Hepatocyte-restricted constitutive activation of PPAR alpha induces hepatoproliferation but not hepatocarcinogenesis

Peroxisome proliferator-activated receptor alpha (PPARalpha) is responsible for peroxisome proliferator-induced pleiotropic responses, including the development of hepatocellular carcinoma in rodents. However, it remains to be determined whether activation of PPARalpha only in hepatocytes is sufficient to induce hepatocellular carcinomas. To address this issue, transgenic mice were generated that target constitutively activated PPARalpha specifically to hepatocytes. The transgenic mice exhibited various responses that mimic wild-type mice treated with peroxisome proliferators, including significantly decreased serum fatty acids and marked induction of PPARalpha target genes encoding fatty acid oxidation enzymes, suggesting that the transgene functions in the same manner as peroxisome proliferators to regulate fatty acid metabolism. However, the transgenic mice did not develop hepatocellular carcinomas, even though they exhibited peroxisome proliferation and hepatocyte proliferation, indicating that these events are not sufficient to induce liver cancer. In contrast to the transgenic mice, peroxisome proliferators activate proliferation of hepatic non-parenchymal cells (NPCs). Thus, activation of hepatic NPCs and/or associated molecular events is an important step in peroxisome proliferators-induced hepatocarcinogenesis.     

Repeated hepatocyte injury promotes hepatic tumorigenesis in hepatitis C virus transgenic mice

Although hepatitis C virus (HCV) is a well-known causative agent of hepatocellular carcinoma (HCC), the mechanism by which HCV induces HCC remains obscure. To elucidate the role of HCV in hepatocarcinogenesis, a model of hepatocyte injury was established using HCV core transgenic mice, which were developed using C57BL/6 mice transfected with the HCV core gene under control of the serum amyloid P component promoter. After 18–24 months, neither steatosis nor hepatic tumors were found in transgenic mice. The extent of hepatocyte injury and tumorigenesis were then examined in transgenic mice following repeated administration of carbon tetrachloride (CCl₄) using various protocols (20%, 1/week; 10%, 2/week and 20%, 2/week). Serum alanine aminotransferase (ALT) levels did not differ among HCV core transgenic mice and non-transgenic littermates; however, after 40 weeks, hepatic adenomas preferentially developed in transgenic mice receiving 20% CCl₄ once weekly. Moreover, HCC was observed in transgenic mice receiving 2 weekly injections of a 20% solution of CCl₄, and was not observed in the non-transgenic control mice. In conclusion, the HCV core protein did not promote hepatic steatosis or tumor development in the absence of hepatotoxicity. However, the HCV core protein promoted adenoma and HCC development in transgenic mice following repeated CCl₄ administration. These results suggest that hepatotoxicity resulting in an increased rate of hepatocyte regeneration enhances hepatocarcinogenesis in HCV-infected livers. Furthermore, this experimental mouse model provides a valuable method with which to investigate hepatocarcinogenesis.     

Cell cross-talk mediates PPARalpha null hepatocyte proliferation after peroxisome proliferator exposure

Peroxisome proliferator activated receptor(alpha) (PPARalpha) mediates the liver's responses to peroxisome proliferator compounds. These responses include induction of specific hepatic enzymes, peroxisome proliferation and hepatocyte proliferation. PPARalpha null mice, which lack receptor in all cells of the body, do not respond to peroxisome proliferators, indicating that hepatocellular proliferation and other responses require the presence of this receptor in at least some cells. To determine if PPARalpha is required specifically in hepatocytes for each response, we used hepatocyte transplantation to generate chimeric livers composed of PPARalpha null and positive hepatocytes in PPARalpha null or positive hosts. Upon exposure to a peroxisome proliferator, peroxisome proliferation and enzyme induction were restricted to receptor positive hepatocytes, indicating that these responses are cell autonomous with respect to hepatocyte receptor status. However, both PPARalpha null and positive hepatocytes in chimeric livers displayed elevated DNA synthesis regardless of host receptor status, as long as at least some hepatocytes contained receptor. These findings indicate that the mitogenic response to peroxisome proliferators does not require PPARalpha in all hepatocytes.     

Evidence that reduction of hepatocyte growth factor (HGF) is not required for peroxisome proliferator-induced hepatocyte proliferation

The mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis are not understood. Because of the uncertainty of human cancer risk associated with peroxisome proliferators, delineating the mechanisms of carcinogenesis by these agents is of great interest. Alterations in liver growth factors were postulated to contribute to the carcinogenic effect of peroxisome proliferators. Administration of these compounds to rodents results in down-regulation of hepatocyte growth factor (HGF) and supplementing culture medium with HGF is reported to suppress cell proliferation of preneoplastic and neoplastic cells from WY-14,643-treated livers. Combined, these observations suggest that reduced levels of hepatic HGF contribute to the mechanisms underlying peroxisome proliferator-induced hepatocarcinogenesis. To determine if HGF can prevent the effects of peroxisome proliferators in liver, the short-term influence of WY-14,643 in two different lines of HGF transgenic mice was examined. Mice were fed either a control diet or one containing 0.1% WY-14-643 for one week. Hepatomegaly was found in both HGF transgenic mouse lines fed WY-14,643 compared with controls. Additionally, hepatic expression of typical mRNA markers of peroxisome proliferation including those encoding peroxisomal fatty acid metabolizing enzymes and cell cycle control proteins were all significantly elevated in HGF transgenic mice fed WY-14,643 compared with controls. Down-regulation of HGF was found to be dependent on PPARalpha since lower levels of HGF mRNA and protein were observed in wild-type mice fed WY-14,643 for 1 week and not in similarly treated PPARalpha-null mice. These results demonstrate that the early increase in hepatic mRNAs associated with peroxisome and cell proliferation induced by WY-14,643 treatment can not be prevented by overexpression of HGF in vivo.     

Overproduction of thrombopoietin by BRAFV600E‐mutated mouse hepatocytes and contribution of thrombopoietin to hepatocarcinogenesis

In hepatocarcinogenesis induced by diethylnitrosamine (DEN) in B6C3F1 mice, the BrafV637E mutation, corresponding to the human BRAFV600E mutation, plays a pivotal role. The livers of transgenic mice with a hepatocyte‐specific human BRAFV600E mutation weighed 4.5 times more than that of normal mice and consisted entirely of hepatocytes, resembling DEN‐induced preneoplastic hepatocytes. However, these transgenic mice spontaneously died 7 wk after birth, therefore this study aimed to clarify the causes of death. In the transgenic mice, the liver showed thrombopoietin (TPO) overexpression, which is associated with eventual megakaryocytosis and thrombocytosis, and activated platelets were deposited in hepatic sinusoids. TPO was also overexpressed in the DEN‐induced hepatic tumors, and sinusoidal platelet deposition was observed in the hepatic tumors of humans and mice. Podoplanin was expressed in some of the Kupffer cells in the liver of the transgenic mice, indicating that platelet activation occurred via the interaction of podoplanin with C‐type lectin receptor 2 (CLEC‐2) on the platelet membrane. Additionally, erythrocyte dyscrasia and glomerulonephropathy/interstitial pneumonia associated with platelet deposition were observed. In the transgenic mice, aspirin (Asp) administration prevented platelet activation, reduced the liver/body weight ratio, decreased the platelet deposition in the liver, kidney, and lung, and prevented erythrocyte dyscrasia and ameliorated the renal/pulmonary changes. Thrombopoietin overproduction by BRAFV600E‐mutated hepatocytes may contribute to hepatocyte proliferation via thrombocytosis, platelet activation, and the interaction of platelets with hepatic sinusoidal cells, while hematologic, renal, and pulmonary disorders due to aberrant platelet activation may lead to spontaneous death in the transgenic mice.     

Molecular mechanism of viral hepatocarcinogenesis

Overwhelming lines of epidemiological evidence have indicated that chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). In the pathogenesis of HCC associated with HBV or HCV, it remains controversial whether these hepatitis viruses play a direct role or merely an indirect role. By virtue of transgenic mice established by us, it has become evident that the product of the HBV X gene (HBx protein) and the core protein of HCV have an oncogenic potential, although the pathways through which these two viral proteins operate may differ. The findings in our studies indicate that HBV and HCV are directly involved in hepatocarcinogenesis, albeit other factors such as continued cell death and regeneration associated with chronic hepatitis may play a role as well. Combined, our results suggest that there might be a mechanism in the development of HCC in persistent infection with hepatitis viruses that is distinct from that in other cancers. Similarly to the pathogenesis of other malignancies represented by colorectal cancer, the accumulation of a set of genetic aberrations may also be necessary for a multistage development of HCC. However, HBx protein and HCV core protein, to which an oncogenic potential is attributed, may allow some of the multiple stages skipped in hepatocarcinogenesis. Unlike for the other cancers, therefore, infection with HBV or HCV may be capable of inducing HCC in the absence of a complete set of genetic aberrations. Such a scenario would explain an unusually high incidence and multicentric nature of HCC developing in chronic hepatitis B or C.     

Diminished hepatocellular proliferation in mice humanized for the nuclear receptor peroxisome proliferator-activated receptor alpha

Lipid-lowering fibrate drugs function as agonists for the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rats and mice. However, humans appear to be resistant to the induction of peroxisome proliferation and the development of liver cancer by fibrate drugs. The molecular basis of this species difference is not known. To examine the mechanism determining species differences in peroxisome proliferator response between mice and humans, a PPARalpha-humanized mouse line was generated in which the human PPARalpha was expressed in liver under control of the tetracycline responsive regulatory system. The PPARalpha-humanized and wild-type mice responded to treatment with the potent PPARalpha ligand Wy-14643 as revealed by induction of genes encoding peroxisomal and mitochondrial fatty acid metabolizing enzymes and resultant decrease of serum triglycerides. However, surprisingly, only the wild-type mice and not the PPARalpha-humanized mice exhibited hepatocellular proliferation as revealed by elevation of cell cycle control genes, increased incorporation of 5-bromo-2'-deoxyuridine into hepatocyte nuclei, and hepatomegaly. These studies establish that following ligand activation, the PPARalpha-mediated pathways controlling lipid metabolism are independent from those controlling the cell proliferation pathways. These findings also suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the development of hepatocarcinomas observed after treatment with fibrates. The PPARalpha-humanized mice should serve as models for use in drug development and human risk assessment and to determine the mechanism of hepatocarcinogenesis of peroxisome proliferators.     

Role of peroxisome proliferator-activated receptor-alpha (PPARalpha) in bezafibrate-induced hepatocarcinogenesis and cholestasis

Prolonged administration of peroxisome proliferators to rodents typically leads to hepatocarcinogenesis. Peroxisome proliferator-activated receptor-alpha (PPARalpha) is required to mediate alterations in PPARalpha target gene expression, repress apoptosis, enhance replicative DNA synthesis, oxidative stress to DNA and hepatocarcinogenesis induced by the relatively specific PPARalpha agonist, Wy-14,643. Interestingly, administration of the less specific PPARalpha agonist, bezafibrate, leads to a modest induction of PPARalpha target genes in the absence of PPARalpha expression. In these studies, the role of PPARalpha in modulating hepatocarcinogenesis induced by long-term feeding of 0.5% bezafibrate was examined in wild-type (+/+) and PPARalpha-null (-/-) mice. The average liver weight was significantly higher in (+/+) and (-/-) mice fed bezafibrate than controls, but this effect was considerably less in (-/-) mice as compared with similarly treated (+/+) mice. Increased levels of mRNA encoding cell cycle regulatory proteins and DNA repair enzymes were found in (+/+) mice fed bezafibrate, and this effect was not found in (-/-) mice. In mice fed bezafibrate for 1 year, preneoplastic foci, adenomas and a hepatocellular carcinoma were found in (+/+) mice, while only a single microscopic adenoma was found in one (-/-) mouse. This effect was observed in both Sv/129 and C57BL/6N strains of mice, although only preneoplastic foci were observed in the latter strain. Interestingly, hepatic cholestasis was observed in 100% of the bezafibrate-fed (-/-) mice, and this was accompanied by significantly elevated hepatic expression of mRNA encoding bile salt export pump and lower expression of mRNA encoding cytochrome P450 7A1, consistent with enhanced activation of the bile acid receptor, farnesoid X receptor. Results from these studies demonstrate that the PPARalpha is required to mediate hepatocarcinogenesis induced by bezafibrate, and that PPARalpha protects against potential cholestasis.     

Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis

The mechanism of hepatocarcinogenesis in hepatitis C virus (HCV) infection is still undefined. One possibility is the involvement of oxidative stress, which can produce genetic mutations as well as gross chromosomal alterations and contribute to cancer development. We recently showed that after a long period, the core protein of HCV induces hepatocellular carcinoma (HCC) in transgenic mice with marked hepatic steatosis but without inflammation, indicating a direct involvement of HCV in hepatocarcinogenesis. To elucidate the biochemical events before the development of HCC, we examined several parameters of oxidative stress and redox homeostasis in a mouse model of HCV-associated HCC. For young mice ages 3-12 months, there was no significant difference in the levels of hydroperoxides of phosphatidylcholine (PCOOH) and phosphatidylethanolamine in liver tissue homogenates between transgenic and nontransgenic control mice. In contrast, the PCOOH level was increased by 180% in old core gene transgenic mice > 16 months old. Concurrently, there was a significant increase in the catalase activity, and there were decreases in the levels of total and reduced glutathione in the same mice. A direct in situ determination by chemiluminescence revealed an increase in hydroperoxide products by 170% even in young transgenic mice, suggesting that hydroperoxides were overproduced but immediately removed by an activated scavenger system in young mice. Electron microscopy revealed lipofuscin granules, secondary lysosomes carrying various cytoplasmic organelles, and disruption of the double membrane structure of mitochondria, and PCR analysis disclosed a deletion in mitochondrial DNA. Interestingly, alcohol caused a marked increase in the PCOOH level in transgenic mice, suggesting synergism between alcohol and HCV in hepatocarcinogenesis. The HCV core protein thus alters the oxidant/antioxidant state in the liver in the absence of inflammation and may thereby contribute to or facilitate, at least in part, the development of HCC in HCV infection.     

Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis

Peroxisome proliferators, such as lipid-lowering fibrate drugs, are agonists for the peroxisome proliferator-activated receptor alpha (PPARalpha). Sustained activation of PPARalpha leads to the development of liver tumors in rodents. Paradoxically, humans appear to be resistant to the induction of peroxisome proliferation and development of liver tumors by peroxisome proliferators. To examine the species differences in response to peroxisome proliferators, a PPARalpha humanized mouse (hPPARalpha) was generated, in which the human PPARalpha was expressed in liver under control of the Tet-OFF system. To evaluate the susceptibility of hPPARalpha mice to peroxisome proliferator-induced hepatocarcinogenesis, a long-term feeding study of Wy-14,643 was carried out. hPPARalpha and wild-type (mPPARalpha) mice were fed either a control diet or one containing 0.1% Wy-14,643 for 44 and 38 weeks, respectively. Gene expression analysis for peroxisomal and mitochondrial fatty acid metabolizing enzymes revealed that both hPPARalpha and mPPARalpha were functional. However, the incidence of liver tumors including hepatocellular carcinoma was 71% in Wy-14,643-treated mPPARalpha mice, and 5% in Wy-14,643-treated hPPARalpha mice. Upregulation of cell cycle regulated genes such as cd1 and Cdks were observed in non-tumorous liver tissue of Wy-14,643-treated mPPARalpha mice, whereas p53 gene expression was increased only in the livers of Wy-14,643-treated hPPARalpha mice. These findings suggest that structural differences between human and mouse PPARalpha are responsible for the differential susceptibility to the peroxisome proliferator-induced hepatocarcinogenesis. This mouse model will be useful for human cancer risk assessment of PPARalpha ligands.     

Hepatocarcinogenesis in hepatitis C: HCV shrewdly exacerbates oxidative stress by modulating both production and scavenging of reactive oxygen species

Persistent infection with hepatitis C virus (HCV) is a major risk for the development of hepatocellular carcinoma (HCC). One of the characteristics of HCV infection is the unusual augmentation of oxidative stress, which is exacerbated by iron accumulation in the liver, as observed frequently in hepatitis C patients. Using a transgenic mouse model, in which HCC develops late in life after the preneoplastic steatosis stage, the core protein of HCV was shown to induce the overproduction of reactive oxygen species (ROS) in the liver. In excessive generation of ROS, HCV affects the steady-state levels of a mitochondrial protein chaperone, i.e. prohibitin, leading to an impaired function of the mitochondrial respiratory chain with the overproduction of ROS. Insulin resistance and hepatic steatosis, which frequently accompany HCV infection, exacerbate ROS production. On the other hand, HCV compromises some of the antioxidant systems, including heme oxygenase-1 and NADH dehydrogenase quinone 1, resulting in the provocation of oxidative stress, together with ROS overproduction, in the liver with HCV infection. Thus, HCV infection not only induces ROS but also hampers the antioxidant system in the liver, thereby exacerbating oxidative stress that would facilitate hepatocarcinogenesis. Combination with the other activated pathway, including an alteration in the intracellular signaling cascade of MAP kinase, along with HCV-associated disturbances in lipid and glucose metabolism would lead to the unusual mode of hepatocarcinogenesis, i.e. very frequent and multicentric development of HCC, in persistent HCV infection.     

Hepatocellular expression of a dominant-negative mutant TGF-beta type II receptor accelerates chemically induced hepatocarcinogenesis

The potent growth-inhibitory activity of cytokines of the transforming growth factor-beta (TGF-beta) superfamily and their widespread expression in epithelia suggest that they may play an important role in the maintenance of epithelial homeostasis. To analyse TGF-beta mediated tumor suppressor activity in the liver, we generated transgenic mice overexpressing a dominant negative type II TGF-beta receptor in hepatocytes under control of the regulatory elements of the human C-reactive protein gene promoter. Transgenic animals exhibited constitutive and liver-specific transgene expression. The functional inactivation of the TGF-beta signaling pathway in transgenic hepatocytes was shown by reduced TGF-beta induced inhibition of DNA synthesis in primary hepatocyte cultures. Liver morphology and spontaneous tumorigenesis were unchanged in transgenic mice suggesting that interruption of the signaling of all three isoforms of TGF-beta in hepatocytes does not disturb tissue homeostasis in the liver under physiological conditions. However, following initiation with the carcinogen diethylnitrosamine and tumor-promotion with phenobarbital transgenic mice exhibited a moderate albeit significant increase in the incidence, size and multiplicity of both preneoplastic tissue lesions in the liver and of hepatocellular carcinomas. These results give in vivo evidence for a tumor suppressor activity of the endogenous TGF-beta system in the liver during chemical hepatocarcinogenesis.     

Hepatocarcinogenesis in Transgenic Mice Carrying Albumin-promoted SV40 T Antigen Gene

We have developed transgenic mice that inherit albumin promoter-regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi-regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.     

Hepatocarcinogenesis in transgenic mice carrying albumin-promoted SV40 T antigen gene.

We have developed transgenic mice that inherit albumin promoter-regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi-regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.     

Tumor necrosis factor alpha is not required for WY14,643-induced cell proliferation

It has been proposed that the cytokine tumor necrosis factor alpha (TNFalpha) stimulates peroxisome proliferator-induced hepatic cell proliferation. To test this hypothesis, induction of peroxisome proliferation and hepatocyte proliferation were compared in wild-type C57Bl/6 and TNFalpha knockout mice. Animals were dosed with either vehicle or 100 mg/kg/day WY14,643 by oral gavage for 4 days. Liver to brain weight ratios increased in both wild-type and TNFalpha knockout animals after WY14,643 administration. In addition, WY14,643-treated wild-type C57Bl/6 and TNFalpha knockout mice displayed marked hepatic induction of fatty acyl-CoA oxidase activity (approximately 8-fold) and mRNA content (approximately 5-fold). Electron microscopic examination confirmed increased numbers of peroxisomes in hepatocytes in both mouse models. Moreover, WY14,643 markedly induced hepatic cell proliferation (approximately 15-fold) in both wild-type C57Bl/6 and TNFalpha knockout mice as measured by bromodeoxyuridine incorporation into hepatocyte nuclei. In addition, a 50% decrease in TNFalpha mRNA was observed in wild-type mice after treatment with WY14,643. These results suggest that the hepatocellular proliferation induced after peroxisome proliferator treatment occurs independently of TNFalpha signaling.     

Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection

BACKGROUND: Hepatic steatosis is one of the histopathologic features of chronic hepatitis C. It was reported recently that the expression of hepatitis C virus (HCV) core protein in transgenic mice induced hepatocellular carcinoma (HCC) in association with steatosis. The objective of this study was to determine the relation between hepatic steatosis and hepatocarcinogenesis in patients with chronic HCV infection. METHODS: The authors studied 161 patients with chronic HCV infection who were diagnosed at Nagasaki University Hospital, Nagasaki, Japan, between January 1980 and December 1999. Age, gender, body mass index (BMI), habitual drinking, diabetes mellitus, serum alanine aminotransferase (ALT) level, HCV serotype, serum level of HCV core protein, interferon (IFN) treatment, hepatic fibrosis inflammation, and hepatic steatosis were studied with regard to their significance in the development of HCC using univariate and multivariate analyses. RESULTS: The cumulative incidence rates of HCC were 24%, 51%, and 63% at 5 years, 10 years, and 15 years, respectively. Multivariate analysis identified hepatic steatosis, together with aging, cirrhosis, and no IFN treatment, as independent and significant risk factors for HCC (P = 0.0135, P = 0.0390, P = 0.0068, and P = 0.0142, respectively). In addition, hepatic steatosis was correlated with BMI, serum ALT levels, and triglyceride levels. CONCLUSIONS: The findings of the current study indicate that hepatic steatosis is a risk factor for HCC in patients with chronic HCV infection. Patients with chronic HCV and hepatic steatosis should be monitored carefully for HCC.     

Compensatory apoptosis in preneoplastic liver of a transgenic mouse model for viral hepatocarcinogenesis

The HBx protein of hepatitis B virus has been shown to induce hepatocellular carcinoma in transgenic mice as direct evidence for its involvement in hepatocarcinogenesis. In these transgenic mice, however, it is not clear why hepatocytes do not acquire a neoplastic phenotype by 13 months old despite the continuous growth stimulation by the HBx protein from 2 months old. In this study, we show that the accelerated proliferation of hepatocytes is counterbalanced by apoptosis, which maintains liver homeostasis. A decrease in the extent of apoptosis seems to precede the emergence of neoplasia in the transgenic mouse liver. The disappearance or block of apoptotic signals, which may be the result of additional genetic or epigenetic aberrations, may result in the preneoplastic hepatocytes becoming neoplastic.