Pro-apoptotic effect of the hepatitis B virus X gene

The hepatitis B virus (HBV) is a common human pathogen that causes acute and chronic liver disease. Persistent HBV infection is strongly associated with the development of hepatocellular carcinoma. The contribution of the viral regulatory protein HBx in liver oncogenesis has been supported by our recent studies in a transgenic mouse model, showing that HBx cooperates with c-myc by accelerating the onset of primary liver tumors. Here we show that liver expression of HBx is associated with increased rates of spontaneous apoptosis in liver cells from two different transgenic lines. In transient transfection assays, overexpression of HBx in the established hepatocyte cell line MMHD3 and in human hepatoma cells HepG2 was found to induce apoptosis in a dose-dependent manner. These data suggest that HBx might trigger an apoptotic process in HBV-infected hepatocytes, in turn possibly favoring liver regeneration and accumulation of genetic alterations, ultimately leading to liver cell transformation in chronically infected patients.     

Altered differentiation of hepatocytes in a transgenic mouse model of hepatocarcinogenesis

Transgenic mice carrying the SV40 T antigen (TAg) gene, which develop hepatocellular and biliary cell tumors by 4 mo of age, show ductular structures in the neonatal liver. Coexpression of c-myc with TAg increases the extent and persistence of ductular lesions and also accelerates tumor development. To analyze possible links between altered gene expression and cell differentiation and to determine the relationship between the ductular structures and tumor development in these mice, ductular cells in single (TAg) and bitransgenic (TAg x c-myc) mice were characterized for biliary and hepatocellular differentiation, transgene expression, and proliferation activity. The results show that the ductular cells in these transgenic mice have characteristics of biliary cells, including basement membrane formation, positive laminin staining, and bile duct-specific lectin (Dolichos biflorus agglutinin and peanut agglutinin) binding, and characteristics of hepatocytes, including albumin expression and ultrastructural features such as round nuclei with 1 or 2 nucleoli and well-developed cytoplasmic organelles. However, differences in transgene expression and cell proliferation between the ductular cells and nonductular hepatocytes were not apparent. Thus, the ductular cells could not be defined as tumor progenitor cells in these mouse livers. However, this model suggests that manipulation of gene expression can alter differentiation of hepatic parenchymal cells.     

Autonomous growth in serum-free medium and production of hepatocellular carcinomas by differentiated hepatocyte lines that overexpress transforming growth factor alpha 1

Transforming growth factor alpha (TGF-alpha) is a polypeptide closely associated with hepatocyte proliferation in vivo and in vitro. In order to investigate the mechanisms by which TGF-alpha contributes to hepatocyte replication and transformation, we isolated hepatocytes from mice bearing a human TGF-alpha transgene and examined their growth properties and gene expression in defined, serum-free culture. The transgenic hepatocytes continued to overexpress human TGF-alpha mRNA and peptide, and were able to proliferate without exogenous growth factors in primary culture, in contrast to nontransgenic mouse hepatocytes. In short-term culture the transgenic hepatocytes underwent 1 wave of DNA replication at 72-96 h in culture before senescing, similar to nontransgenic hepatocytes supplemented with epidermal growth factor. Constitutive expression of TGF-alpha rendered the transgenic hepatocytes unresponsive to further growth stimulation by exogenous TGF-alpha, as well as other mitogens such as epidermal growth factor and hepatocyte growth factor. However, it did not alter their sensitivity to growth inhibition by TGF beta 1, 2 and 3. The addition of nicotinamide to the culture medium enabled both transgenic and epidermal growth factor-supplemented normal hepatocytes to replicate repeatedly and survive for > or = 2 months in primary culture while maintaining differentiated traits. From these long-term primary cultures of transgenic and nontransgenic hepatocytes, we established immortalized cell lines (designated TAMH and NMH lines, respectively). Both lines continued to express differentiated adult hepatocytic markers such as albumin, alpha-1-antitrypsin, transferrin, and connexin 26 and 32 mRNAs, but also expressed mRNAs for the oncofetal markers alpha-fetoprotein and insulin-like growth factor II. Unlike the near-diploid NMH hepatocyte line, the transgenic TAMH hepatocyte line was quasi-tetraploid, strongly expressed human TGF-alpha mRNA, and was highly tumorigenic in nude mice. Well-differentiated hepatocellular carcinomas developed in nude mice given injections of the TAMH line, and these appeared similar to the primary liver tumors seen in TGF-alpha transgenic mice with regard to histology and strong expression of mouse and human TGF-alpha, insulin-like growth factor II, and alpha-fetoprotein mRNAs. Our data show that TGF-alpha overexpression causes autonomous hepatocyte proliferation and contributes to neoplasia but that additional cellular alterations must occur for carcinogenesis. Inappropriate expression of insulin-like growth factor II may constitute one of these steps. The TGF-alpha transgenic mouse hepatocyte line TAMH appears to undergo transformation in a similar manner to that of hepatocytes overexpressing TGF-alpha in vivo, and should serve as an ideal system in which to study hepatocarcinogenesis.     

Role of glutathione, glutathione S-transferases and multidrug resistance-related proteins in cisplatin sensitivity of head and neck cancer cell lines

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-ras gene (rasH2). The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-ras codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively. No point mutations in the human c-H-ras transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-ras gene, but no point mutations were detected in codon 61 of the mouse c-H-ras gene. No mutations in human or mouse c-H-ras codon 61 were detected in altered cell foci or hepatocellular adenoma. These results indicate that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rasH2 Tg mice.     

Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.     

Phenobarbital mechanistic data and risk assessment: enzyme induction, enhanced cell proliferation, and tumor promotion

Chronic exposure to high doses of phenobarbital (PB) causes hepatocellular adenomas in both mice and rats and hepatocellular carcinomas in some strains of mice. Long-term PB therapy has not been found to cause human tumors. PB is not DNA reactive, and most genotoxicity tests have yielded negative results. PB has been extensively studied as an epigenetic, rodent liver tumor promoter. At exposures causing rodent liver tumors, PB has measurable effects on hepatocytes: PB inhibits cell-to-cell communication; PB induces enzymes, including P450 cytochromes; PB stimulates proliferation and inhibits apoptosis of hepatocytes in neoplastic foci. Threshold exposures for some of these endpoints coincide with the threshold exposure for tumorigenesis.     

In vitro transformation by hepatitis B virus DNA

There is strong epidemiological evidence that the hepatitis B virus (HBV) contributes to the development of hepatocellular carcinoma (HCC). In several immortalized cell lines, an in vitro transforming activity of HBV DNA and expression vectors for the viral protein X (HBx) has now been demonstrated. Furthermore, it appears as if still unknown parts of the HBV genome other than HBx contribute to the transforming activity of HBV DNA in vitro. Only one of several studies found that HBx-transgenic mouse lines develop HCC. A mouse line transgenic for the large surface protein of HBV develops HCC due to concomitant necroinflammatory infection. Growing evidence shows the importance of recombination of integrated viral DNA and cellular DNA for HCC development. A direct transforming potential of one of these viral integrates has been demonstrated. Chemical carcinogens are more effective in HBV-containing cell lines or transgenic mice.     

Disruption of redox homeostasis in the transforming growth factor-alpha/c-myc transgenic mouse model of accelerated hepatocarcinogenesis

In previous studies we have demonstrated that transforming growth factor (TGF)-alpha/c-myc double transgenic mice exhibit an enhanced rate of cell proliferation, accumulate extensive DNA damage, and develop multiple liver tumors between 4 and 8 months of age. To clarify the biochemical events that may be responsible for the genotoxic and carcinogenic effects observed in this transgenic model, several parameters of redox homeostasis in the liver were examined prior to development of hepatic tumors. By 2 months of age, production of reactive oxygen species, determined by the peroxidation-sensitive fluorescent dye, 2',7'-dichlorofluorescin diacetate, was significantly elevated in TGF-alpha/c-myc transgenic hepatocytes versus either wild type or c-myc single transgenic cells, and occurred in parallel with an increase in lipid peroxidation. Concomitantly with a rise in oxidant levels, antioxidant defenses were decreased, including total glutathione content and the activity of glutathione peroxidase, whereas thioredoxin reductase activity was not changed. However, hepatic tumors which developed in TGF-alpha/c-myc mice exhibited an increase in thioredoxin reductase activity and a very low activity of glutathione peroxidase. Furthermore, specific deletions were detected in mtDNA as early as 5 weeks of age in the transgenic mice. These data provide experimental evidence that co-expression of TGF-alpha and c-myc transgenes in mouse liver promotes overproduction of reactive oxygen species and thus creates an oxidative stress environment. This phenomenon may account for the massive DNA damage and acceleration of hepatocarcinogenesis observed in the TGF-alpha/c-myc mouse model.     

Inhibition of hepatocellular carcinoma development in hepatitis B virus transfected mice by low dietary casein

In a comprehensive human ecological study, primary liver cancer has been shown to be highly significantly associated with 1) the prevalence of persistent infection with hepatitis B virus (HBV) and 2) plasma cholesterol concentrations that are, in turn, associated with the consumption of animal based foods. In rat studies, aflatoxin-induced hepatocellular carcinoma is substantially prevented by decreasing the intake of animal based protein (casein), a hypercholesterolemic nutrient. Thus the development of primary liver cancer associated with persistent HBV infection or with aflatoxin exposure may be controlled by reduced intake of animal-based proteins. Transgenic mice transfected with an HBV gene fragment containing the viral transactivator of hepatis B virus, HBx, which induces the formation of hepatocellular carcinoma, were used to examine the ability of dietary casein to modify tumor formation. Reducing the concentration of dietary casein to 6% from the traditional level of 22% markedly inhibited (by 75%) hepatic tumor formation in these transgenic mice. Tumor development also was substantially altered by interchanging dietary casein concentration well after tumor development had begun (at 8 months), increasing by 173% from the expected yield when casein intake was increased and decreasing by 99% when casein was reduced. These findings suggest that the development of liver tumor formation among individuals persistently infected with HBV may be controlled by minimizing or eliminating the intake of animal protein-based foods.     

Hepatitis B injury, male gender, aflatoxin, and p53 expression each contribute to hepatocarcinogenesis in transgenic mice

The major risk factors for human liver cancer: hepatitis B virus (HBV) related liver injury, male gender, aflatoxin exposure, and p53 expression, are evaluated and compared in experimental transgenic mouse models. Transgenic mice that express hepatitis B surface antigen (HBsAg) in their liver and develop liver tumors at 18 months of age (HBV+ mice) were bred to p53 null mice (p53-/-) to produce mice p53+/-, HBV+ mice. These mice and control littermates ([p53+/+, HBV+], [p53+/-, HBV-], and [p53+/+, HBV-) were divided into groups that did or did not receive an injection of aflatoxin at 1 week of age. At sacrifice at 13 months of age, 100% (7/7) of male mice with each of the three risk factors (p53+/-, HBV+, AFB1+) developed high-grade hepatocellular carcinomas (HCC). If any one of the risk factors was absent, the incidence drops: if both p53 alleles are present, 62% (10/16); if HBsAg is not expressed, 14% (1/7); if AFB1 is not given, 25% (2/8). If only one of the risk factors is present no tumors above grade I are found. Similar results were observed in female mice except that HCC incidence in each group is less than in male mice. Some of the tumors in mice with more than one risk factor are of unusual histological types, such as hepatocholangio-carcinomas, adenocarcinomas and undifferentiated carcinomas that are not usually seen in HBV transgenic C57BL/6 mice. No loss or mutation of the p53 gene is detected in any of the tumors. Possibilities of how the four major risk factors for HCC interact to produce malignant liver tumors in these transgenic mouse models of hepatocarcinogenesis are discussed.     

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.     

A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides -6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progresses rapidly to local invasion. Metastases to lymph nodes, liver, lung, and bone are common by 6 months. Tumorigenesis is not dependent on androgens. This model indicates that the neuroendocrine cell lineage of the prostate is exquisitely sensitive to transformation and provides insights about the significance of neuroendocrine differentiation in human prostate cancer.     

Interaction of enteropathogenic Escherichia coli with host epithelial cells

Transfer of genetic material into recipient cells by transfection has been used successfully to isolate genes responsible for particular phenotypic traits. By using this strategy, DNA fragments were isolated that when transfected into appropriate uncommitted cells will commit the recipient cells to undergo adipocyte differentiation. Transgenic mice were generated with one of the active DNA clones, Clone B. The transgenic mice were expected to display an adipocyte-related phenotype; however, the animals developed melanin containing tumors at a young age. Insertion of Clone B into the mouse DNA probably interrupted a gene(s) that is involved in the regulation of cell growth, specifically regulation of cell growth in melanin-producing cells. Histopathologic analysis of these mice showed dark spots on the ear lobes of the animals as early as 10-12 d of age. By 3 mo, in addition to the ear lobes, pigmented tumors could be observed in other organs. A significant number of these transgenic mice died within 1 y of age. The melanomas developed spontaneously in these animals in the absence of any known chemical carcinogen or ultraviolet radiation. This line of mice provides a way of identifying genes involved in regulation of cell growth control and differentiation. These mice also serve as a model system to investigate the molecular, genetic, and phenotypic characterization and development of melanomas.     

Transforming growth factor-alpha in carcinogen-induced F344 rat hepatic foci

Transforming growth factor-alpha (TGF alpha) is a positive growth regulator in epithelial cells, including hepatocytes. Overexpression of TGF alpha has been associated with increased growth and malignancy of end-stage cancers in humans and rodents. The overall aim of this study was to characterize TGF alpha staining in diethylnitrosamine-induced hepatic foci from male F344 rats with the hematoxylin and eosin (H and E) histological phenotype. The association between the individual focal DNA synthesis labeling index and the presence of TGF alpha was also examined. Hepatic foci were identified as eosinophilic, basophilic, clear cell, or mixed cell. Of these foci, 37.5% labeled positive for TGF alpha. There were distinct differences in the pattern of TGF alpha labeling between the different H and E histological phenotypes. Intense, uniform TGF alpha labeling was observed in eosinophilic foci. Basophilic foci labeled for TGF alpha diffusely uniform throughout the cytoplasm. In clear-cell foci, TGF alpha labeling occurred primarily along the periphery of the cell membrane. In mixed-cell foci, labeling occurred both along the periphery and diffusely throughout the cytoplasm. On those slides stained, glutathione-S-transferase (placental; GSTP) was detected in almost all eosinophilic and mixed-cell foci, whereas approximately half of the basophilic and clear-cell foci stained for GSTP. The presence of GSTP in a focus was not always associated with the presence of increased TGF alpha protein. All rat hepatic adenomas and the one carcinoma labeled positive for TGF alpha. Increased levels of TGF alpha protein were associated with increased DNA synthesis labeling index. The number of TGF alpha-positive foci with the highest DNA synthesis labeling indices were statistically higher than those with lower levels of DNA synthesis labeling. Although characteristic staining patterns for TGF alpha were associated with specific histological subtype, the role that TGF alpha plays in the progression of focal lesions to neoplasia requires further definition. High levels of TGF alpha protein appear to be acquired sometime during the hepatocarcinogenic process. It may be that early lesions that acquire high levels of TGF alpha are the ones to develop into hepatocellular carcinoma (e.g., hepatocellular carcinoma is determined very early in the carcinogenic process). It is apparent that further work is needed to delineate the role of TGF alpha in both rodent and human hepatocarcinogenesis.     

Coexpression of C-myc and transforming growth factor alfa in the liver promotes early replicative senescence and diminishes regenerative capacity after partial hepatectomy in transgenic mice

We have recently shown that overexpression of c-myc and transforming growth factor alpha (TGF-alpha) in the liver of double-transgenic mice results in severe DNA damage, aberrant hepatic growth, and development of tumors at a much younger age than that observed in c-myc single-transgenic mice. We now report that double-transgenic TGF-alpha/c-myc hepatocytes rapidly lose their ability to proliferate upon mitogenic stimulation following partial hepatectomy (PH). At 4 weeks of age, the overall rate of bromodeoxyuridine (BrdU) incorporation following PH was comparable in c-myc and TGF-alpha/c-myc livers and exceeded that seen in wild-type (WT) mice. However, by 10 weeks of age, c-myc single-transgenic hepatocytes showed proliferative advantages over the WT cells, whereas TGF-alpha/c-myc double-transgenic hepatocytes had a decreased capacity to proliferate upon mitogenic stimulation. This decreased proliferative response was accompanied by a reduction in the total fraction of proliferating hepatocytes, as well as by a decline in the induction of cyclin A, cyclin B, and cdc2 gene expression. These data show that constitutive coexpression of c-myc and TGF-alpha accelerates age-related loss in the regenerative potential following PH, and suggest that early replicative senescence of differentiated hepatocytes may have a role in providing a selective growth advantage to initiated cell populations in this model.     

Differences in phenotype and cell replicative behavior of hepatic tumors induced by dichloroacetate (DCA) and trichloroacetate (TCA)

Dichloroacetate (DCA) and trichloroacetate (TCA) are two hepatocarcinogenic by-products of water chlorination. To compare the effects of DCA and TCA on cell replication in the nodules and tumors they induce, male B6C3F1 mice were administered 2.0 g/L DCA or TCA in their drinking water for 38 or 50 weeks, respectively. The pretreated mice were then given water containing 0, 0.02, 0.5, 1.0, or 2.0 g/L DCA or TCA for two additional weeks to determine whether cell proliferation in the normal liver or tumors that had been induced by DCA or TCA was dependent on continued treatment. Prior to sacrifice the mice were subcutaneously implanted with mini-osmotic pumps to label DNA in dividing cells with 5-bromo-2'-deoxyuridine (BrdU). Serial sections of nodules/tumors and normal liver were stained immunohistochemically for BrdU, the oncoproteins c-Jun and c-Fos, and hematoxylin and eosin (H & E); or with Periodic acid-Schiff (PAS) stain, BrdU, and H & E, respectively. DCA and TCA transiently stimulated the division of normal hepatocytes relative to rates observed in the livers of control mice. However, at 40 and 52 weeks of treatment, replication of normal hepatocytes was substantially inhibited by DCA and TCA, respectively. Cell division within DCA-induced lesions that were identified macroscopically was significantly higher with increasing dose of DCA administered in the last 2 weeks of the experiment. DCA-induced lesions were found to display immunoreactivity to anti-c-Jun and anti-c-Fos antibodies, were predominantly basophilic, and contained very little glycogen relative to surrounding hepatocytes. In contrast, rates of cell division within TCA-induced altered hepatic foci and tumors were very high and appeared to be independent of continued treatment. TCA-induced lesions did not display immunoreactivity to either c-Jun or c-Fos antibodies. Results from this study suggest that the mechanisms by which DCA and TCA induce hepatocarcinogenesis in the male B6C3F1 mouse differ.