Transgenic Rats Carrying Human c‐Ha‐ras Proto‐oncogene Are Highly Susceptible to N‐Nitrosomethylbenzylamine Induction of Esophageal Tumorigenesis

Abstract

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.Key words: Transgenic rats -c-Ha-ras -Esophageal tumors -N-Nitrosomethylbenzylamine Transgenic mice are widely used for analysis of gene function and as animal models for various diseases. In the field of chemical carcinogenesis, transgenic mice harboring a human c-Ha-ras proto-oncogene, 1, 2) v-Ha-ras transgenic mice (Tg.AC mice),3) pim-1 transgenic mice 4) and p53 knockout mice 5) have been shown to be highly susceptible to tumor induction by certain carcinogens, with indication that the transgene is actively involved. However, for studies of chemical carcinogenesis, 6) rats have been more frequently used, so that abundant information has accumulated regarding various biological characteristics of preneoplasias. 7,8) Therefore the Hras128 rats generated in our laboratory have great potential for studies of neoplasia. 9,10) For esophageal tumors, rats are preferable to mice 11,12) due to the availability of the organotropic carcinogens and the larger size of the organ.Human esophageal squamous cell carcinomas are presumed to be caused by ...

Possible enhancing effects of atrazine and nonylphenol on 7,12‐dimethylbenz[a]anthracene‐induced mammary tumor development in human c‐Ha‐ras proto‐oncogene transgenic rats

“…N-nitrosomethylbenzylamine esophageal, 35) and DMBA skin carcinogenesis. 36) We recently showed that the transgenic (Tg) rats have a potential for use in medium-term bioassay models to test for the modifying effects of estrogenic environmental compounds on mammary tumor development.…”

“…Most of the tumor cells contained mutated transgene, but not endogenous Hras gene, indicating that activation of the transgene plays an important role in such rapid development of tumors. The animals have also been found to be susceptible to N-butyl-N-(4-hydroxybutyl)nitrosamine urinary bladder, 34) N-nitrosomethylbenzylamine esophageal, 35) and DMBA skin carcinogenesis. 36) We recently showed that the transgenic (Tg) rats have a potential for use in medium-term bioassay models to test for the modifying effects of estrogenic environmental compounds on mammary tumor development.…”

Preferential mammary carcinogenic effects of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP) in human c‐Ha‐ras proto‐oncogene transgenic rats

“…7) In humans, Ki-ras mutations are preferentially found in colon, 8,9) pancreatic duct 10) and bile duct carcinomas, 11) and N-ras mutations are typically seen in myeloid leukemia. 12) We have generated a transgenic rat line, harboring copies of a human c-Ha-ras proto-oncogene (Hras128 rat), and have shown that these animals are highly susceptible to mammary, 13,14) bladder 15) and esophageal 16) carcinogens.…”

Chemoprevention of esophageal squamous cell carcinoma

“…These data suggest that mutational activation of the H-ras gene is important for progression of the premalignant lesions to papillomas. Additional evidence in support of this concept is the observation that transgenic rats carrying the human c-Ha-ras proto-oncogene are highly susceptible to the induction of esophageal tumors with NMBA (Asamoto et al, 2002). As has been found in human esophageal tumors, G:C→ A:T transition mutations have been observed in the p53 tumor suppressor gene in ∼30 % of rat esophageal papillomas (Lozano et al, 1994;.…”