The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligandactivated 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.
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. Here we show that the levels of several cytochrome P450 (CYP) isoenzymes are also altered in GS-positive liver tumors. Immunohistochemical and western blotting analyses demonstrated that GS-positive, catnb-mutated tumors showed levels of CYP1A, CYP2B, CYP2C and CYP2E1, which were similar or slightly enhanced in comparison with non-tumoral liver tissue. This contrasts with tumors without catnb mutations, which exhibited decreased levels of these CYP isoforms. Real-time RT-PCR revealed that the differences in CYP levels in the tumors corresponded to changes in the respective mRNAs. Mouse hepatoma cells were transiently transfected with an expression vector encoding an S33Y-mutated beta-catenin protein, which was functional with regard to transactivation of a beta-catenin/TCF-responsive (topflash) reporter construct. Co-transfected with luciferase reporter vectors containing either the regulatory upstream sequence of the CYP2B1 gene or three dioxin-responsive core elements were activated by S33Y-beta-catenin. These results indicate that mutation of catnb leads to transcriptional activation of CYP isoenzymes in mouse liver tumors. As CYPs are involved in both the activation and the inactivation of several clinically important anticancer drugs, our findings may be relevant for chemotherapy of human cancers, where activation of beta-catenin-dependent signaling by mutation of the gene or alternative mechanisms is frequently observed.
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