Transgenic rats carrying human c-Ha-ras proto-oncogene are highly susceptible to N-nitrosomethylbenzylamine induction of esophageal tumorigenesis

A transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene, including its own promoter region, has been established in our laboratory (Hras128 rats). and shown to be highly susceptible to induction of mammary and urinary bladder tumors. Mutation analysis of induced lesions indicated the majority to contain some but not all cells with transgene mutation. In the present study, the susceptibility of Hras128 rats to N-nitrosomethylbenzylamine (NMBA) induction of esophageal tumors was examined with a similar mutation analysis of the transgenes. Male 6-week-old Hras128 and wild littermate rats were treated with NMBA, 0.5 mg/kg subcutaneously, 3 times a week for 5 weeks and then maintained for 5 weeks without any further treatment. Multiple esophageal tumors, squamous cell papillomas and carcinomas, rapidly developed within this 10-week experimental period in Hras128 rats (11.05+/-7.83/rat). In contrast, wild-type littermates had only small numbers of mostly benign tumors (1.67+/-2.06/rat). The Hras128 rats had no other tumors or abnormalities. In their esophageal lesions, codon 12 GGC to GAC mutations of the transgene were frequently detected by restriction fragment length polymorphisms (RFLP) and subsequent direct sequencing analysis (19/25, 76%). In the endogenous rat c-Ha-ras gene they were less frequent (2/25, 8%), than in wild-type rats (8/14, 57.1%). The densities of mutated bands in the RFLP analysis indicated that mutated cells were major populations in tumors, in contrast to the case with mammary and urinary bladder lesions. Furthermore, activated ras protein, detected by binding to raf protein, was clearly increased in tumors as compared to surrounding epithelium or the normal esophagus of untreated rats. The results show that Hras128 rats are highly susceptible to NMBA esophageal carcinogenesis, as well as induction of mammary and urinary bladder tumors, but that tissue-specific characteristics exist for the roles of transgene ras mutations.