Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986

Abstract

In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.

Ha-ras mutations inN-nitrosomorpholine-induced lesions and inhibition of hepatocarcinogenesis by antisense sequences in rat liver

“…One molecular pathway of rat hepatocarcinogenesis induced by NNM has been classified using antisense techniques. Determination of mutation frequencies in hepatic lesions has proved that mutations in codon 61 of Ha-ras are early events in spontaneous murine hepatocarcinogenesis (Richardson et al, 1992). However, our data show that a mutated Ha-ras antisense oligonucleotide (AS-Leu) decreases cell proliferation in both preneoplastic lesions and hepatocellular carcinomas.…”

“…In murine hepatic tumors, however, a single point mutation at codon 61 of Ha-ras is common. For example, Richardson et al (1992) examined genetic alterations in codon 61 of Ha-ras oncogenes isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in B6C3F1 mice, which have a high incidence of spontaneous liver tumors. They found that 41% of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations in codon 61, being mostly CAA-CGA transversion, followed by CAA-CTA transversion.…”

“…Mutation frequencies were determined with the RFLP assay procedure modified by Richardson et al (1992). A Ha-ras sequence of 167 base pairs (bp), including codon 61 of exon II, was amplified with a Perkin-Elmer Cetus (Norwalk, CT) Amplitaq kit and a DNA thermal cycler.…”

“…Single mutations in codon 61 of Ha-ras can be detected in spontaneous and chemically-induced B6C3F1 liver lesions in mice (Richardson et al, 1992) and also in vinyl chloride-induced liver tumors in rats (Froment et al, 1994). Therefore, to determine whether therapy using the mutated Ha-ras antisense oligonucleotide can help to prevent the occurrence and development of liver tumors in rats, we examined the types and frequencies of mutations in codon 61 of Ha-ras in hepatic lesions induced by N-nitrosomorpholine (NNM), and then administered an antisense oligonucleotide directed against Ha-ras mRNA containing a mutation in codon 61 to inhibit this type of hepatocarcinogenesis.…”

Point Mutations of the c-H-ras Gene in Spontaneous Liver Tumors of Transgenic Mice Carrying the Human c-H-ras Gene

“…The incidence and pattern of the mutations are similar to those in spontaneous benign and malignant hepatocellular tumors in some non-Tg mouse strains such as C3H and B6C3Fl. Mouse c-H-ras activation has been believed to be a relatively early event in spontaneous mouse liver carcinogenesis (6,15). Although the number of benign lesions examined in this study is not sufficient to draw any definite conclusion, the absence of mutations in the 4 foci of cellular alteration and in the 1 hepatocellular adenoma from rusH2 Tg mice may suggest that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in this Tg mouse.…”