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-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.

p21Ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between Ras -mutated and Ras -wild-type lesions

“…To allow for the analysis of ras mutations, tumor material was punched out from parallel tissue sections as previously described. [2][3][4] Since no mutations other than at Ha-ras codon 61 had been detected in the DEN-induced mouse liver tumors described above (Table 1) HEPATOLOGY Vol. 27, No.…”

“…The observation that ras mutations can be detected at a very early stage during mouse hepatocarcinogenesis strongly indicates that they represent initial genetic alterations that confer a selective advantage to the mutated cells which drives their clonal expansion. [2][3][4] During the last couple of years, the role of p21 Ras proteins as part of signal transduction pathways that mediate signals from transmembrane receptors into the nucleus has been unraveled. [5][6][7][8][9][10][11] The functional Ras signaling pathway is essential for many signals initiated by growth factors and is also required for the expression of the transforming potential of oncogenic proteins upstream of p21 Ras .…”

Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: correlation with susceptibility to hepatocarcinogenesis.

“…1). In the liver system, activated ras genes have been detected in a high percentage of spontaneously occurring and carcinogen-induced liver tumors of the B6C3F1 mouse, where the predominant types of mutations were found to be specific single-base substitutions at different bases ofcodon 61 ofthe c-Ha-ras gene (2)(3)(4)(5)(6). In contrast to the B6C3F1 mouse, liver tumors from rats analyzed so far did not contain any mutations within the Ha-ras gene (5).…”

“…Sections (10 ,um) were prepared from frozen liver tissue with a cryostat microtome, mounted on dialysis bags, and enzyme-histochemically stained for glucose-6phosphatase (G-6Pase) activity (18). Thereafter, small tissue samples were taken with punching cannulas both from G-6-Pase-deficient liver lesions and from normal parts of the liver sections and used for polymerase chain reaction (PCR) without isolation of DNA as described (6 (6). On the basis ofthe mouse c-Ha-ras sequence (20), the primers used for amplification of Ha-ras codon 61 sequences were Amp61/1A (5'-CTAAGCCTG1TGTITGCAGGAC-3') and Amp61/2A (5'-GTGCGCATGTACTGGTCCCGCAT-3') or Amp61/1B (5'-GAGACATGTCTACTGGACATCTT-3') and Amp61/2B (5'-GTGTTGTTGATGGCAAATACACAGAGG-3'), yielding PCR products of 130 base pairs (primer set 1A/2A) and 116 base pairs (primer set 1B/2B), respectively.…”

“…Mutation analysis by allele-specific oligonucleotide hybridization was performed as described (6). In brief, [1][2][3][4][5] ,l of the PCRamplified reaction mixtures was spotted onto BioTrace RP nylon membranes, hybridized with 32P-labeled oligonucleotide probes (for sequences, see ref.…”