We developed hepatitis C virus (HCV) core-E1-E2 and HCV core transgenic mice on a common genetic background to assess the contribution of HCV structural proteins to hepatocarcinogenesis. Eight-week-old core-E1-E2, core, and nontransgenic mice inbred on the FVB×C57Bl/6 background were treated with diethylnitrosamine (DEN) and sacrificed at 32 weeks old. Proliferation and apoptosis were assessed by immunohistochemistry. The effect of viral proteins on apoptosis was evaluated in HepG2 cells in which apoptosis was induced by anti-Fas antibody. HCCs were identified at 32 weeks in the majority of DEN-treated mice from all three groups. The mean size of HCCs was significantly larger in core-E1-E2 transgenic (4.63 ± 1.48 mm), compared with core transgenic (0.78 ± 0.26 mm, P = .01), and nontransgenic (1.0 ± 0.19 mm, P = .002) mice. While there were no differences in proliferation, the apoptotic index in core-E1-E2 transgenic HCCs was significantly lower than those found in core and non-transgenic HCCs. Core-E1-E2 transfected HepG2 cells demonstrated a significantly lower apoptotic index (0.35 ± 0.11) compared with that of core transfected cells (0.74 ± 0.07, P = .0103). Analysis of a Fas-induced apoptosis model in HCV transgenic mice confirmed that core-E1-E2 transgenic liver underwent significantly less apoptosis than transgenic tissue expressing core only. In conclusion, HCV core-E1-E2 transgenic mice develop significantly larger tumors than transgenic mice expressing core alone or nontransgenic mice. The accelerated tumor phenotype is attributable to suppression of apoptosis rather than enhanced proliferation. These data implicate HCV E1 and/or E2 in conjunction with core as antiapoptotic, tumor accelerator proteins. (HEPATOLOGY 2005; 41:660–667.)
Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, and 2.7 million people in the United States harbor active HCV infection.1 Chronic HCV infection has been independently established as a leading cause of hepatocellular carcinoma (HCC). Unlike hepatitis B virus (HBV), the RNA genome of HCV does not integrate into the host chromosome. HCV-related hepatocarcinogenesis is, therefore, not likely to involve insertional mutagenesis. Rather, it has been hypothesized that HCV produces HCC through the cumulative effects of chronic infection, injury and repair. Whether HCV proteins are directly oncogenic has not been established. Unfortunately, the lack of an appropriate small animal model of HCV has impeded progress in defining the molecular mechanisms of HCV-induced carcinogenesis.
We originally developed an HCV transgenic mouse model encoding the core, E1, and E2 structural proteins under the control of the albumin promoter on the FVB background; despite high-level protein expression, this model did not develop hepatic pathology.2 However, more recently, Moriya et al. developed a transgenic mouse model on the C57Bl/6 background that overexpresses HCV core protein under the control of the HBV enhancer that is capable of inducing HCC in a subset of animals following the development of steatosis.3–6 These apparently contradictory findings could be due to inherent differences in the transgenes, HCV core dose effect, mouse genetic background, or a combination of these.
To reconcile these findings, we expressed the HCV core and HCV core-E1-E2 transgenes on a common mouse genetic background (FVB×C57Bl/6). We additionally studied whether HCV acts as a cocarcinogen in a diethylnitrosamine (DEN)-induced model of hepatocarcinogenesis.
HCV, hepatitis C virus; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; AI, apoptotic index; PI, proliferation index.
Materials and Methods
Alb-core-E1-E2 Transgenic (Core-E1-E2 Tg).
The PvuI-NsiI fragment from pAlb-HCV, containing the core, E1, and E2 regions, was purified and microinjected into mouse oocytes from FVB-inbred mice (Taconic, Germantown, NY) as previously described.2 Transgenic mice were identified by subjecting 1 μg of tail DNA to PCR amplification using the HCV core primers (5′-ATGAGCACAAATCCTAAACCTC-3′ and 5′-CAAGCGGAATGTACCCCATGAG-3′). The resulting 418- bp fragment was visualized on 1.2% agarose gels. From the original seven lines created, we selected the line with the strongest core protein expression, AC 1-0,2 as the founder. This line was then mated with wild-type C57Bl/6 mice (Taconic) to produce an HCV core-E1-E2 line on a hybrid background (FVB×C57Bl/6).
HBV enh-core Transgenic (Core Tg).
A 1.2-kb KpnI-HindIII fragment containing HCV core transgenic construct (pBEP39), previously used in the study associating HCV core with HCC6 was generously provided by Dr. Kazuhiko Koike (University of Tokyo, Tokyo, Japan).3–6 Seven transgenic mouse lines were created on the C57Bl/6 background, and HCV core protein expression was confirmed by Western blotting using a mouse anti-hepatitis C core monoclonal antibody (gift from Dr. Michinori Kohara, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan),7 and one line with the strongest core expression was selected as the founder. Transgenic mice were identified by PCR using HCV core primers (5′-GCCCACAGGACGTTAAGTTC-3′ and 5′-TAGTTCACGCCGTCCTCCAG-3′) yielding a 438 bp fragment. Again, animals from the founder line were mated with wild type FVB mice (Taconic) to produce an HCV core line on a hybrid background (FVB×C57Bl/6). Mice were housed in a controlled environment and fed regular sterile mouse chow and water. All procedures involving animals were approved by the Institutional Animal Care and Use Committee.
Diethylnitrosamine (DEN) Treatment
Eight-week-old male Alb-core-E1-E2 transgenic (core-E1-E2 Tg), HBV enh-core transgenic (core Tg), and nontransgenic (non-Tg) mice on the FVB×C57Bl/6 background were divided into 6 groups: (1) core-E1-E2 Tg with DEN (n = 15); (2) core Tg with DEN (n = 8); (3) non-Tg with DEN (n = 18); (4) Alb-core-E1-E2 Tg without DEN (n = 3); (5) HBV enh-core Tg without DEN (n = 5); and (6) non-Tg without DEN (n = 4) (Table 1). Animals from groups 1, 2, and 3 were injected intraperitoneally with 75 mg/kg body weight of DEN weekly for 3 weeks and then 100 mg/kg of DEN weekly for 3 weeks according to the protocol of Shiota.8 As controls for DEN effects, animals from groups 4, 5, and 6 received saline intraperitoneal injections.
Table 1. Schedule of Treatments
NOTE. Animals were injected intraperitoneally with 75 mg/kg body weight of DEN weekly for 3 weeks and then 100 mg/kg of DEN weekly for 3 weeks. Control groups received saline intraperitoneal injections.
DEN 75 mg/kg
DEN 75 mg/kg
DEN 75 mg/kg
DEN 100 mg/kg
DEN 100 mg/kg
DEN 100 mg/kg
DEN-treated mice were sacrificed at 16, 24, and 32 weeks of age. Control animals, treated with saline only, were sacrificed at 32 weeks to determine the background HCC rate. HCC tissues and matching nonmalignant tissue from the animals in groups 1, 2, and 3 were isolated and immediately frozen in OCT cryostat embedding compound (Tissue-Tek, Torrance, CA) or placed in 10% formalin for histological analysis and a portion of each tissue was stored frozen at −80°C for protein analysis. Histological evaluation of the formalin-fixed liver tissues by H&E staining was also carried out by a pathologist (L.Z.) in a blinded manner.
Immunohistochemical Analysis of Tumor Cell Proliferation
Proliferation index was evaluated by immunohistochemical analysis of fresh frozen liver tissue sections using anti-Ki67 antibody. Cryostat sections (6μ) of the unfixed frozen samples were cut, air dried for 1 hour, and fixed in cold acetone for 10 minutes. Samples were then incubated with blocking buffer (1% bovine serum albumin) for 30 minutes at room temperature. Endogenous peroxidase activity was blocked by immersing in 0.3% H2O2 and methanol for 30 minutes. Mouse anti-Ki67 polyclonal antibody (Novocastra Laboratories Ltd., Newcastle, UK) was applied at a 1:500 dilution for two nights at 4°C. After washing, specimens were incubated with a peroxidase-labeled anti-mouse goat IgG F(ab′)2 fragment (DakoCytomation, Carpinteria, CA) at 1:500 dilution for 3 hours at room temperature, followed by 3,3′-diaminobenzidine tetrahydrochloride (DAB; DakoCytomation) as chromogen. Nuclei were counterstained with methylgreen. All positive and negative cells were counted in each of four randomly selected high power fields (40×) for each tumor and nontumor tissue. The difference in cell number between groups was compared using two-tailed unpaired Student t test. P values less than .05 were considered to be significant.
In situ detection of DNA fragmentation was carried out using a terminal deoxyribonucleotidyl transferase mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay (ApopTag In Situ Apoptosis Detection kit; Serological Corp., Norcross, GA). Cryostat sections (6 μm) of the fresh frozen samples were cut, air dried for 1 hour, and fixed in 4% paraformaldehyde at 4°C for 10 minutes. Apoptotic cells were counted in each of four randomly selected high power fields (40×).
The apoptotic index (AI) was calculated as the number of apoptosis-positive hepatocytes divided by the number of all hepatocytes in each high-power field. The difference in AI between groups was compared using the two-tailed unpaired Student t test. P values less than .05 were considered significant.
Western Blots of HCV Core Protein
Nontumor tissues from each group were homogenized in RIPA buffer and protein expression was determined by Western blotting using a mouse anti–hepatitis C core monoclonal antibody from Dr. Kohara.7 Equal quantities of protein were utilized for HCV core protein determinations.
Apoptotic Index in HCV Core and Core-E1-E2 Transfected Cells
To elucidate the effects of HCV E1-E2 protein on apoptosis, apoptotic index was determined in HCV core-expressing cells. Approximately 5 × 104 HepG2 cells per well of a 24-well plate were seeded 24 hours prior to transfection. Cells were transfected using Lipofectamine (Invitrogen, Carlsbad, CA) with 0.4 μg of plasmids (pAlb-HCV containing the core-E1-E2 regions and pBEP39 containing the core region) and incubated for 8 hours. To induce apoptosis, transfected cells were then exposed to 0.5 μg/mL of anti-Fas agonistic antibody (EOS9.1 monoclonal Ab, eBioscience, San Diego, CA) for 12 hours.
Cells were trypsinized and fixed with 1% paraformaldehyde in PBS for 10 minutes at room temperature and dried on microscope slides. HCV core expression was stained by mouse anti-hepatitis C core monoclonal antibody from Dr. Kohara7 followed by FITC-F(ab′)2 goat anti-mouse IgG as secondary antibody (Zymed Laboratories Inc., San Francisco, CA). A second stain for detection of DNA fragmentation was carried out using a rhodamine labeled TUNEL assay (ApopTag Red In Situ Apoptosis Detection kit; Serological Corp.). Apoptotic cells were counted in each of ten randomly selected high-power fields (40×). For cell lines, the AI was calculated as the number of apoptosis-positive cells divided by the number of HCV core protein-positive cells in each high-power field. The difference in AI between groups was compared using two-tailed unpaired Student t test. P values less than .05 were considered to be significant.
Apoptotic Index in Fas-Induced Apoptosis in HCV Transgenic Mouse Models
To further confirm the effect of E1 and/or E2 protein in apoptosis, AI was assessed in a Fas-induced apoptosis model using HCV core-E1-E2 transgenic, core transgenic, and non-transgenic mice. Two micrograms of anti-Fas agonistic antibody (monoclonal Ab clone Jo2, BD Biosciences, Palo Alto, CA) in 0.5 mL of normal saline was injected intraperitoneally into mice of 8 months of age in three groups: (1) core-E1-E2 Tg (n = 3), (2) core Tg (n = 3), and (3) non-Tg (n = 3). The animals were sacrificed 4 hours later, and liver samples were fixed in 10% formaldehyde. Apoptosis detection was carried out using a peroxidase labeled TUNEL assay (ApopTag In Situ Apoptosis Detection kit; Serological Corp.). Apoptotic cells were counted in each of four randomly selected high-power fields (40×). The AI was calculated as the number of apoptosis-positive hepatocytes divided by the number of hepatocytes in each high-power field. The difference in AI between groups was compared using two-tailed unpaired Student t test. P values less than .05 were considered to be significant.
Phenotype of Transgenic Mice.
We observed no spontaneous hepatocellular carcinomas or adenomas in mice bearing either transgene when they were both expressed on the FVB×C57Bl/6 background. These mice were observed for as long as 21 months of age. To test the ability of these transgenes to accelerate tumorigenesis after an initial hepatic injury, we then compared the effects of the two transgenes on tumorigenesis after treatment with DEN. Saline-treated HCV-core-E1-E2 and HCV-core transgenic mice were phenotypically normal with no apparent delays in growth or development. In contrast, all three lines of mice (HCV-core-E1-E2, HCV-core, and nontransgenic) treated with DEN experienced growth retardation at 20 weeks of age. There was neither obvious inflammatory cellular infiltrate in the liver nor significant change in serum ALT levels in any of these three lines (data not shown).
Development of Hepatocellular Carcinoma.
HCC did not develop prior to 24 weeks of age in any of the mice examined. HCC developed at 32 weeks of age in all DEN-treated mice, but not in saline-treated mice. The mean number of adenomas and HCCs was 1.00 ± 0.38/mouse (core-E1-E2), 0.75 ± 0.25/mouse (core), and 0.92 ± 0.35/mouse (nontransgenic). These differences were not significant (Fig. 1A). The mean size of adenomas and HCCs was 4.18 ± 1.36 mm (core-E1-E2), 0.87 ± 0.23 mm (core), and 1.1 ± 0.16 mm (nontransgenic). The difference between core-E1-E2 and core mice (P = .008), and between core-E1-E2 and nontransgenic mice (P = .003) was statistically significant (Fig. 1B). The mean number of HCCs was 0.87 ± 0.40/mouse (core-E1-E2), 0.50 ± 0.27/mouse (core), and 0.75 ± 0.37/mouse (nontransgenic). These differences were not statistically significant. The mean size of HCCs was 4.63 ± 1.48 mm (core-E1-E2), 0.78 ± 0.26 mm (core), and 1.00 ± 0.19 mm (nontransgenic). The difference between core-E1-E2 and core mice (P = .01) or core-E1-E2 and nontransgenic mice was statistically significant (P = .002, Fig. 1C).
The morphology of HCCs was variable. Most HCCs consisted of a well-differentiated hepatic cord with proliferating hepatocytes, while some contained thickened trabeculae or acini of dysplastic hepatocytes (Fig. 2). Mild steatosis was observed in less than 5% of the hepatocytes in all mice studied. No fibrosis or inflammation was observed in any of the mice studied.
No Difference in Proliferation Between HCCs Was Observed.
Ki67 proliferation index (PI) in HCC tissue was 68.1 ± 3.7 for core-E1-E2 transgenic, 58.3 ± 4.5 for core transgenic, and 63.2 ± 3.8 for nontransgenic mice. The PI in nontumor tissue was 42.4 ± 5.4 for core-E1-E2 transgenic, 37.5 ± 3.6 for core transgenic, and 35.8 ± 3.9 for nontransgenic mice. There were no significant differences within HCCs or within nontumor tissues according to group; however, PI was significantly different between HCC and nontumor tissues for each group of mice (P = .0005, P = .0003, P = .002 in core-E1-E2, core, nontransgenic, respectively). (Fig. 3).
Apoptosis Is Suppressed in HCV Core-E1-E2 HCCs.
The mean apoptotic index (AI) of HCC tissue was 19.9 ± 3.2 in core-E1-E2 transgenic mice, 52.0 ± 7.6 in core transgenic mice, and 47.0 ± 2.3 in nontransgenic mice. The mean AI of nontumor was 6.6 ± 1.0 in core-E1-E2 transgenic mice, 36.0 ± 3.0 in core transgenic mice, and 27.8 ± 1.0 in nontransgenic mice. There was a significant difference in AI of HCC between core-E1-E2 and core (P < .0001), and core-E1-E2 and nontransgenic (P < .0001) animals. A significant difference in AI was also found in nontumor tissues between core-E1-E2 and core (P < .0001), and core-E1-E2 and nontransgenic mice (P < .0001) (Fig. 4A-G). These data indicate that a global suppression of apoptosis occurs in both tumor and nontumor tissues in HCV core-E1-E2 transgenic mice.
HCV core protein was expressed at equivalent levels in core-E1-E2 transgenic and core transgenic mice, confirming that the observed differences cannot be attributable to an HCV core dose effect (Fig. 5).
Apoptosis Is Suppressed in HCV Core-E1-E2 Transfected Hepatocytes.
To confirm that the observed differences in apoptosis were attributable to expression of the respective HCV transgenes, we performed transfection studies in HepG2 hepatocytes using individual HCV gene constructs. We performed double staining for apoptosis and viral protein expression in a model of apoptosis induced by the anti-Fas agonistic antibody. AI was scored among cells expressing HCV core to determine whether core-E1-E2 expression was antiapoptotic (i.e., where apoptosis was suppressed). Representative fields demonstrating the feasibility of the double staining are shown in Fig 6A-B for HCV core-E1-E2- and HCV core–transfected cells, respectively. The mean AI of ten high power fields was significantly lower in cells transfected with core-E1-E2 (0.35 ± 0.11) compared to core alone (0.74 ± 0.07, P = .01) (Fig. 6C). The mean AI in nontransfected cells was identical to that of core-transfected cells (data not shown). These data implicate E1 and/or E2 expression in suppression of apoptosis.
Fas-Induced Apoptosis Is Suppressed in HCV Core-E1-E2 Transgenic Liver Tissue.
To further confirm the suppressive effect of HCV E1 and/or E2 proteins on apoptosis, we analyzed AI in a Fas-induced model of apoptosis in core-E1-E2 Tg, core Tg, and non-Tg liver tissue. As shown in representative fields (Fig. 7A-F), core-E1-E2 Tg liver tissue underwent significantly less apoptosis than liver tissue expressing core only or non-Tg tissue. The mean AI of four high-power fields of core-E1-E2 Tg liver (40.3 ± 2.6) was significantly lower than that seen in core Tg (75.2 ± 2.1, P < .001) or non-Tg (79.8 ± 2.6, P < .001) liver. As a comparison, mesenchymal cells, which do not express HCV proteins by staining (data not shown), underwent diffuse apoptosis in each group analyzed, including the core-E1-E2 group. These data indicate that the suppression of apoptosis occurred only in core-E1-E2 expressing hepatocytes and suggest that E1 and/or E2 have a suppressive effect on Fas-induced apoptosis in vivo.
HBV and HCV are important risk factors for hepatocellular carcinoma. However, the precise role of each agent in carcinogenesis has not been well defined. In HBV-associated HCC, exposure to aflatoxin B1 (i.e., chemical hepatocarcinogenesis) early in life is believed to play a key role in carcinogenesis, as is integration of HBV into the host genome (i.e., insertional mutagenesis). In HCV-associated HCC, chronic injury and regeneration have been postulated to underlie transformation; however, growing evidence suggests that HCV replication itself may directly lead to carcinogenesis.
HCV contains structural (core, E1, E2) and regulatory proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B). The core nucleocapsid packages the viral genomic RNA; it may also promote apoptosis and cell proliferation through its physical interaction with p53.1 Additionally, two regions of the envelope protein E2, designated hypervariable regions 1 and 2, have an extremely high rate of mutation, believed to be the result of selective pressure imposed by the humoral immune system. To date, a role of the structural proteins in hepatocarcinogenesis has not been suggested.
To better define the respective role(s) of the HCV core, E1, E2, and nonstructural proteins in hepatocarcinogenesis, various mouse transgenic models utilizing core, E1+E2, E2, core+E1+E2, as well as the entire HCV genome have been developed by several groups. These transgenic mice have exhibited two phenotypes: those with and those without hepatic tumors. The tumor phenotype has been reported to occur in transgenic mice for core,6 the entire region,9 or core+E1+E2.9 However, in different investigations mice transgenic for core,10 E2,10 E1+E2,3 and core+E1+E2,2 did not exhibit the tumor phenotype.
Among HCV transgenic mice without tumor phenotype, two models expressing core have been reported by Pasquinelli et al.10 and our group.2 Neither model showed histologically recognizable steatosis, adenoma, or HCC. Although it remains unclear why these two models did not develop HCC, possible explanations include differences in genetic background of the mice, lengths of observation, outcome measures, transgene constructs, and levels of HCV RNA and/or protein expression. The data from Pasquinelli et al. differ from others in transgene construct, in that MUP was used as the promoter.10 These may have led to a level of protein insufficient for developing HCC.
The experiment by our group was unique in that we used FVB transgenic mice.2 We used the albumin promoter as did Lerat et al.9 The transgenic mice developed by Kawamura et al. demonstrated high core expression,2 whereas those by Lerat et al. showed much lower core protein expression, suggesting that levels of core protein expression alone do not explain the HCC phenotype, and that the FVB background may have a protective role in hepatocarcinogenesis. Indeed, saline-treated control animals expressing the core and core-E1-E2 construction on the FVB×C57Bl/6 background both failed to develop HCC, unless they were exposed to a chemical carcinogen. Thus, it appears that mouse genetic background plays a critical role in explaining the disparate outcomes between these transgenic models.
The present study demonstrates that DEN initiates tumor development and that HCV core+E1+E2 proteins act to further accelerate tumor growth. Sell et al. reported a similar function of HBV in hepatocarcinogenesis.11 They showed that mice transgenic for HBV large envelope protein are at increased risk for adenoma and HCC if exposed to DEN at several months of age. These data suggest that DEN- and HBs antigen-induced liver damage act synergistically to produce HCC in transgenic mice. Nontransgenic mice exposed to DEN at the same age showed no morphological alterations. Subsequently, Huang et al. suggested that a strong and sustained proliferative response in hepatocytes in mice transgenic for HBV occurs after the onset of hepatocellular injury and precedes the development of HCC.12 Our data differ from that of Huang et al. in that hepatocyte mitotic and proliferation indices were not increased in transgenic mice exposed to DEN. However, the apoptotic index of hepatocytes in DEN-treated HCV core+E1+E2 transgenic mice was significantly reduced compared to DEN-treated core transgenic mice or nontransgenic mice. Together, these findings suggest that HCV and HBV envelope proteins work synergistically with chemical carcinogens to induce HCCs through different mechanisms.
The relationship between HCV E1 and E2 proteins and apoptosis was confirmed by double staining of core-expressing, apoptotic hepatocytes and by our in vivo studies demonstrating the suppressive effect of HCV core-E1-E2 on Fas-induced apoptosis. There are contradictory reports regarding the effect of HCV envelope proteins on apoptosis. Honda et al. reported that liver samples from HCV core-E1-E2 transgenic mice showed higher Fas-mediated cell damage compared with non transgenic mice.13 Dumoulin et al. reported that HCV core protein or HCV E2 protein individually do not prevent TNF-α or Fas induced-apoptosis in transient transfected HepG2 cells.14 Lasarte et al. reported that a recombinant adenovirus encoding HCV core and E1 proteins protects liver cells from cytokine-induced hepatocellular damage in experimental models of TNF-mediated hepatic injury.15 These data suggest the possibility that E1 with or without E2 has an antiapoptotic effect.
Notably, none of our mouse lines developed significant steatosis. Our findings suggested that HCV related HCC does not exclusively employ steatosis as a precondition; rather, HCV only acts as a “second hit” on the backdrop of chemically induced genetic injury. Given data that DEN can generate reactive oxygen species (ROS) and enhance oxidative stress,16–19 it is tempting to speculate that DEN and steatosis, another known cause of ROS, may act in similar manners to predispose hepatocytes to genotoxic injury and malignant transformation.
In summary, HCV E1 and/or E2, possibly in conjunction with core protein, act as tumor accelerators in a DEN-based model of hepatocarcinogenesis and appear to do so by suppressing apoptosis. These data suggest a previously unrecognized and unexpected property of the viral envelope glycoproteins.