Satoshi Yamamoto's research while affiliated with Keio University and other places

2,3,5-Trimethyl-6-(12-hydroxy-5, 10-dodecadiynyl)-1.4-benzoquinone (AA861) strongly inhibited epidermal lipoxygenase activity which was determined by the formation of [14 C]12-hydroxy-5,8,10,14-eicosatetraenoic acid by incubating [l4 C]arachidonic acid with cytosol fraction of epidermal homogenate of ***CD-1 mice. AA861 failed to inhibit epidermal cyclooxygenase activity. The present results indicate that AA861 is a potent inhibitor of 12-lipoxygenase in mouse epidermis (IC50 1.9 μM).

In primary cultured mouse epidermal cells, protein kinase C isozyme zeta (PKCzeta) consists of multiple forms, for example, low-salt eluted PKCzeta (1-PKCzeta; 79 and 85 kDa) and high-salt eluted PKCzeta (h-PKCzeta; 79 and 85 kDa) on anion-exchange column chromatography. In this study, biochemical and biophysical differences between 1-PKCzeta and h-PKCzeta were examined by using carcinogen-initiated mouse epidermal cell-line WYF31 cells, whose growth is stimulated by tumour promoter phorbol 12-myristate 13-acetate (PMA). The binding efficiency of h-PKCzeta to anti-PKCzeta antibody-affinity column was 10 times higher than that of 1-PKCzeta. T7-tagged rat PKCzeta overexpressed in WYF31 cells was recovered only in the high-salt eluted area on the anion-exchange column. Furthermore, when rat PKCzeta was stably overexpressed in WYF31 cells, the content of h-PKCzeta increased 4 to 5 times compared to that of parental cells, but the content of 1-PKCzeta was not altered. All of these results indicate that h-PKCzeta is the product of the PKCzeta gene (referred to as PKCzeta) and that 1-PKCzeta is closely related but different from PKCzeta (referred to as PKCzeta-related kinase). Interestingly, serum starvation of WYF31 cells caused a marked increase of the content of PKCzeta-related kinase with a concomitant decrease of PKCzeta content. These changes were reversed by stimulating the cell growth with 10% foetal calf serum. Prolonged treatment of starved cells with PMA, which induces the proliferation of WYF31 cells, also caused the downregulation of PKCzeta-related kinase. These results suggest that the expression levels of PKCzeta-related kinase and PKCzeta are differently regulated, and that the increased expression of PKCzeta-related kinase might play a significant role in the growth-suppression processes of WYF31 cells.

Studies were conducted to validate the transgenic (Tg) mice harboring human prototype c-Ha-ras gene, namely the rasH2 mice (CB6F1), as a model for rapid carcinogenicity testing. Short-term (26 weeks) carcinogenicity testing of 18 mutagenic (Salmonella) trans-species carcinogens, two mutagenic single-species (mouse-only) carcinogens six non-mutagenic trans-species carcinogens, one non-mutagenic single-species (mouse-only) carcinogen, four mutagenic non-carcinogens and four non-mutagenic non-carcinogens were completed. The studies revealed that the Tg mice are able to detect various types of mutagenic carcinogens and may also detect various non-mutagenic carcinogens within 26 weeks. Dose-dependent tumor responses were observed with various carcinogens except for a few equivocal cases. The validation studies also revealed that the Tg mice are generally much more susceptible to both mutagenic and non-mutagenic carcinogens than control non-Tg mice. Most of the malignant tumors were observed in the carcinogen-treated Tg mice and only very few or none in the corresponding non-Tg mice. Most of the carcinogens tested induced some of the target organ tumors observed in B6C3F1 mice in a 2-year bioassay as well as certain types of tumors specific to the Tg mice, i.e. lung alveolar epithelial tumors, spleen hemangiosarcomas, forestomach squamous cell tumors. No significant tumor induction has been observed in the Tg mice either with mutagenic or non-mutagenic non-carcinogens. Although further validation studies are still required, the rasH2 mouse seems to be a promising candidate as an animal model for the development of a rapid carcinogenicity testing system.

To validate the transgenic (Tg) mouse carrying a human prototype c-Ha-ras gene (rasH2 mouse) as a model for short or medium-term carcinogenicity testing, 6-mo carcinogenicity studies using more than 30 chemicals, including carcinogens and noncarcinogens, have been performed. The results obtained so far indicate that rasH2 mice are generally much more susceptible to both mutagenic and nonmutagenic carcinogens than are non-Tg mice, pointing to advantageous application for detection of carcinogenic potential. In this review, histopathological features and diagnostic criteria for spontaneous and induced-tumors observed in our 6-mo carcinogenicity studies are described. Incidences of spontaneous tumors were generally low in rasH2 mice during the 6-mo studies, although values for lung adenomas and splenic hemangiosarcomas were higher than those in the control non-Tg mice. A few forestomach papillomas and skin papillomas were also observed in the control rasH2 mice. The target organs in rasH2 mice treated with known carcinogens were not always identical to those in the treated B6C3F1 mice in 2-yr carcinogenicity bioassays, with forestomach squamous cell tumors, lung alveolar epithelial tumors and/or hemangiosarcomas in the spleen observed in addition to some but not all of the lesions in target organs observed in non-Tg mice in long-term carcinogenicity bioassays. The results of the present histological study suggest that the lung, spleen and/or forestomach, where tumors are induced in rasH2 mice treated with known carcinogens, should be regarded as informative target organs in addition to the target organs reported in previous long-term carcinogenicity bioassays in rats and mice.

Carcinogenicity testing is indispensable for identifying environmental carcinogens and for evaluating the safety of drugs in the process of development. Conventional 2-year rodent bioassays are one of the most resource-consuming tests in terms of animals, time, and costs. Development of rapid carcinogenicity testing systems that can assess carcinogenicity within a short period has become a social demand and is essential to improve efficacy in the identification of environmental carcinogens as well as in the development of new drugs. In this review we introduce the rapid carcinogenicity testing system using transgenic (Tg) mice carrying the human prototype c-Ha-ras gene, namely rasH2 mouse (CB6F1-TgHras2 mouse is the same mouse). The studies have been conducted to validate the rasH2 mouse as a model for the rapid carcinogenicity testing system. Our current validation studies revealed that rasH2 mice are able to detect various types of mutagenic carcinogens within 6 months. The rasH2 mice may also be able to detect various nonmutagenic carcinogens. The validation studies also revealed that rasH2 mice are generally much more susceptible to both mutagenic and nonmutagenic carcinogens than control non-Tg mice. No significant tumor induction has been observed in rasH2 mice with either mutagenic or nonmutagenic noncarcinogens. More rapid onset and higher incidence of more malignant tumors can be expected with a high probability after treatment with various carcinogens in the rasH2 mice than in control non-Tg mice. The rasH2 mouse appears to be a promising candidate as an animal model for development of a rapid carcinogenicity testing system.

To determine if hemizygous transgenic mice carrying the human c-Ha-ras gene (CB6F1-Tg Hras2 mice (Hras2 mice)) are susceptible to the carcinogenic potential of known murine carcinogens, male and female Hras2 mice and their non-transgenic CB6F1 littermates (non-Tg mice) were each given a single intraperitoneal injection of 60 mg of vinyl carbamate (VC)/kg body weight or saline (vehicle control) and monitored for 16 wk without further treatment. At necropsy, grossly visible tumors were fixed for histopathologic diagnosis and, when of sufficient size, portions were frozen for subsequent molecular analysis. Nine of 31 male and nine of 29 female Hras2 mice treated with VC died within 16 wk as a result of lung tumor burden. At the termination of the study, lung tumors (alveolar-bronchiolar epithelial neoplasms and hemangiosarcomas) and focal alveolar-bronchiolar hyperplasias were present in both sexes of Hras2 and non-Tg mice treated with VC; there were significantly more proliferative lung lesions in Hras2 than non-Tg mice. Splenic hemangiosarcomas and squamous cell tumors of the forestomach were induced in male and female VC-treated Hras2 mice but not in VC-treated non-Tg mice. Polymerase chain reaction–single-strand conformation polymorphism analysis and DNA sequencing of the induced lung tumors revealed point mutations at codon 61 of the transgene in two of 29 lung tumors (one of 16 in males and one of 13 in females) from VC-treated Hras2 mice; no mutations in murine Ki-ras were found in these tumors. Point mutations at codons 12 and 61 of the murine Ki-ras gene were observed, however, in one of 10 and six of 10 lung tumors respectively, from VC-treated non-Tg mice. These findings indicate that Hras2 mice are highly sensitive to pulmonary neoplasms and splenic and lung hemangiosarcomas after treatment with VC. The molecular analyses suggest that point mutations of the transgene and the murine Ki-ras gene do not play a major role in VC induction of pulmonary neoplasms in these transgenic mice. Mol. Carcinog. 20:298–307, 1997. © 1997 Wiley-Liss, Inc.

In primary cultured mouse epidermal cells, protein kinase C (PKC) zeta consists of multiple forms: a low salt-eluted PKC zeta (1-PKC zeta, 79 and 85 kDa) and a high salt-eluted PKC zeta (h-PKC zeta, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca(2+)-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca2+, 79-kDa 1-PKC zeta in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKC zeta was increased in association with the shift. These results suggest the pivotal role of 1-PKC zeta in the particulate fraction in the Ca(2+)-induced epidermal cell differentiation processes.

In primary cultured mouse epidermal cells, protein kinase C (PKC) ζ consists of multiple forms: a low salt-eluted PKCζ (1-PKCζ, 79 and 85 kDa) and a high salt-eluted PKCζ (h-PKCζ, 79 and 85 kDa) by anion-exchange column chromatography (K. Nishikawa et al., Cell. Signal. 7, 491-504, 1995). In the present study, PKC isozyme-specific responses during terminal differentiation of epidermal cells, which were induced by the increase of Ca²⁺-concentration in culture medium, were examined. After 24 hr-treatment with 1.8 mM Ca²⁺, 79-kDa 1-PKCζ in the particulate fraction was apparently shifted to the 85-kDa form. The phosphatidylserine-dependent kinase activity of this 1-PKCζ was increased in association with the shift. These results suggest the pivotal role of 1-PKCζ in the particulate fraction in the Ca²⁺-induced epidermal cell differentiation processes.

In this study, we investigated the carcinogenic response of transgenic mice carrying the human prototype c-Ha-ras gene, namely Tg rasH2/CB6F1 mice, to various genotoxic carcinogens and compared it with that of control non-transgenic CB6F1 mice (non-Tg mice). The present studies were conducted as the first step in the evaluation of the Tg rasH2/CB6F1 mouse as a model for the rapid carcinogenicity testing system. Short-term (< or = 6 months) rapid carcinogenicity tests of various genotoxic carcinogens, 4-nitroquinoline-1-oxide, cyclophosphamide, N,N-diethylnitrosamine, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol, revealed that Tg rasH2/CB6F1 mice are more susceptible to these genotoxic carcinogens than control non-Tg mice. Tg rasH2/CB6F1 mice developed tumors more rapidly compared with non-Tg mice. Malignant tumors were observed only in the carcinogen-treated Tg rasH2/CB6F1 mice, but not in non-Tg mice treated with the same carcinogens. Each carcinogen induced tumors in corresponding target tissues of the Tg rasH2/CB6F1 mice. Only a very few lung adenomas but no other tumors were seen as spontaneous tumors during the 6 months of carcinogenicity tests. These results demonstrate that more rapid onset and higher incidence of more malignant tumors can be expected with high probability after treatment with various genotoxic carcinogens in the Tg rasH2/CB6F1 mice than in control non-Tg mice. The Tg rasH2/CB6F1 mouse seems to be a promising candidate as an animal model for the development of a rapid carcinogenicity testing system.

Oral administration of TMK688 (1-([5'-(3"-methoxy-4"-ethoxycarbonyloxyphenyl)-2',4'-pentadien oyl] aminoethyl)-4-diphenylmethoxypiperidine: 30 mg/kg) at 6 h and 30 min prior to and at 30 min after a single topical application of 7,12-dimethylbenz[a]anthracene (DMBA) to dorsal skins of mice reduced both tumor incidence and number of tumors per mouse in the DMBA-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage mouse skin carcinogenesis. TMK688 and its active metabolite TMK777 (1-([5'-(3"-methoxy-4"- hydroxyphenyl)-2',4'-pentadienoyl]-aminoethyl)-4-diphenylmethox y piperidine) inhibited 3-methylchol-anthrene (MC)-induced epidermal aryl hydrocarbon hydroxylase (AHH) activity in a concentration-dependent manner. IC50 of TMK688 and TMK777 was 0.18 and 0.01 mumol/l, respectively. Oral administration of TMK688 (30 mg/kg) almost completely suppressed Cyp1a1 mRNA levels in mouse epidermis induced by a topical application of MC (40 mg/kg) or benzo[a]pyrene (200 nmol) to mouse skin. Oral administration of TMK688 (30 mg/kg) also almost completely inhibited induction of epidermal AHH activity caused by a topical application of MC. These results indicate that oral administration of TMK688 inhibited DMBA-caused skin tumor initiation at least in part by inhibiting Cyp1a1 mRNA induction and epidermal AHH activity.