Tetsuya Hamaguchi

High susceptibility of human c-Ha-ras proto-oncogene transgenic rats to carcinogenesis: A cancer-prone animal model

Transgenic animals carrying human c‐Ha‐ras proto‐oncogene, v‐Ha‐ras transgenic mice, pim‐1 transgenic mice and several knockout mice deficient of tumor suppressor genes, such as p53, have been shown to exhibit increased carcinogen susceptibility. As a result, studies into practical application and medium‐term screening of environmental carcinogens are under way. Given the advantages of rat models characterized by larger organ size, abundant information regarding preneoplasias and virus‐free constitution, we have concentrated on the generation of transgenic rats bearing copies of the human c‐Ha‐ras proto‐oncogene and shown the Hras128 strain to be extremely sensitive to the induction of mammary carcinomas, and to a lesser extent, lesions in the urinary bladder, esophagus and skin. In most, if not all, the mammary cancers mutations of the transgene but not the endogenous H‐ras gene are present, appearing to occur early in the process of tumorigenesis, which involves proliferation of cells in TEB and intraductal hyperplasia before carcinomas arise. Preliminary findings suggest that this is independent of endogenous ovarian hormones, although inhibited by soy isoflavones and promoted by atrazine and nonylphenols. Although further studies of the mechanisms are clearly necessary, the model appears to have great potential for screening purposes, not only for modifiers active in the breast, but also other organs where tumors characterized by ras gene mutations develop. (Cancer Sci 2005; 96: 309–316)

Rapid induction of skin and mammary tumors in human c‐Ha‐ras proto‐oncogene transgenic rats by treatment with 7,12‐dimethylbenz[a]anthracene followed by 12‐O‐tetradecanoylphorbol 13‐acetate

We have established a transgenic rat line carrying 3 copies of the human c‐Ha‐ras proto‐oncogene with its own promoter region (Jcl/SD‐TgN(HrasGen)128Ncc) (Hras128 rat), expression being detectable in almost all organs. We have already demonstrated that the rat is highly sensitive to mammary, esophagus and bladder carcinogenesis. In the present study, male and female transgenic and wild‐type littermates were topically treated with 2.5 mg of 7,12‐dimethylbenz[a]anthracene (DMBA) dissolved in 1.0 ml of acetone on the back skin at 50 days after birth. Starting 1 week thereafter, they were again topically treated with 100 nmol of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) dissolved in 0.5 ml of acetone 3 times weekly for the following 31 weeks. In males treated with DMBA and/or TPA, skin tumors, including both squamous cell papillomas (SCP) and carcinomas (SCC), were preferentially induced at the DMBA‐TPA painting sites: DMBA‐TPA, 15/15 (100%); DMBA, 6/8 (75%); TPA, 1/6 (16.7%). They were also, unexpectedly, induced on remote scrotal skin: DMBA‐TPA, 13/15 (86.7%); DMBA, 5/8 (62.5%); TPA, 0/6 (0%). Lesions were thus more frequent in the DMBA‐TPA group than with DMBA or TPA alone. In females, adenomas and adenocarcinomas of the mammary glands were preferentially induced: DMBA‐TPA, 12/14 (85.7%); DMBA, 6/8 (75%); TPA, 3/6 (50%), with only a few small skin papillomas at painting sites. Incidences and numbers of the mammary and skin tumors were much greater in Hras128 rats than in their wild‐type counterparts. PCR‐RFLP analysis of the transgene indicated that the percentage of the cell populations harboring a mutation in codons 12 and/or 61 ranged from 2% to 60% in individual tumors; skin tumors showed more mutations in codon 61 in the DMBA‐treated groups. In contrast, no mutations were detected in the endogenous rat c‐Ha‐ras gene. These results indicate that the Hras128 rat is highly susceptible to DMBA‐TPA skin and mammary carcinogenesis, thus providing a unique painting model for skin as well as mammary gland carcinogenesis, that would be suitable for investigating the role of transgene mutations.

Rapid Emergence of Mammary Preneoplastic and Malignant Lesions in Human c-Ha-ras Proto-Oncogene Transgenic Rats: Possible Application for Screening of Chemopreventive Agents

For comparison of mammary gland whole mounts with examination of 2 histologic sections of mammary gland, 56 Hras128 rats were intravenously injected with 50 mg/kg body weight of N-methyl-N-nitrosourea at 50 days of age and then sacrificed at days 5, 10, 15, 20, 25, and 56. Comparison of detection sensitivity between the whole mounts and histologic sections revealed no lesions apparent in whole mounts on day 10, although intraductal proliferation was clearly detected in histologic sections in 44% of treated rats. Proliferative lesions were first detected in whole mounts at a 44% incidence on day 15, while intraductal proliferations and atypical hyperplasias were apparent in the sections at 89% and 44% incidences, respectively. On day 20, atypical hyperplasias and small adenocarcinomas in histologic sections were found in almost all animals. In conclusion, examination of 2 histologic sections from mammary tissues was found to be practical for detection of small malignant lesions as early as 15 days after MNU injection, and suppressive effects of soy isoflavones were clearly evident within 20 days after carcinogen exposure. These results suggest that this model has practical utility for short-term screening of chemopreventive agents for mammary carcinogenesis.

Terminal Endbuds and Acini as the Respective Major Targets for Chemical and Sporadic Carcinogenesis in the Mammary Glands of Human c-Ha-ras Protooncogene Transgenic Rats

A rat strain carrying the human c-Ha-ras protooncogene, established by our laboratory, is highly susceptible to mammary chemical carcinogens. The transgenic rats exhibit increased number of terminal endbuds (TEBs) at the tips of developing ducts in the mammary gland compared to non-transgenic littermates. Confocal microscopy revealed the level of active mitogen-activated protein kinase to be elevated in these TEBs, and a close correlation between their numbers and tumorigenic response initiated by 7,12-dimethylbenz[a]anthracene was confirmed. Single injections of N-methyl-N-nitrosourea into the transgenic rats caused mutations in codon 12 of human c-Ha-ras transgene in TEBs before tumor development, supporting the conclusion that these structures are the major targets of chemical carcinogens. In contrast, with spontaneous development of lesions, alveolar hyperplasia with elevated expression levels of rat and human c-Ha-ras protooncogenes is the first morphological alteration which becomes apparent. Some but not all hyperplastic alveolar nodules were found to harbor mutations in the transgene. The results indicate that elevated expression of c-Ha-ras protooncogene is sufficient in itself to cause a highly proliferative phenotype of mammary alveoli. Our data suggest that TEBs and acini are the major targets for chemical and sporadic carcinogenesis, respectively, in the mammary glands of human c-Ha-ras protooncogene transgenic rats.

Molecular analysis of rat mammary carcinogenesis: an approach from carcinogenesis research to cancer prevention

A rat strain carrying the human c-Ha-ras proto-oncogene is highly susceptible to chemically induced mammary carcinogenesis. All the transgenic rats develop preneoplastic mammary lesions within 20 days of an injection of N-methyl-N-nitrosourea, and mammary carcinomas appear within 8 weeks of treatment with a variety of chemical carcinogens. In this review, we summarize molecular aspects of mammary carcinogenesis in transgenic rats and the potential application of this model for studies of breast cancer prevention.

Pneumatosis intestinalis and necrotizing enterocolitis associated with liver cirrhosis

We report a 58-year-old woman with pneumatosis intestinalis and necrotizing enterocolitis associated with liver cirrhosis. She was receiving treatment for liver cirrhosis and hepatic failure when sudden severe right upper abdominal pain and fever developed. Abdominal radiograph disclosed pneumatosis intestinalis, involving the ascending colon, and small collections of free air along the right hemidiaphragm. The pneumatosis intestinalis appeared in both cystic and linear form. Autopsy revealed necrosis and multiple gas-filled mural cysts in the ascending colon. To our knowledge, this is the first case to be reported of pneumatosis intestinalis and necrotizing enterocolitis associated with liver cirrhosis.