Mutations at codon 61 of the Ha-ras proto-oncogene in precancerous liver lesions of the B6C3F1 mouse.
Liver tumors of the B6C3F1 mouse frequently contain mutations at specific sites of codon 61 of the Ha-ras proto-oncogene. To address whether these mutations occur early or late during carcinogenesis, we analyzed mutations in the Ha-ras gene in small precancerous liver lesions of the B6C3F1 mouse. For this purpose, 10-microns frozen liver sections were prepared and stained for glucose-6-phosphatase activity. Using punching cannuli, we then took small tissue samples of approximately 5-30 micrograms from enzyme-deficient liver lesions and from normal parts of the liver. These tissue samples were analyzed for mutations in the Ha-ras gene by in vitro amplification of DNA via the polymerase chain reaction combined with selective oligonucleotide hybridization. By this approach we were able to analyze mutations in the Ha-ras gene within lesions with diameters of less than 0.5 mm. Our results demonstrate that approximately 15% of the glucose-6-phosphatase-negative lesions that occurred 24-28 wk after a single injection of diethylnitrosamine contain either C----A transversions at the first base or A----G transitions and A----T transversions at the second base of codon 61 of the Ha-ras gene. The same types of mutations, although with a somewhat higher frequency (33%), were found in liver tumors taken 68 wk after diethylnitrosamine treatment. These findings demonstrate that Ha-ras mutations can be detected even in very small precancerous liver lesions, suggesting that these mutations may be an early, perhaps even the first, critical event during murine hepatocarcinogenesis.
Buchmann
A
A
Institute of Biochemistry, German Cancer Research Centre, Heidelberg, Federal Republic of Germany.
Mahr
J
J
Bauer-Hofmann
R
R
Schwarz
M
M
D000818:Animals; D001483:Base Sequence; D003062:Codon / Q000235:genetics*; D004052:Diethylnitrosamine / Q000633:toxicity; D011905:Genes, ras; D008099:Liver / Q000187:drug effects / Q000473:pathology; D008113:Liver Neoplasms / Q000139:chemically induced / Q000235:genetics*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D009154:Mutation; D010634:Phenobarbital / Q000633:toxicity; D016133:Polymerase Chain Reaction; D011230:Precancerous Conditions / Q000139:chemically induced / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*
D003062:Codon; D012333:RNA, Messenger; D004052:Diethylnitrosamine; D010634:Phenobarbital