Volume 133, Issue 5, Pages 1649-1659 (November 2007) Hepatitis C Virus Triggers Apoptosis of a Newly Developed Hepatoma Cell Line Through Antiviral Defense System  Haizhen Zhu, Huijia Dong, Erika Eksioglu, Alan Hemming, Mengde Cao, James M. Crawford, David R. Nelson, Chen Liu  Gastroenterology  Volume 133, Issue 5, Pages 1649-1659 (November 2007) DOI: 10.1053/j.gastro.2007.09.017 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 HCV infection in human hepatoma cell lines. (A) JFH-1 HCV particles or controls (JFH-1/GND) were used to infect Huh-7.5 cells. At day 7 after infection, cells were immunostained with mouse monoclonal anti-NS5A antibody. DAPI was used for nuclear counterstaining. (B) H&E section of the well-differentiated hepatocellular carcinoma from a surgical resection specimen. It highlights the thickened trabecular hepatic cords. (C) Morphology of the LH86 cell line derived from hepatocellular carcinoma. The cells form pseudoacinar (left panel) or trabecular (right panel) cell aggregate, resembling the original tumor. (D) JFH-1 HCV particles or controls (JFH-1/GND) were inoculated into LH86 culture, followed by immunostaining with anti-NS5A antibody. DAPI was used for nuclear counterstaining. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 HCV RNA replication in LH86 cells. (A) Viral RNA kinetics determined by real-time RT-PCR analysis in LH86 cells infected with HCV. The viral replication is represented by the HCV genome equivalence (Geq) per microgram total RNA. (B) Viral RNA in cell culture medium of HCV-infected LH86 cells as determined by real-time RT-PCR. The relative viral RNA is calculated as Geq per milliliter medium using a standard curve generated by in vitro transcribed full-length HCV RNA. (C) IFN inhibits HCV RNA replication in LH86 cells in a dose-dependent manner. The data represent the relative RNA levels after 2 days of IFN treatment. (D) Anti-CD81 antibody blocks HCV infection in LH86 cells. Cells were first treated with various concentrations of anti-CD81 antibody for 4 hours before viral inoculation. Viral RNA was analyzed by real-time RT-PCR 5 days after infection. The data are representative of 3 repeats. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 HCV infection causes LH86 cell apoptosis. (A) Huh-7.5 or LH86 cells were incubated with 0.5 MOI JFH1 HCV or JFH-1/GND control for 7 days. The cells were harvested and stained with DAPI. As shown in the figure, apparent nuclear condensation and fragmentation are only seen in LH86 cells infected with HCV. (B) Annexin V expression as determined by flow cytometric analysis. All the cells were harvested for flow cytometric analysis at day 5 postinfection. (C) DNA fragmentation in HCV-infected LH86 cells. Total genomic DNA was extracted from LH86 control cells and HCV-infected cells after 5 days. Approximately 2 μg DNA from control LH86 cells and 2 μg and 1 μg of DNA from infected cells were resolved in 1% agarose gel. The arrows highlight the bands resulting from DNA fragmentation. (D) Kinetics of apoptosis in HCV-infected LH86 cells. LH86 cells were harvested every 24 hours for Annexin V expression determined by flow cytometric analysis. The percentage of cell death is plotted. The data represent the means of 3 experiments. (E) Blocking viral entry or suppression of viral replication reduces LH86 cell apoptosis. LH86 cells were treated with anti-CD81 antibody or 100 IU/mL IFN before viral inoculation. The cells were harvested at day 5 after infection and subjected to Annexin V analysis. The graph shows the percentage of cell death, which represents the mean of 2 experiments. *P = .00013; **P = .0002; ***P = .000019. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 HCV activates the TRAIL death pathway in LH86 cells. HCV induces the expression of (A and B) TRAIL and (C) its receptors, DR4 and DR5, in LH86 cells. Total RNA was isolated from the cells and followed by (A) quantitative RT-PCR analysis or (B and C) real-time PCR analysis. The data were normalized with internal control glyceraldehyde-3-phosphate dehydrogenase. (D) TRAIL knockdown with TRAIL-specific siRNA reduces HCV-induced apoptosis. The graph shows the percentage of cell death determined by Annexin V expression in comparison between HCV-infected LH86 cells with control siRNA and the TRAIL-specific siRNA. The image shows the effectiveness of TRAIL-specific siRNA on knocking down messenger RNA, as determined by RT-PCR analysis with TRAIL-specific primers (E). Forced expression of Bcl-2 in LH86 cells protects HCV-induced apoptosis. LH86 cells were transfected by either control plasmid or Bcl-2 expression construct, followed by HCV infection. The cells were harvested at day 5 after infection and subjected to Annexin V expression analysis. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 HCV infection activates IRF-3 in LH86 cells. The activation of IRF-3 was determined by detection of the dimeric form of IRF-3 by (A) Western blot analysis or (B) immunostaining. LH86 cells were infected with 0.5 MOI HCV. The cells were harvested every 24 hours, and the protein extracts were used for Western blot analysis using an anti–IRF-3 antibody. (B) The cells harvested at day 2 were fixed and used for immunostaining. The arrows highlight the nuclear localization of IRF-3 in the immunofluorescence stain as well as the DAPI counterstain. The 2 images in B were taken from the same area, representing the “mirror image.” Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 HCV infection induces type I IFN production in LH86 cells. LH86 cells were infected with 0.5 MOI. Cells were harvested for total RNA extraction every 24 hours. The kinetics of IFN-β induction was analyzed by real-time RT-PCR assay using IFN-β–specific primers. The data were normalized with internal control glyceraldehyde-3-phosphate dehydrogenase. The data represent the means of 3 independent experiments. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 IRF-3 directly regulates TRAIL expression in HCV-infected LH86 cells. LH86 cells were first transfected with control siRNA or IRF-3–specific siRNA for 24 hours, followed by JFH-1 HCV infection. (A) The cells were harvested for RNA extraction and RT-PCR analysis using TRAIL-specific primers or (B) the cells were harvested for flow cytometric analysis. The data show that knockdown of IRF-3 would reduce TRAIL expression and apoptosis in HCV-infected LH86 cells. The knockdown efficiency was determined by Western blot analysis using (C) anti–IRF-3 antibody. (D) Poly (I:C) induces apoptosis in LH86 cells. LH86 cells were treated with 10 μg/mL (poly I:C) for 3 days, followed by Annexin V analysis. Gastroenterology 2007 133, 1649-1659DOI: (10.1053/j.gastro.2007.09.017) Copyright © 2007 AGA Institute Terms and Conditions