Phenobarbital-mediated promotion of hepatocarcinogensis in mice depends on connexin32 and leads to clonal outgrowth of β-catenin-mutated tumors.

O Moennikes Dr. a, H Aydenlik a, T D Nguyen a, A Buchmann Dr.a, K Willecke Prof.b and M Schwarz Prof.a

a Institut für Toxikologie, Wilhelmstr. 56, 72074 Tübingen, Germany; and b Institut für Genetik, 53117 Bonn, Germany

AIMS Many tumor-promoting agents, including phenobarbital (PB), block gap junctional communication in vitro. The relevance of this effect was tested in vivo by analyzing the efficacy of PB as a liver tumor promoter in male Cx32-wild-typ and Cx32-null mice. Addition studies were undertaken aimed to clarify the mechanism of tumor promoting action of PB. METHODS Liver tumors were generated by single administration of diethylnitrosmamine (DEN) to 6 weeks old Cx32-null and Cx32-wildtyp mice followed by feeding of PB (0.05 %) containing or control diet for 39 weeks. Number and size of macroscopically visible tumors were monitored and (pre)neoplastic lesions identified by their deficiency in glucose-6-phosphatase staining. Mutations at Ha-ras codon 61 were screened by allele-specific oligonucleotide hybridization; β-catenin mutations were detected by direct sequencing of PCR products spanning exon 2. RESULTS Phenobarbital led to a 5-fold increase in the volume fraction occupied by liver lesions in Cx32-wildtyp mice, whereas there was no such increase in Cx32-null mice. The occurrence of numerous large hepatomas in Cx32-wildtyp mice was clearly promoted by PB, while no such effect was seen in Cx32-null mice. In tumors from wild-type mice treated with DEN alone, the prevalence of activating mutations in the Ha-ras protooncogene was 30 % (6/20), while no mutations in the β-catenin gene (0/13) were detectable. By contrast, Ha-ras mutations were undetectable in tumors from wild-type mice treated with DEN/PB (0/32), while 80 % (37/46) of tumors from this group showed β-catenin mutations. CONCLUSIONS These results demonstrate (i) that functional Cx32 protein is required for tumor promotion by Pb and (ii) that the tumor promoter acts by positive selection for β-catenin-mutated hepatocytes.

KEY WORDS: phenobarbital, tumor promotion, Ha-ras, &beta, -catenin.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Carcinogenesis.