After incubation for 2 hours at 37°C in a 5% CO2 incubator, absorbance was measured at 450 nm by using a microplate reader (Bio-Rad, Richmond, CA). A lactate dehydrogenase (LDH) release assay (Promega, Madison, WI) was also carried out according to the manufacturer's protocol.
Cell lysates were separated by standard 10% glycine/sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and probed with antibodies against IKK, IκB, JNK, B-cell lymphoma—extra large (xL), XIAP, c-FLIP, FLAG, GAPDH, and β-actin. Blottings were developed using enhanced chemiluminescence (AbFrontier, Seoul, Korea). Images were captured and band intensities were quantified by the Kodak Image Station (Eastman Kodak, Rochester, NY).
Cells grown in a four-well chamber slides were fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.15% Triton X-100 (Sigma-Aldrich, St. Louis, MO) for 15 minutes, and blocked with 1.5% bovine serum albumin (BSA) for 1 hour. Slides were then incubated with polyclonal anti-p65 or anti-HCV core antibody. After washing with PBS, slides were incubated with FITC or rhodamine-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). Slides were observed under a fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany).
Subcellular Fractionation and Measurement of DNA-Binding Activity of NF-κB.
Huh-7.5 cells were harvested and fractionated into nuclear and cytoplasmic fractions using a nuclear/cytosol fractionation kit (BioVision, Mountain View, CA), according to the manufacturer's protocols. NF-κB activity was monitored using an enzyme-linked immunosorbent assay (ELISA)-based colorimetric TransAM NF-κB p65 kit (Active Motif, Carlsbad, CA), containing a 96-well plate with immobilized oligonucleotides encoding an NF-κB consensus site (5′-GGGACTTTCC-3′). The amount of immobilized NF-κB was determined by colorimetric reaction and absorbance at 450 nm.
Electrophoretic Mobility Shift Assays.
For the binding reaction, 5 μg of nuclear extract was incubated at room temperature for 30 minutes with probe in binding buffer containing 10 mM of Tris-Cl (pH 7.5), 100 mM of KCl, 1 mM of dithiothreitol, 1 mM of ethylene diamine tetraacetic acid, 0.2 mM of phenylmethanesulfonyl fluoride, 1 g/L of BSA, and 5% glycerol. For competition and supershift experiments, nuclear extracts were pretreated with a 100-molar excess of cold oligonucleotide or 1 μg of NF-κB (p50) antibody (Santa Cruz Biotechnology) for 30 minutes before the addition of the labeled probe. Reaction mixtures were analyzed in a 6% polyacrylamide gel and by autoradiography. The sequence of the oligonucleotide used as the probe was 5′-AGTTGAGGGGACTTTCCCAGGC-3′.
Liver tissues of 5 patients with HCV-associated HCC were included in the present study. During the surgical resection of tumor, nontumorous HCV-infected tissues were obtained and frozen at −70°C for RNA extraction. Part of these samples was dissected, formalin-fixed, and paraffin-embedded for immunohistochemistry (IHC). These specimens were provided by the National Biobank of Korea (PNUH, Busan, Korea). Six liver tissues without viral hepatitis were also included in the study. These tissues were obtained during operations, such as cholecystectomy, adrenalectomy, and partial liver resection for intrahepatic duct stones, under the approvement of the institutional review board (Daejeon St. Mary's Hospital, Daejeon, Korea) and the agreement of the patients. Paraffin-embedded tissues were used for IHC to evaluate the expression of XIAP, c-FLIP, and Bcl-xL.
Extraction of RNA, Complementary DNA synthesis, and TaqMan Real-Time Polymerase Chain Reaction.
Total RNA was isolated from liver tissues using the RNeasy Mini Kit (Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized from 800-1,000 ng of total RNA with the First-Strand cDNA Synthesis Kit (Marligen Biosciences, Ijamsville, MD). TaqMan real-time PCR was performed in duplicate to determine mRNA levels of Bcl-xL, XIAP, and c-FLIP using TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA). Target mRNA levels were normalized to an endogenous reference (β-actin).
Cloning of HCV Genes and Transfection.
Genes for individual HCV proteins (i.e., core, E1, E2, NS2, NS3/4A, NS4B, NS5A, and NS5B) were amplified by PCR from a plasmid encoding the full genome of JFH-1 HCV. PCR products were then digested with restriction endonucleases and ligated into the pCMV-3Tag-3A plasmid vector (Stratagene, La Jolla, CA). The nucleotide sequence of each HCV gene was confirmed by DNA sequencing. Transient transfection was carried out using Lipofectamine 2000 (Invitrogen), and transfection efficiency was assessed by immunoblotting for FLAG-tag.
NF-κB Reporter Assay.
Cells were transfected with the luciferase reporter plasmids containing NF-κB responsive elements using Lipofectamine 2000. The pRL-CMV vector (Promega) was used as a control reporter for normalization. Twenty-four hours post-transfection, cells were treated with TNF-α for 6 hours. Cells were lysed, and luciferase activity was determined using the dual-luciferase assay system (Promega), according to the manufacturer's instructions. Luminescence was measured with a Wallac multilabel counter (PerkinElmer Wallac, Gaithersburg, MD).
IKK Activity Assay.
IKK activity was measured using the CycLex IKK-α/β assay kit (MBL International, Woburn, MA), which is a single-site–binding immunoassay. Plates are precoated with a substrate corresponding to recombinant IκB-α, which contains two serine residues that are phosphorylated by IKK-α and IKK-β. We used a peroxidase-coupled anti-phospho-IκB-α S32 monoclonal antibody as a reporter molecule in a 96-well ELISA format.
Data are presented as the mean ± standard error of the mean (SEM). Level of significance for comparisons between two independent samples was determined using Mann-Whitney's test. Groups were compared by one- or two-way analysis of variance (ANOVA), with Tukey's post-hoc test or Bonferroni's post-hoc test applied to significant main effects.