Transforming c-Ki-ras mutation is a preneoplastic event in mouse mammary carcinogenesis induced in vitro by N-methyl-N-nitrosourea.

Abstract

Mouse mammary epithelial cells can be transformed in primary cultures to preneoplastic and neoplastic states when treated with N-methyl-N-nitrosourea (MNU). Mammary carcinomas arising from MNU-induced hyperplastic alveolar nodules (a type of mouse mammary preneoplastic lesion) contained transforming c-Ki-ras genes when examined by the NIH 3T3 focus assay. Hybridization of allele-specific oligonucleotides to c-Ki-ras sequences amplified by the polymerase chain reaction demonstrated the presence of a specific G-35--->A-35 point mutation in codon 12 in each of the NIH 3T3 foci as well as the mammary carcinomas. This mutation resulted in the substitution of the normal glycine with an aspartic acid. Furthermore, this mutation in the c-Ki-ras proto-oncogenes was also detected in 9 of 10 hyperplastic alveolar nodules. These results demonstrate that the specific c-Ki-ras mutation is a preneoplastic event in MNU-induced mouse mammary carcinogenesis.Cancer is recognized as a disease arising from a perturbation of gene function. Over 30 different cellular genes, called proto-oncogenes, have been investigated intensely because of their involvement in tumorigenesis. Of the proto-oncogenes identified in human and animal tumors, members of the ras gene family, c-Ha-ras, c-Ki-ras, and N-ras, are by far the most frequent (1, 2, 19). The ras genes are commonly activated by single point mutations in codons 12, 13, and 61 (1, 2). In rodent tumors induced by chemical or physical carcinogens, the nature of the mutational activation of ras proto-oncogenes has shown a remarkable correlation with the known mutagenic properties of the causative agents (1). For example, mammary tumors in rats induced by Nmethyl-N-nitrosourea (MNU) contain c-Ha-ras proto-oncogenes activated by G--A transitions (26,29). Mouse hepatic neoplasms induced by a variety of carcinogens contain c-Ha-ras proto-oncogenes activated by an alteration corresponding to the mutational specificity of each carcinogen (27). Therefore, the identification of the critical gene mutations directly involved in cancer is becoming feasible by use of animal model systems.We have recently developed a transformation system in which mouse mammary epithelial cells in serum-free primary collagen gel cultures are transformed by treatment with chemical carcinogens (11,18). Cells treated with carcinogens form mammary lesions when transplanted into a cleared mammary fat pad, an anatomically natural transplantation site from which mammary epithelial cells have been removed surgically (6). This in vitro-in vivo system facilitates a stepwise study of the transformation process at the molecular level as the transformants progress through morphologically well-defined preneoplastic stages (11,17,18). Furthermore, the molecular effects upon transformation by various factors that control growth and differentiation of mammary cells can be directly studied under well-defined conditions. In the present studies, in vitro MNU-induced mouse mammary preneoplasias and neoplasias were analyzed for the * Corres...

Isoform-Specific Ras Activation and Oncogene Dependence during MYC- and Wnt-Induced Mammary Tumorigenesis

“…Therefore, we hypothesized that c-MYC overexpression in the mammary gland might select for the outgrowth of Kras2-mutant cells, whereas Wnt1 might select for the outgrowth of Hras1-mutant cells. To address this hypothesis, we treated female MTB/TOM and MTB/TWNT mice with MNU, an agent with a 10-min biological half-life that induces mammary tumors harboring Hras1 and Kras2 mutations in mice and rats (15,16,25,35). One week following MNU treatment, expression of either c-MYC or Wnt1 was induced in the mammary epithelium by doxycycline administration.…”

Distinct requirements for Ras oncogenesis in human versus mouse cells

“…Additionally, a divergent association of Ras mutations with distinct neoplasms is also seen when compared in humans and rodents. For example, whereas Ras mutations are strongly associated with the carcinogen-induced mammary carcinomas in mice and rats (Zarbl et al 1985;Miyamoto et al 1990), such mutations are rare in human breast cancers (Bos 1989). One interpretation of these results is that Rasmediated tumorigenic growth may display subtle, but important differences between mice and humans.…”

A Method to Detect and Quantitate the Expression of Normal Versus Mutant H-Ras Transcripts at Codon-12

“…Activation through point mutations of ras genes has been found to be an early and influential event during the initiation and progression of many cancers (1)(2)(3)(4). However, recent work has emphasised the importance of gene dosage on the phenotype of cells bearing ras oncogenes (5).…”