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Mutations in the p53 gene are frequent genetic alterations in human hepatocellular carcinomas. We have examined, by single-strand conformation polymorphism analysis of polymerase chain reaction products, a total of 93 carcinogen-induced liver tumors from mice of three different strains (C3H/He, C57BL/6J, and B6C3F1) for the presence of p53 aberrations. Hepatoma lines, established from 12 liver tumors, were also included in the analysis. While structural aberrations of the p53 gene were not detected in any of the primary mouse liver tumors analyzed, single-base substitutions occurred at different locations within the p53 gene in three of the cell lines during in vitro propagation. One hepatoma line carried two point mutations on separate alleles. All four mutations were either G:C----C:G or C:G----G:C transversions. Mutations at codon 61 of the c-Ha-ras gene, which were frequent in primary liver tumors from C3H/He and B6C3F1 mice, were not detected in the hepatoma lines. Our data indicate (i) that c-Ha-ras but not p53 mutations play an important role during the early stages of mouse hepatocarcinogenesis and (ii) that p53 mutations confer a selective growth advantage to the mutated hepatoma cells in vitro.

Mutations in codon 61 of the Ha-ras proto-oncogene have been shown to occur with a high frequency in both spontaneous and carcinogen-induced liver tumours of the B6C3F1 mouse. In the present study we analysed the frequency and pattern of Ha-ras mutations in liver tumours in a different strain of mice, namely the CF1 mouse. These liver tumours occurred spontaneously or after administration of either aflatoxin B1, phenobarbital or dieldrin. Mutation analysis was performed by in vitro amplification of DNA using the polymerase chain reaction combined with selective oligonucleotide hybridization. Our results demonstrate the presence of mutations in the Ha-ras gene in liver tumours of the CF1 strain. In total, 6 out of 35 liver tumours of male CF1 mice, two of them occurring after treatment with the tumour promoter phenobarbital solely, contained mutations at either the first or second base of codon 61 of the Ha-ras gene. The types of mutations found in liver tumours of the CF1 mouse were very similar to those described in the B6C3F1 mouse, indicating that mutations in the Ha-ras proto-oncogene may represent a critical event in mouse hepatocarcinogenesis.

Liver tumors of certain strains of mice frequently contain mutations at codon 61 of the c-Ha-ras gene. In our study, we investigated the significance of these mutations in the carcinogenic process. Male C3H/He mice received a single injection of diethylnitrosamine (DEN) on day 15 after birth, and groups of animals were killed at various time intervals between 11 and 52 wk after treatment. At the earlier time points (11-29 wk), we analyzed microdissected tissue from precancerous glucose-6-phosphatase-deficient liver lesions larger than approximately 200 microns in diameter, for the presence and pattern of c-Ha-ras codon 61 mutations. In parallel, the growth characteristics of these liver lesions were studied by pulse labeling with [3H]thymidine and by determining the size distribution of the lesions. At the later time points (42-52 wk after DEN treatment), liver tumors were dissected and also analyzed for the presence of c-Ha-ras mutations. We found mutations to be already present in some of the enzyme-altered liver lesions at weeks 11-29, suggesting that the mutations occurred early in the carcinogenic process. Whereas about 10% of the precancerous focal liver lesions showed mutations in the c-Ha-ras gene, the mutation frequency was increased to about 50% in the later-appearing hepatocellular adenomas and carcinomas, suggesting that c-Ha-ras codon 61 mutations may provide a selective advantage to the mutated cell clones.

Activation of cellular proto-oncogenes has been found to play an important role in the development of different human and animal tumors1. Such activation can result from a variety of genetic alterations including chromosomal translocation or rearrangement, gene amplification or gene mutation. Amongst the best studied mechanisms of activation are single point mutations at either codon 12,13 or 61 of the different members of the ras gene family2,3. Activating mutations in the Harvey-ras (H-ras) pproto-oncogene have been described in rat mammary tumors4,5, mouse skin tumors6,7 and mouse liver tumors 8–11. To better understand the significance of these mutations for tumor development it would be of importance to know whether they occur at an early or at a later stage of the carcinogenic process. To address this question we have analyzed mutations in the H-ras proto-oncogene in precancerous liver foci of the B6C3F1 mouse which were induced by a single administration of diethylnitrosamine (DEN) A detailed description of these experiments has been reported previously12.

The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C----A transversions at the first base and A----T transversions or A----G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, c-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains.

The B6C3F1 mouse is characterised by a high spontaneous liver tumour frequency and a high susceptibility to chemical carcinogens. Both spontaneous and carcinogen- induced liver tumours of this mouse strain have been shown to contain specific mutations at either the first or second base of codon 61 of the H- ras proto-oncogene (Reynolds et al. 1986; 1987; Wiseman et al. 1986; Stowers et al. 1988). No such mutations, however, have been reported sofar in liver tumours of rats or other mouse strains. The CF1 mouse is very similar to the B6C3F1 mouse with respect to spontaneous liver tumour formation and sensitivity to chemical carcinogens. We were now interested in whether liver tumours of the CF1 mouse also possess mutations in the H-ras proto-oncogene. For this purpose we analysed samples of liver tumours from the CF1 mouse which occurred spontaneously or after carcinogen or promoter treatment for mutations at codon 61 of the H-.. ras gene. Since the CF1 liver tumours were available to us only as formalin- fixed, paraffin-embedded specimens, we first tested whether this material could be used as a source for our analyses. For this purpose we used fixed and embedded material from tumours with known mutations in the H-ras proto-oncogene as a model system.

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.