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.

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.

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

The gene CTNNB1 encoding β-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 β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes.

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/β-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 β-catenin(S33Y) in the absence of AhR-activating compounds but repressed after knockout of β-catenin.

Tumor formation in liver of conditional β-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 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.

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

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.

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.

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.

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.

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.

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.

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.

Proteome analysis of chemically induced mouse liver tumors with different genotype.

Mouse liver tumors frequently harbor mutations in Ha-ras, B-raf, or Ctnnb1 (encoding beta-catenin). We conducted a proteome analysis with protein extracts from normal mouse liver and from liver tumors which were induced by a single injection of N-nitrosodiethylamine (DEN) as initiator followed by multiple injections of two different polychlorinated biphenyls (PCBs) as tumor promoters, or corn oil as a control. Liver tumors were stratified into two classes: they were either mutated in Ctnnb1 and positive for the marker glutamine synthetase (GS(+)), or they lacked Ctnnb1 mutations and were therefore GS-negative (GS(-)).

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.

Global gene expression in Ha-ras and B-raf mutated mouse liver tumors.

Chemically-induced mouse liver tumors harbor mutations in different oncogenes. About 50% of tumors contain activating mutations in the Ha-ras gene contain and about 20% of tumors show point mutations in the B-raf oncogene. We have investigated the gene expression profiles in tumors of the 2 genotypes by microarray analysis.

Zonal gene expression in mouse liver resembles expression patterns of Ha-ras and beta-catenin mutated hepatomas.

Hepatocytes of the periportal and perivenous zones of the liver lobule differ in their levels and activities of various enzymes and other proteins. We have recently suggested that beta-catenin- and Ras-dependent signaling pathways play an important role in the regulation of perivenous and periportal gene expression profiles. This hypothesis was primarily based on similarities in zonal differences in gene expression of hepatocytes from normal liver with gene expression patterns of liver tumors: several proteins and mRNAs preferentially expressed in periportal hepatocytes were often overexpressed in Ha-ras mutated mouse liver tumors, whereas perivenous markers were overexpressed in Ctnnb1 (encoding beta-catenin) mutated tumors.

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.

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.

Zonal gene expression in murine liver: lessons from tumors.

Gene expression in hepatocytes within the liver lobule is differentially regulated along the portal to central axis; however, the mechanisms governing the processes of zonation within the lobule are unknown. A model for zonal heterogeneity in normal liver is proposed, based on observations of differential expression of genes in liver tumors from mice that harbor activating mutations in either Catnb (which codes for beta-catenin) or Ha-ras. According to the model, the regulatory control consists of two opposing signals, one delivered by endothelial cells of the central veins activating a beta-catenin-dependent pathway (retrograde signal), the other by blood-borne molecules activating Ras-dependent downstream cascades (anterograde signal).

Genotype-phenotype relationships in hepatocellular tumors from mice and man.

Experimentally induced liver tumors in mice harbor activating mutations in either Catnb (beta-catenin) or Ha-ras, according to the carcinogenic treatment. We have now investigated by microarray analysis the gene expression profiles in tumors of the two genotypes. In total, 364 genes or expressed sequences with aberrant expression relative to normal liver were identified, but only 30 of these demonstrated unidirectional changes in both tumor types.

Human p53 knock-in (hupki) mice do not differ in liver tumor response from their counterparts with murine p53.

Mouse models are important tools in toxicologic research. Differences between species in pathways contributing to tumor development, however, raise the question in how far mouse models are valid for human risk assessment. One striking difference relates to the frequency of spontaneous liver cancer which is high in certain mouse strains but rather low in humans.

Effect of the tumor promoter phenobarbital on the pattern of global gene expression in liver of connexin32-wild-type and connexin32-deficient mice.

The antiepileptic drug phenobarbital (PB) is used frequently as a model tumor promoter in rodent liver. It is believed to increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. The molecular mechanism underlying this process is only partly understood but seems to require the function of connexin32 (Cx32), one of the 2 gap junction proteins expressed in hepatocytes.

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.

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.

A beta-catenin-dependent pathway regulates expression of cytochrome P450 isoforms in mouse liver tumors.

Phenobarbital (PB) is a model tumor promoter in the rodent liver. In the mouse, the promotional effect of PB results from a selective stimulation of clonal outgrowth of hepatocytes harboring activating mutations in the beta-catenin (catnb) gene. Glutamine synthetase (GS), a downstream target in the Wnt/beta-catenin/T-cell factor (TCF) signaling pathway, is strongly up-regulated in catnb-mutated mouse liver tumors and may serve as a marker for their identification.