Selective pressure during tumor promotion by phenobarbital leads to clonaloutgrowth of beta-catenin-mutated mouse liver tumors

Tumor promoters are non-mutagenic chemicals which increase the probability of cancer by accelerating the clonal expansion of cells transformed during tumor initiation. Phenobarbital (PB) is an antiepileptic drug which promote s hepatocarcinogenesis in rodents when administered subsequent to an initia ting carcinogen like diethylnitrosamine (DEN). Here we have investigated th e prevalence and patterns of mutations in two genes, Haras and beta -cateni n, both known mutational targets in mouse hepatocarcinogenesis. Liver tumor s were generated by a single administration of DEN to 6 week old mice follo wed by feeding of PB (0.05%) containing or control diet for 39 weeks. Mutat ions at Ha-ras codon 61 were screened by allele-specific oligonucleotide hy bridization; beta -catenin mutations were detected by direct sequencing of PCR products spanning exon 2. In tumors from mice treated with DEN alone, t he prevalence of Ha-ras mutations was similar to 30% (6/20), while no beta -catenin mutations (0/13) were detectable in tumors of this treatment group . By contrast, Ha-ras mutations were undetectable in tumors from mice treat ed with DEN/PB (0/32), while similar to 80% (37/46) of tumors from this gro up showed beta -catenin mutations. These results demonstrate that PB strong ly affects the prevalence of mutations in the two cancer-related genes, pre sumably by positive and negative selection for cells harboring the respecti ve mutation.