p16(INK4a) and beta-catenin alterations in rat liver tumors induced by NNK

Inactivation of the p16(INK4a) (p16) tumor suppressor gene by promoter hype rmethylation and mutation within exon 3 of beta -catenin represent two of t he more common gene alterations in human hepatocellular carcinoma (HCC), On e exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liv er parenchyma. Treatment of rats with high doses of the tobacco-specific ni trosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes li ver toxicity and a high incidence of tumors. The purpose of the current inv estigation was to define the prevalence of genetic alterations in p16 and b eta -catenin in NNK-induced rat liver cancer to determine if the molecular mechanisms seen in human tumors are the same in this animal model. DNA isol ated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR, p16 methylation was detected in five of 15 ad enomas acid eight of 14 carcinomas (45% of all tumors), Methylation of p16 was extensive within the 5'-untranslated region and exon la, areas shown to correlate with loss of gene transcription. Liver tumors were also screened for mutations within exon 3 of beta -catenin. single strand conformation p olymorphism and DNA sequencing revealed five mutations in four of 29 tumors (14%). Mutations were present in three adenomas and one carcinoma and were located within codons 33, 36 or 37, All mutations resulted in amino acid s ubstitutions; three of these mutations occurred at potential serine phospho rylation sites, Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common g enetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic altera tions critical to the initiation of HCC.