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 β-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 β-catenin signaling and (ii) absence of β-catenin enhances carcinogen-induced hepatocarcinogenesis and induces a pre-cirrhotic phenotype in mouse liver.
Signaling through the Wnt/β-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 β-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 β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-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 β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-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 β-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 β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.
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.
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.
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