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

Abstract

Spontaneous proliferative liver lesions were found in 15 (13 males and 2 females) of 244 (122 of each sex) transgenic (Tg) mice carrying the human prototype c-H-rus gene (msH2). The liver lesions included 3 foci of cellular alteration, 1 hcpatocellular adenoma, 5 hepatocellular carcinomas. and 4 hepatic hemangiosarcomas in the males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in the females. The mutation patterns of the human and endogenous mouse c-H-rus codon 61 in these proliferative liver lesions were analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-H-rus gene: 3, 1. and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61. a CAA to CTA transversion, and a CAA to CGA transition at the second base of codon 61. respectively. No point mutations in the human c-H-rus transgene were detected in any hepatocellular carcinoma. All 4 hepatic hemangiosarcomas had a CAG to CTG transversion at codon 61 of the human c-H-rus gene, but no point mutations were detected in codon 61 of the mouse c-H-rus gene. No mutations in human or mouse c-Hrus codon 61 were detected in altered cell foci or hepatocellular,adenoma. These results indicate that spontaneous liver tumors in rusH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosarcomas). The absence of similar mutations in foci of cellular alteration and the hepatocellular adenoma suggests that the occurrence of codon 61 point mutations is a late event in the progression of hepatocellular neoplasia in rusH2 Tg mice.Keywords. Human prototype c-H-rus transgenic (rusH2 Tg) mouse; hepatocellular carcinoma; hepatic hemangiosarcoma; polymerase chain reaction. single-strand conformation polymorphism (PCR-SSCP); nonradioactive oligonucleotide dot blot hybridization INTRODUC~IONThe current Japanese, United States Food and Drug Administration, European Union, and other nationayinternational guidelines specify the use of 2 species of rodents (usually mice and rats) in long-term (>l.S yr for mice and 2 yr for rats) carcinogenicity testing for approval of new drug applications (5,14,19,24). In view of the high cost of testing, the questionable relevance of mouse tumor responses to human health, and the extensive use of animals (usually >400 animals per study) in the present protocol, some toxicologists, regulatory authorities, and international committees have suggested that the use of vaIidated alternative in vivo tests of chemical carcinogenicity in at least 1 species would not significantly compromise hazard identification and human risk assessment (9,10,21,26,27).The usefulness of short-term carcinogenicity testing

Modifying Effects of Genistein, 4-Nonylphenol and Methoxychlor on <i>N</i>-ethyl-<i>N</i>-nitrosourea-induced Uterine Carcinogenesis in RasH2 Transgenic Mice Harboring a Human Prototype C-Ha-<i>ras</i> Gene

“…With regard to the mechanism on the tumorigenesis in rasH2 mice, as reported in the previous studies [45][46][47][48][49][50][51] , somatic mutations of codon-12, -13, and -61 of c-Ha-ras transgene were detected in spontaneous or chemically induced tumors in rasH2 mice. However, the incidences of point mutations of the transgene appeared to vary in tumors of rasH2 mice, and some carcinogens induced extremely low incidences of transgene mutations 45,52,53 .…”

Analysis of Gene Expression Profile on Uterine Tumorigenesis Initiated with N-ethyl-N-nitrosourea and Inhibited by Ethinylestradiol in rasH2 Mice

“…These data may suggest a possible functional disorder of the cell cycle only in the uterine epithelium of rasH2 mice. However, with regard to the enhanced carcinogenesis in various organs of rasH2 mice treated with genotoxic carcinogens, it has been reported that somatic mutations of codons 12, 13 and 61 of the c-Ha-ras transgene were detected in spontaneous or chemically induced tumors in rasH2 mice [21][22][23][24][25][26][27] . On the other hand, the incidences of point mutations of the transgene appeared to vary in tumors of rasH2 mice, and some carcinogens induced extremely low incidences of transgene mutations 21,28,29 .…”

Possible Mechanism on Enhanced Carcinogenesis of Genotoxic Carcinogens and Unsolved Mechanisms on Lesser Carcinogenic Susceptibility to Some Carcinogens in Rash2 Mice.

“…Based on these results, his group suggested that definite somatic point mutation of the human c-Ha-ras transgenes in certain cell types may be a causative event in tumorigenesis in these transgenic mice. Hayashi et al (1998) performed mutation analyses (DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR- The liver lesions included 3 foci of cellular alteration, 1 hepatocellular adenoma, 5 hepatocellular carcinomas, and 4 hepatic hemangiosarcomas in males and 1 focus of cellular alteration and 1 hepatocellular carcinoma in females. The hepatocellular carcinomas in 4 males and 1 female contained a point mutation in the mouse c-Ha-ras gene: 3, 1, and 1 carcinomas had a CAA to AAA transversion at the first base of codon 61, a CAA to CTA transversions, and a CAA to CGA transition at the second base of codon 61, respectively (Table 2).…”

“…No mutations in human or mouse c-Ha-ras codon 61 were detected in altered cell foci or hepatocellular adenomas. Based on these results, Hayashi et al (1998) pointed out that spontaneous liver tumors in rasH2 Tg mice contain different mutation patterns depending on the histologic type or cell origin of the tumors (i.e., hepatocellular carcinomas or hepatic hemangiosacomas). The mutation patterns of the human c-Ha-ras codon 61 and endogenous mouse c-Ki-ras codons 12, 13, and 61 in spontaneous lung proliferative lesions [3 bronchiolo-alveolar hyperplasias (BAHs), 2 bronchiolo-alveolar adenomas (BAAs), and 5 bronchiolo-alveolar adenocarcinomas (BACs)] observed in 10 of 244 rasH2 mice have been analyzed by DNA amplification using polymerase chain reaction, single-strand conformation polymorphism (PCR-SSCP), and oligonucleotide dot blot hybridization (Mori et al, 2000).…”