Fischer 344/Ncr rats of both sexes were subjected to partial
hepatectomy and then initiated 21-24 h later by a single injection of
methyl(acetoxymethyl)nitrosamine at 0.1 mmol/kg
body weight via the portal vein. Beginning 3 weeks later, development of hepatocellular
neoplasms in initiated rats was promoted by feeding 0.05%
phenobarbital (PB) in the diet. Not only intrahepatic lesions but also a variety of extrahepatic
tumors were induced. High-molecular-weight DNAs were prepared from 67 samples of grossly normal liver containing multiple preneoplastic foci/areas of microscopic dimensions, 137
hepatocellular adenomas (nodules), 93
hepatocellular carcinomas (HCC), 10
cholangiomas, and 25 extrahepatic
tumors in 95 rats and tested for transforming activity in the NIH 3T3 transfection assay.
DNA preparations from 7 of 93 HCCs, 2 of 10
cholangiomas, 2 of 137 nodules, 1
histiocytic sarcoma, and 1
thyroid carcinoma were positive in the transfection assay. Southern blot analysis showed that NIH 3T3 transformants induced by
DNA from 5 HCCs, 1
hepatocellular adenoma, 1
cholangioma, 1
histiocytic sarcoma, and 1
thyroid carcinoma contained an activated K-ras gene of rat origin. Rat-derived H-ras was identified in transformants from 2 additional HCCs and rat c-raf from 1
hepatocellular adenoma. The transforming gene from one
cholangioma showed no sequence homology to the ras genes, neu, or c-raf. Immunoprecipitation analysis of ras Mr 21,000
protein in 11 transformants indicated that, based upon
protein electrophoretic mobilities, activation of the ras genes consistently resulted from mutations in
codon 12 of these genes. Selective
oligonucleotide analysis revealed that a G----A transition in the second base of
codon 12 of K-ras was present in the 9 K-ras-positive transformants and also in DNAs prepared from the original
tumors. In contrast,
oligonucleotide hybridization experiments with DNAs from 35 hepatocellular
tumors that were negative in transfection assays revealed the presence of mutant K-ras in 1 of 15 HCCs; no mutation could be detected in 20 transfection-negative
adenomas. The infrequency of detection of a specific oncogene, more frequent detection of oncogenes in malignant
tumors, and failure to observe activated oncogenes in preneoplastic lesions suggest that activation of ras oncogenes may occur as a late and infrequent event in the evolution of some rat hepatocellular
neoplasms and that mutation of a specific ras locus is not an obligatory early event in the genesis of these
neoplasms.