MUTATIONAL ACTIVATION OF C-HA-RAS GENES IN INTRADUCTAL PROLIFERATION INDUCED BY N-NITROSO-N-METHYLUREA IN RAT MAMMARY-GLANDS

Although administration of a single dose of N-nitroso-N-methylurea (NMU) to young virgin rats induces a high rate of mammary carcinomas, precise histogenesis of the carcinomas has not been well characterized. In this study, we investigated the alterations of H-ras gene in early focal lesions as well as carcinomas in the mammary glands of F344 rats treated with NMU. At 2 weeks after treatment, intraductal proliferation (IDP) was occasionally observed, and mammary carcinomas emerged at 12 and 36 weeks. The individual lesions of IDP and carcinomas were scooped out from the tissue sections under a stereomicroscope, and the DNA-sequence-spanning codon 12 of H-ras gene was amplified from the tissue sections by polymerase chain reaction (PCR). The analysis of amplified DNA by oligonucleotide hybridization revealed that 65% (11/17) of IDP and 89% (16/18) of carcinomas had a point mutation (G-to-A transition) at the 2nd position of H-ras codon 12. However, the DNA amplified from the areas, which appear histologically normal, never showed such mutation. These results indicate that IDP is a very early change for NMU-induced mammary carcinogenesis.