Mutations at codon 61 of the Ha‐ras proto‐oncogene in precancerous liver lesions of the B6C3F1 mouse

Abstract

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‐μm 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 μg 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.