Chemically induced mouse liver tumors are resistant to treatment with atorvastatin.

BACKGROUND: Atorvastatin is a potent inhibitor of the mevalonate pathway and widely used as a hypolipidemic drug. Some epidemiological studies and animal experiments indicate that the long-term use of atorvastatin and structurally related drugs might be associated with a reduced risk of developing hepatocellular carcinoma (HCC), the most common hepatocellular malignancy in humans. However, the potential of atorvastatin to inhibit HCC formation is controversially discussed. METHODS: Hepatocellular tumors were chemically induced by treatment of C3H/He mice with 10 µg/g body weight N-nitrosodiethylamine and the ability of atorvastatin to interfere with tumor formation was investigated by treatment of mice with 0.1% atorvastatin in the diet for 6 months. Tumor size and tumor multiplicity were analyzed, as were tissue levels of cholesterol and atorvastatin. RESULTS: Atorvastatin treatment efficiently reduced serum cholesterol levels. However, the growth of tumors driven by activated MAPK (mitogen-activated protein kinase) signaling was not attenuated by the presence of the drug, as evidenced by a lack of reduction of tumor volume or tumor multiplicity by atorvastatin. Levels of the atorvastatin uptake transporters Oatp1a4 and Oatp1b2 were down-regulated at the mRNA and protein levels in chemically induced mouse liver tumors, but without striking effects on atorvastatin concentrations in the tumor tissue. CONCLUSION: In summary, the present data provide substantial evidence that atorvastatin does not beneficially influence tumor growth in mouse liver and thereby challenge the hypothesis that statin use might protect against hepatocellular cancer.

Tumor formation in liver of conditional ß-catenin-deficient mice exposed to a diethylnitrosamine/phenobarbital tumor promotion regimen.

The antiepileptic drug phenobarbital (PB) is a potent tumor promoter in mouse liver, where it stimulates the selective outgrowth of tumor populations harboring activating mutations in Ctnnb1, encoding ß-catenin. A tumor initiation-promotion study was conducted in mice with conditional hepatocyte-specific knockout (KO) of Ctnnb1 and in Ctnnb1 wild-type controls. Mice received a single injection of N-nitrosodiethylamine (DEN) at the age of 6 weeks followed by continuous administration of PB given in the diet (0.05%) for 27 weeks. Metabolic activation of DEN in hepatocytes from both Ctnnb1 wild-type and KO mice was demonstrated. PB strongly enhanced liver tumor formation in Ctnnb1 wild-type mice, and 90% of the PB-promoted tumors were Ctnnb1-mutated. A similar increase in carcinogenic response was seen when using glucose-6-phosphatase and glutamine synthetase as tumor markers. The prevalence of tumors in Ctnnb1 KO mice was ∼7-fold higher than in wild-type mice, suggesting an enhancing effect of the gene KO on liver tumor development. However, in strong contrast to wild-type mice, PB did not promote tumor formation in the Ctnnb1 KO mice. Livers of KO mice, particularly from the PB treatment group, demonstrated fibrosis and massive infiltration of immune cells, an effect not seen in wild-type mice. In summary, our data demonstrate that (i) liver tumor promotion by PB requires functional ß-catenin signaling and (ii) absence of ß-catenin enhances carcinogen-induced hepatocarcinogenesis and induces a pre-cirrhotic phenotype in mouse liver.

Phenotype and growth behavior of residual ß-catenin-positive hepatocytes in livers of ß-catenin-deficient mice.

Signaling through the Wnt/ß-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding ß-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining ß-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the ß-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated ß-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in ß-catenin-deficient liver. Clusters of ß-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, ß-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of ß-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of ß-catenin, and that ß-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.

Hepatocarcinogenesis in mice with a conditional knockout of the hepatocyte growth factor receptor c-Met.

The receptor for the hepatocyte growth factor/scatter factor (HGF/SF), c-Met, plays a role in tumour promotion, progression and metastasis. In this study, we analysed chemically induced hepatocarcinogenesis in mice lacking a functional HGF receptor in their liver. Control and c-Met deficient mice were injected with a single dose of N-nitrosodiethylamine (DEN, 90 mICROg/g b.wt.) at 6 weeks of age and mice were subsequently kept on a phenobarbital (PB) containing diet (0.05%) for 35 weeks or on control diet. At the end of the experiment, the carcinogenic response in liver of the animals was monitored. Conditional c-met knockout (KO) mice showed a higher prevalence of macroscopically visible liver tumours and of glutamine synthetase positive and glucose-6-phosphatase deficient lesions in liver. Tumour promotion by PB led to significant increases in the number of preneoplastic and neoplastic lesions in liver of both wild-type and c-met knockout mice, with only minor differences in response. Our results indicate that a defect in c-Met-mediated signaling increases chemically induced tumour initiation in liver but does not significantly affect PB-mediated tumour promotion.

The glycogen synthase kinase inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) is a partial agonist of the aryl hydrocarbon receptor.

Kinase inhibitors are frequently used tools in signal transduction research. 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), a potent inhibitor of glycogen synthase kinase 3beta (GSK3beta), is frequently used to activate beta-catenin signaling by mimicking the action of Wnt molecules. beta-Catenin is a crucial player in the regulation of hepatic drug metabolism. Thus, it is of particular importance to know whether the tools used to study the effects of beta-catenin signaling may affect the respective drug-metabolizing target enzymes in an unwanted manner. In this study, we show that SB216763 is able to induce cytochrome P450 1a1 (Cyp1a1) expression in a dose-dependent manner in mouse hepatoma cells. Moreover, SB216763 is able to inhibit Cyp1a1 induction by the prototype aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachloro-p-dibenzodioxin. Cyp1a1 induction by SB216763 is independent of GSK3beta and the beta-catenin pathway. Instead, SB216763 induces Cyp1a1 by activation of AhR-mediated transcription. The present results suggest that SB216763 acts as a partial agonist of the AhR.

Differential selection for B-raf and Ha-ras mutated liver tumors in mice with high and low susceptibility to hepatocarcinogenesis.

Activation of the Ras/Raf/MEK/ERK pathway is frequently observed in animal and human tumors. In our study, we analyzed B-raf codon 637 (formerly 624) and Ha-ras codon 61 mutations in liver tumors from C3H, B6C3F1 and C56BL mice which differ considerably with regard to their susceptibility to hepatocarcinogenesis. In total, 73% (102/140) of tumors induced by a single application of N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene contained either B-raf or Ha-ras mutations and only <3% (4/140) were mutated in both genes. In addition, B-raf mutations were present in 76% (19/25) of early precancerous liver lesions. The prevalence of Ha-ras mutated tumors was significantly higher in the susceptible C3H and B6C3F1 mouse strains (39-50%) than in the comparatively resistant C57BL mouse (7%). B-raf mutated tumors, by contrast, were more frequent in C57BL mice (68%) than in the other two strains (17-45%). Taken together, our findings indicate that alterations affecting the Ras/Raf/MEK/ERK signalling pathway are a hallmark of carcinogen-induced liver tumors in mice. Moreover, our results show that mutational activation of B-raf in liver tumors of different mouse strains is, by contrast to Ha-ras, inversely related to their susceptibility to hepatocarcinogenesis. Although activated Ras and Raf proteins are assumed to have similar biological effects because they feed into the same signalling pathway, there seem to be subtle strain-specific differences in selection processes favouring the preferential outgrowth of either B-raf or Ha-ras mutated tumor populations in mouse liver.

Serum components and activated Ha-ras antagonize expression of perivenous marker genes stimulated by beta-catenin signaling in mouse hepatocytes.

Hepatocytes of the periportal and perivenous zones of the liver lobule show marked differences in the contents and activities of many enzymes and other proteins. Previous studies from our and other groups have pointed towards an important role of beta-catenin-dependent signaling in the regulation of expression of genes encoding proteins with preferential perivenous localization, whereas, in contrast, signaling through Ras-dependent pathway(s) may induce a 'periportal' phenotype. We have now conducted a series of experiments to further investigate this hypothesis. In transgenic mice with scattered expression of an activated Ha-ras (Ha-ras(G12V)) mutant in liver, expression of the perivenous markers glutamine synthetase and two cytochrome P450 isoforms was completely abolished in those hepatocytes demonstrating constitutively activated extracellular signal-regulated kinase activity, even though they were located directly adjacent to central veins. Similarly, incubation of primary hepatocytes or hepatoma cells with increasing amounts of serum caused a concentration-dependent attenuation of expression of perivenous marker mRNAs, whereas the expression of periportal markers was increased. The inhibitory effect of high amounts of serum on the expression of perivenous markers was also observed if their expression was stimulated by activation of beta-catenin signaling, and comparable inhibitory effects were seen in cells stably transfected with a T-cell factor/lymphoid-enhancing factor-driven luciferase reporter. Epidermal growth factor could partly mimic serum effects in hepatoma cells, and its effect could be blocked by an inhibitor of extracellular signal-regulated kinase activity. These data suggest that activation of the Ras/mitogen-activated protein kinase (extracellular signal-regulated kinase) pathway favors periportal gene expression while simultaneously antagonizing a perivenous phenotype of hepatocytes.

B-raf and Ha-ras mutations in chemically induced mouse liver tumors.

The mitogen-activated protein kinase signalling pathway is a central regulator of tumor growth, which is constitutively activated in chemically induced mouse liver tumors. In about 30-50% of cases this effect can be related to activation of the Ha-ras gene by point mutations, whereas in the remaining cases mutations may occur in other members within this pathway, such as Raf kinases. Recently, B-raf has been shown to be frequently mutated in human melanomas and certain other cancers, with a V599E amino-acid change representing the most predominant mutation type. We now screened 82 N-nitrosodiethylamine-induced liver tumors from C3H/He mice for mutations within the hotspot positions in the Ha-ras and B-raf genes. About 50% (39/82) of tumors showed Ha-ras codon 61 mutations and 16 tumors ( approximately 20%) harbored mutations at codon 624 of the B-raf gene, which corresponds to codon 599 in human B-raf. None of the tumors was mutated in both Ha-ras and B-raf. The high prevalence of Ha-ras and B-raf mutations in mouse liver tumors is in striking contrast to human hepatocellular cancers which very infrequently harbor mutations in the two genes. These fundamental differences between the biology of liver tumors in mice and man may be of toxicological relevance.

Differential gene expression in periportal and perivenous mouse hepatocytes.

Hepatocytes located in the periportal and perivenous zones of the liver lobule show remarkable differences in the levels and activities of various enzymes and other proteins. To analyze global gene expression patterns of periportal and perivenous hepatocytes, enriched populations of the two cell types were isolated by combined collagenase/digitonin perfusion from mouse liver and used for microarray analysis. In total, 198 genes and expressed sequences were identified that demonstrated a >/= 2-fold difference in expression between hepatocytes from the two different zones of the liver. A subset of 20 genes was additionally analyzed by real-time RT-PCR, validating the results obtained by the microarray analysis. Several of the differentially expressed genes encoded key enzymes of intermediary metabolism, including those involved in glycolysis and gluconeogenesis, fatty acid degradation, cholesterol and bile acid metabolism, amino acid degradation and ammonia utilization. In addition, several enzymes of phase I and phase II of xenobiotic metabolism were differentially expressed in periportal and perivenous hepatocytes. Our results confirm previous findings on metabolic zonation in liver, and extend our knowledge of the regulatory mechanisms at the transcriptional level.

PCB 153, a non-dioxin-like tumor promoter, selects for beta-catenin (Catnb)-mutated mouse liver tumors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental toxicants which act as liver tumor promoters in rodents and can be classified as either dioxin-like or non-dioxin (phenobarbital [PB])-like inducers of cytochrome P-450. Since we have previously shown that tumor promotion by PB leads to clonal outgrowth of beta-catenin (Catnb)-mutated but not Ha-ras-mutated mouse liver tumors, we were interested to know whether the non-dioxin-like tumor promoter 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) shows the same selective pressure during tumor promotion. Male B6129SF2/J mice were given a single injection of N-nitrosodiethylamine (90 mg/kg body weight) at 9 weeks of age, followed by 39 weeks of treatment with PCB 153 (20 biweekly ip injections of 300 mumol/kg body weight) or corn oil as a control. Animals were killed 15 weeks after the last PCB 153 injection and liver tumors were identified by immunohistochemical staining of glutamine synthetase (GS) and analyzed for Catnb, Ha-ras, and B-raf mutations. Quantitative analyses revealed that GS-positive tumors were much larger and more frequent in livers from PCB 153-treated mice than in control animals, whereas GS-negative tumors were similar in both groups. Almost 90% (34/38) of all tumors from PCB 153-treated animals contained Catnb mutations, which compares to approximately 45% (17/37) of tumors in the control group. Ha-ras- and B-raf-mutated liver tumors were rare and not significantly different between treatment groups. These results clearly indicate that PCB 153 strongly selects for Catnb-mutated, GS-positive liver tumors, which is similar to the known action of PB, a prototypical tumor promoter in rodent liver.

Overexpression of glutamine synthetase is associated with beta-catenin-mutations in mouse liver tumors during promotion of hepatocarcinogenesis by phenobarbital.

Phenobarbital (PB) is an antiepileptic drug that promotes hepatocarcinogenesis in rodents when administered subsequent to an initiating carcinogen like N-nitrosodiethylamine (DEN). In the mouse, the promotional effect of PB on liver tumor development results from a selective stimulation of clonal outgrowth of hepatocytes harboring activating mutations in the beta-catenin gene. Because glutamine synthetase (GS) has recently been shown to be a putative transcriptional target of beta-catenin, expression of GS during PB-mediated promotion of mouse hepatocarcinogenesis was investigated. Preneoplastic and neoplastic liver lesions were induced in 6-week-old male mice by a single injection of 90 micro g/g body weight of DEN, and groups of mice were subsequently kept on PB-containing (0.05%) or control diet for 39 weeks. In PB-treated mice, 46 of 51 lesions ( approximately 90%) were GS-positive in contrast to only 16 of 46 ( approximately 35%) in mice not treated with PB. Approximately 33% of liver was occupied by neoplastic tissue in PB-treated mice, of which >80% was GS positive. By contrast, only approximately 3.5% of liver consisted of neoplastic tissue in mice treated with DEN only, and approximately 25% of this was GS positive. We have previously shown that beta-catenin mutations are present in approximately 80% of liver tumors from PB-treated mice but are absent in liver tumors from mice treated with DEN only. By analyzing a panel of larger liver tumors, we now observed that tumors harboring beta-catenin mutations were GS positive, whereas tumors without beta-catenin mutations were GS negative. Similarly, tumors from an additional mouse carcinogenicity experiment where PB inhibited rather than promoted hepatocarcinogenesis were mostly GS negative. These data suggest that promotion of hepatocarcinogenesis by PB confers beta-catenin-mutated tumor cells with a selective advantage by up-regulation of GS expression.

A constitutively active dioxin/aryl hydrocarbon receptor promotes hepatocarcinogenesis in mice.

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding enzymes involved in drug metabolism. Known ligands include polycyclic aromatic hydrocarbons, certain polychlorinated biphenyls, and the polyhalogenated dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both polyhalogenated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin are potent promoters of rodent hepatocarcinogenesis in two-stage initiation-promotion experiments. Although several lines of evidence indicate the involvement of the AhR in toxic effects mediated by polyhalogenated biphenyls and dioxins, its involvement in tumor promotion has not been unequivocally proven. In the present study, a transgenic mouse line expressing a constitutively active AhR (CA-AhR) has been used to investigate the role of the AhR in hepatocarcinogenesis. Male AhR wild-type and CA-AhR-transgenic B6C3F1-mice were treated with a single injection of the hepatocarcinogen N-nitrosodiethylamine at 6 weeks of age and were subsequently kept untreated on control diet. Thirty five weeks after carcinogen treatment, mice were sacrificed, and the prevalence and multiplicity of liver tumors were determined. Whereas only 1 small liver tumor was observed in 15 AhR-wild-type mice, 19 tumors (two >1 cm in diameter) were present in 18 CA-AhR-transgenic mice. This result demonstrates the oncogenic potential of the activated AhR and implicates an important role of the receptor in promotion of hepatocarcinogenesis. A microarray-based gene expression-profiling analysis revealed down-regulation in the liver of CA-AhR-transgenic mice of a cluster of genes encoding heat shock proteins, including GRP78/BiP, Herp1, Hsp90, DnaJ (Hsp40) homologue B1, and Hsp105, which are important for protein folding and quality control.

Modulation of liver tumorigenesis in Connexin32-deficient mouse.

Connexin32 (Cx32) is the major gap junction forming protein in liver. Mice deficient in Cx32 demonstrate enhanced liver tumor formation, but are resistant to promotion of hepatocarcinogenesis by the model tumor promoter phenobarbital (PB). Here, we re-evaluate data on the number and sizes of glucose-6-phosphatase (G6Pase)-deficient liver lesions, both in Cx32-wildtype (WT) and Cx32-null male mice, obtained from two earlier experiments with similar protocols but paradoxical outcomes. In these experiments, enzyme-altered lesions were induced in mice of both strains by a single injection of N-nitrosodiethylamine (DEN) at age 6 weeks with a dose of 90 microg/g body weight (experiment 1) or at age 2 weeks with 10 microg/g body weight (experiment 2). Three weeks after DEN treatment groups of mice (sub-divided by Cx32 status) were also started on a PB-containing (0.05%) diet to test the responsiveness of the lesions to the tumor promoter. Additionally, for experiment 1, tumors were analyzed for the presence of Ha-ras and beta-catenin mutations. Based on the mutational analysis and the mathematical analysis of the G6Pase-deficient lesions, the two studies are consistent with the hypothesis of two types of lesions, 'late-type' lesions which are mainly characterized by beta-catenin mutations, and 'early-type' lesions that are frequently (but not exclusively) Ha-ras mutated. This concept affords an explanation as to the differential response seen in the two experiments with regard to Cx32 status and the role of PB as a tumor promoter (experiment 1) or inhibitor (as in experiment 2). Our findings also underscore the importance of the timing (6 weeks versus 2 weeks) of the genotoxic insult in relation to the developmental stage of the liver and the importance of clonal selection during tumor promotion.

Insulin and dexamethasone inhibit TGF-beta-induced apoptosis of hepatoma cells upstream of the caspase activation cascade.

Insulin and dexamethasone are potent inhibitors of apoptosis induced by transforming growth factor-beta1 (TGF-beta) in hepatoma cells. Using FTO-2B rat hepatoma cells, we determined whether the anti-apoptotic effects of these agents result from interference within or upstream of the TGF-beta-induced caspase cascade. Activation of different initiator and effector caspases, Bax and Bcl-xL expression, mitochondrial cytochrome c release and activation of PKB/Akt were analyzed by use of synthetic caspase substrates and Western blotting, respectively. TGF-beta-induced apoptosis was characterized by release of cytochrome c from mitochondria and activation of caspases-3, -7, -8 and -9. These effects were observable as early as 8-12 h after start of treatment and increased with time of observation. Inhibition of TGF-beta-induced apoptosis by insulin and dexamethasone was paralleled by a strong reduction of caspase-3-like activity. Caspase-8 activation was almost completely suppressed by these agents, and caspase-9 activity was decreased to levels within or slightly above unstimulated control cells. In addition, cytochrome c release from mitochondria was efficiently repressed, which was associated with upregulation of Bcl-xL by dexamethasone and activation of PKB/Akt by insulin. Thus, both anti-apoptotic compounds exert their inhibitory effects through modulation of anti-apoptotic signalling pathways involved in regulation of cytochrome c release and activation of the caspase machinery.

Biological and tumor-promoting effects of dioxin-like and non-dioxin-like polychlorinated biphenyls in mouse liver after single or combined treatment.

To assess the impact of a mixture containing dioxin-like and non-dioxin-like polychlorinated biphenyls (PCBs), male mice were initiated with N-nitroso-diethylamine and subsequently treated with PCB126, an Ah-Receptor agonist, and PCB153, acting via activation of the constitutive androstane receptor. The two congeners were given at two dose levels: the low dose was adjusted to induce ~150-fold increases in cytochrome P450 (Cyp)1a1 (PCB126) and Cyp2b10 mRNAs (PCB153), and the high dose was chosen as twice the low dose. To keep the liver PCB levels constant, mice were given initial loading doses followed by weekly maintenance doses calculated on the basis of the PCBs' half-lives. Mice were treated with the individual congeners (low and high dose) or with a mixture consisting of the low doses of the 2 PCBs. The following results were obtained: (1) the 2 PCBs produced dose-dependent increases in Cyp1a1 and Cyp2b10 mRNA, protein, and activity when given individually; (2) combined treatment caused more than additive effects on Cyp1a1 mRNA expression, protein level, and ethoxyresurofin activity; (3) changes in the levels of several proteins were detected by proteome analysis in livers of PCB-treated mice; (4) besides these biological responses, the individual PCBs caused no significant increase in the number of glucose-6-phospatase (G6Pase)-deficient neoplastic lesions in liver, whereas a moderate significant effect occurred in the combination group. These results suggest weak but significant response-additive effects of the 2 PCBs when given in combination. They also suggest that the Cyp biomarkers tend to overestimate the carcinogenic response produced by the PCBs in mouse liver.

Coordinate regulation of cytochrome P450 1a1 expression in mouse liver by the aryl hydrocarbon receptor and the beta-catenin pathway.

The expression of cytochrome P450 (CYP) 1a1 and other drug-metabolizing enzymes is controlled by the aryl hydrocarbon receptor (AhR), which is activated by dioxin-type inducers leading to transcriptional induction of target genes. Here, we show that a second level of transcriptional control exists in hepatocytes, which is tightly linked to the Wnt/ß-catenin/T-cell factor (TCF) signaling pathway. In transgenic mice, hepatic expression of CYP1A (and other CYP isoforms) is stimulated by the expression of mutationally activated ß-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of ß-catenin. These effects were further analyzed in vitro, and the stimulatory role of ß-catenin was ascribed to a TCF-binding site within the CYP1A1 promoter. Moreover, ß-catenin signaling acted cooperatively with AhR agonists via AhR-binding sites on the DNA during the induction of Cyp1a1 in vivo and in vitro. Activation of ß-catenin enhanced the transactivation potential of ligand-activated AhR at its DNA-binding sites without altering the total amount of DNA-bound AhR. Coimmunoprecipitation demonstrated a physical interaction between AhR and ß-catenin. Furthermore, the present results suggest that transcriptional induction of the AhR by ß-catenin does not play a major role in ß-catenin-dependent regulation of Cyp1a1 expression and that inhibition of ß-catenin signaling by ligand-activated AhR, as recently observed in the intestine does not occur in mouse liver. In conclusion, signaling through ß-catenin activates basal CYP1A1 expression and augments CYP1A1 induction by AhR ligands through enhancement of the transactivation potential of the AhR.

Phenotype of single hepatocytes expressing an activated version of ß-catenin in liver of transgenic mice.

The gene CTNNB1 encoding ß-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of ß-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where ß-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated ß-catenin (human ß-catenin(S33Y)) was established using the Cre/loxP system. Expression of several downstream targets of ß-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the ß-catenin(S33Y) transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated ß-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated ß-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of ß-catenin in clusters of hepatocytes, suggesting a role of cell-cell communication in this process.

Wnt/beta-catenin signaling activates and determines hepatic zonal expression of glutathione S-transferases in mouse liver.

Glutathione S-transferases (GSTs) play an essential role in the elimination of xenobiotic-derived electrophilic metabolites and also catalyze certain steps in the conversion of endogenous molecules. Their expression is controlled by different transcription factors, such as the antioxidant-activated Nrf2 or the constitutive androstane receptor. Here, we show that the Wnt/beta-catenin pathway is also involved in the transcriptional regulation of GSTs: GSTm2, GSTm3, and GSTm6 are overexpressed in mouse hepatomas with activating Ctnnb1 (encoding beta-catenin) mutations and in transgenic hepatocytes expressing activated beta-catenin. Inversely, GSTm expression is reduced in mice with hepatocyte-specific knock out of Ctnnb1. Activation of beta-catenin-dependent signaling stimulates GSTm expression in vitro. Activation of beta-catenin in mouse hepatoma cells activates GSTm3 promoter-driven reporter activity, independently of beta-catenin/T-cell factor sites, via a retinoid X receptor-binding site. By contrast, GSTm expression is inhibited upon Ras activation in mouse liver tumors and transgenic hepatocytes. Recent studies by different groups have shown that beta-catenin-dependent signaling is involved in the transcriptional control of "perivenous" expression of various cytochrome P450s in mouse liver, whereas Ras signaling was hypothesized to antagonize the perivenous hepatocyte phenotype. In synopsis with our present results, it now appears that the Wnt/beta-catenin pathway functions as a master regulator of the expression of both phase I and phase II drug-metabolizing enzymes in perivenous hepatocytes from mouse liver.

Comparative transcriptome and proteome analysis of Ha-ras and B-raf mutated mouse liver tumors.

Mouse liver tumors frequently harbor activating mutations in the Ha-ras protooncogene. In addition, mutations are also found in the B-raf gene leading to constitutive activation of the B-Raf kinase. In two previous studies, we have investigated by microarray analysis the effect of the mutations on the mRNA expression patterns of the respective tumors. In the present study, we analyzed proteome changes in Ha-ras and B-raf mutated liver tumors by 2-D gel-electrophoretic separation of proteins followed by their identification by mass spectrometry. In total, 104 significantly altered protein spots were identified in Ha-ras mutated tumors and 111 in B-raf mutated tumors when compared to the corresponding normal liver tissue. The changes in protein expression patterns were highly correlated between Ha-ras and B-raf mutated tumors, and in the majority of the cases, both tumor types showed the respective alteration. Most of the tumor-specific changes in protein expression were reflected by similar changes in their mRNAs except for some up-regulated proteins without accompanying changes in mRNA levels. Interestingly, Ha-ras but not B-raf mutated tumors showed high levels of the phosphorylated (activated) form of the Ras/Raf/MEK effector kinase ERK which was, however, not associated with any detectable difference in the transcriptome or protein setup of the tumors.

Tumor promotion in liver of mice with a conditional Cx26 knockout.

Connexin (Cx) 26 and 32 are the major gap junction proteins in liver. We recently demonstrated that Cx32 is essential for phenobarbital (PB)-mediated tumor promotion in mouse liver. To investigate whether Cx26 plays a similar role, an initiation-promotion experiment was conducted using mice with a liver-specific knockout of Cx26. Control and Cx26-deficient mice were injected a single dose of N-nitrosodiethylamine (DEN, 90 microg/g b.wt.) at 6 weeks of age and groups of mice were subsequently kept on a PB (0.05%) containing or control diet for 35 weeks. At the end of the experiment, the carcinogenic response in the liver was monitored. Mice from PB treatment groups showed strongly increased liver weights compared with mice treated with DEN alone, which was mostly due to a much higher tumor burden. The tumor response in PB-treated mice of both strains was quite similar, but the number of smaller tumors and of enzyme-altered neoplastic lesions was somewhat larger in PB-treated Cx26 knockout (Cx26 KO) compared with wild-type mice, whereas the volume fraction of enzyme-altered lesions was slightly reduced in PB-treated Cx26-deficient mice. There was no significant difference in tumor prevalence between Cx26 KO and wild-type mice. Altogether our present data show that elimination of Cx26 has only minor effects on chemically induced mouse hepatocarcinogenesis, in striking contrast to the effects seen in Cx32 KO mice.