p16INK4a and beta-catenin alterations in rat liver tumors induced by NNK.
Inactivation of the p16INK4a (p16) tumor suppressor gene by promoter hypermethylation and mutation within exon 3 of beta-catenin represent two of the more common gene alterations in human hepatocellular carcinoma (HCC). One exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liver parenchyma. Treatment of rats with high doses of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes liver toxicity and a high incidence of tumors. The purpose of the current investigation was to define the prevalence of genetic alterations in p16 and beta-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 isolated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR. p16 methylation was detected in five of 15 adenomas and eight of 14 carcinomas (45% of all tumors). Methylation of p16 was extensive within the 5'-untranslated region and exon 1alpha, 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 polymorphism 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 substitutions; three of these mutations occurred at potential serine phosphorylation sites. Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common genetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic alterations critical to the initiation of HCC.
Lovelace Respiratory Research Institute, PO Box 5890, Albuquerque, NM 87185, USA.
L C
LC
Pulling
D M
DM
Klinge
S A
SA
Belinsky
D000818:Animals; D001483:Base Sequence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D003598:Cytoskeletal Proteins / Q000235:genetics*; D019175:DNA Methylation; D006801:Humans; D008114:Liver Neoplasms, Experimental / Q000139:chemically induced / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation; D009602:Nitrosamines / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D051381:Rats; D011916:Rats, Inbred F344; D015534:Trans-Activators; D051176:beta Catenin
C495270:CTNNB1 protein, human; C495268:Ctnnb1 protein, rat; D019941:Cyclin-Dependent Kinase Inhibitor p16; D003598:Cytoskeletal Proteins; D009602:Nitrosamines; D015534:Trans-Activators; D051176:beta Catenin; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone