Details of a Researcher - ICHIMIYA Shingo

アトピー性皮膚炎は本邦の小児の約1割が罹患し，罹患者の生活の質を著しく低下させる．さらにアトピー性皮膚炎自体が気管支喘息やアレルギー性結膜炎など種々の慢性アレルギー疾患の根底にある可能性が分子免疫学的にも支持されつつあり，その病態の機序の解明が急がれている．近年，アトピー性皮膚炎の患部のケラチノサイトがthymic stromal lymphopoietin（TSLP）を高産生することが明らかとなった．TSLPはTSLP受容体（TSLPR）を発現する樹状細胞を介してTh0細胞をTh2細胞へと分化させることで，アトピー・アレルギー疾患の病態形成に中心的役割を果たすと考えられている．我々は今回，表皮ケラチノサイトにおけるTSLPRの発現を明らかとした．また，表皮ケラチノサイト初代培養細胞および表皮モデルHaCaT細胞を用いた検討において，TSLPRの発現は表皮幹細胞因子p63を介した自然免疫経路によって制御されていた．興味深いことに表皮に対するTSLP刺激は更なるTSLP産生を惹起し，その他の炎症性サイトカインの発現も促進していた．これらの結果からTSLPはオートクラインあるいはパラクラインによってケラチノサイト自身にも作用するループを形成し，アトピー性皮膚炎患部表皮の炎症環境の形成，維持に関与していると考えられる．

DOI CiNii

O58-3 アトピー型気管支喘息患者における血清IL-9の検討(サイトカイン・ケモカイン1,口演,第62回日本アレルギー学会秋季学術大会)

重原克則, 伊藤進, 井上祐二, 一宮慎吾

アレルギー ( 一般社団法人日本アレルギー学会 ) 61 ( 9-10 ) 1541 2012

DOI CiNii

症例解説-リンパ節マントル細胞リンパ腫の病理組織診断

一宮慎吾

病理と臨床 27 1094 - 1095 2009

JNK特異的阻害剤による免疫抑制の可能性とその機序

宮島正博, 森川雅之, 田畑哲寿, 一宮慎吾, 佐藤昇志, 安倍十三夫

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 107 ( 2 ) 709 - 709 2006.03

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Clinical significance of vascular endothelial growth factor-C and vascular endothelial growth factor receptor 3 in patients with T1 lung adenocarcinoma

H Kojima, N Shijubo, G Yamada, S Ichimiya, S Abe, M Satoh, N Sato

CANCER ( JOHN WILEY & SONS INC ) 104 ( 8 ) 1668 - 1677 2005.10

BACKGROUND. Vascular endothelial growth factor-C (VEGF-C) plays an important role in lymphangiogenesis and activates VEGF receptor-3 (VEGFR-3). Lymphatic spread is an important prognostic factor in patients with lung adenocarcinoma. The aim of the current study was to determine whether the expression of VEGF-C and VEGFR-3 correlates with clinicopathologic factors and prognosis in patients with TNM classification T1 lung adenocarcinoma. METHODS. The authors conducted a retrospective review of 129 consecutive patients who underwent complete resection for T1 lung adenocarcinoma. Immuno- histochernical staining for VEGF-C, VEGF, VEGFR-3, CD34 (microvessels), tryptase (mast cells), and CD68 (macrophages) was performed to statistically analyze clin- icopathologic implications of VEGF-C and VEGFR-3 status. RESULTS. Of 129 patients with T1 lung adenocarcinoma, 56 (43.3%) patients were positive for tumor-cell VEGF-C and 73 (56.6%) and 69 (53.5%) patients were positive for tumor-cell and endothelial-cell VEGFR-3, respectively. Patients with positive staining for tumor-cell VEGF-C showed significantly less favorable survival rates than patients with negative staining (P = 0.031). The survival rates of patients with positive staining for tumor-cell and endothelial-cell VEGFR-3 were significantly cantly lower than those with negative staining (P = 0.0034 and P = 0.0020, respectively). Patients with positive staining for both tumor-cell VEGF-C and endothelial-cell VEGFR-3 exhibited the most unfavorable prognoses. Multivariate analysis demonstrated that coexpression of tumor-cell VEGF-C and endothelialcell VEGFR-3 was an independent negative prognostic factor (P = 0.0129) as well as N factor (P = 0.0020). CONCLUSIONS. VEGF-C and VEGFR-3 status may be indicative of survival rates for patients with T1 lung adenocarcinoma.

DOI

重症筋無力症の胸腺におけるCHRNA9遺伝子の発現(縦隔腫瘍 (3), 第22回日本呼吸器外科学会総会)

小柳哲也, 渡辺敦, 馬渡徹, 安倍十三夫, 一宮慎吾

日本呼吸器外科学会雑誌 ( 特定非営利活動法人日本呼吸器外科学会 ) 19 ( 3 ) 374 - 374 2005.05

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HLA-DR- and CD11c-positive dendritic cells penetrate beyond well-developed epithelial tight junctions in human nasal mucosa of allergic rhinitis

K Takano, T Kojima, M Go, M Murata, S Ichimiya, T Himi, N Sawada

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 53 ( 5 ) 611 - 619 2005.05

The epithelial barrier of the upper respiratory tract plays a crucial role in host defense. In this study, to elucidate whether there is antigen monitoring by dendritic cells (DCs) beyond the epithelial tight-junction barrier in allergic rhinitis, we investigated the expression and function of tight junctions and characterized DCs in the epithelium of nasal mucosa from patients with allergic rhinitis. In reverse transcription-polymerase chain reaction, mRNAs of tight-junction proteins occludin, JAM-1, ZO-1, and claudin-1, -4, -7, -8, -12, -13, and -14 were detected in the nasal mucosa. Occludin, JAM-1, and ZO-1 were colocalized in the uppermost layer in the pseudostratified epithelium of the nasal mucosa, whereas claudin-1, -4, and -7 were found throughout the epithelium. in freeze-fracture replicas of the nasal mucosa, continuous tight-junction strands formed well-developed networks. Epithelial barrier function measured by a dye tracer was well maintained in occludin-positive tight junctions in the epithelium of the nasal mucosa. HLA-DR- and CD11c-positive DCs expressed claudin-1 and penetrated beyond occludin in the epithelium of the nasal mucosa with, but not without, allergic rhinitis. These results indicate that DCs may easily access antigens beyond epithelial tight junctions in the human nasal mucosa of allergic rhinitis.

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Effects of inhaled FK 506 on the suppression of acute rejection after lung transplantation: Use of a rat orthotopic lung transplantation model

A Ingu, K Komatsu, S Ichimiya, N Sato, Y Hirayama, M Morikawa, T Abe

JOURNAL OF HEART AND LUNG TRANSPLANTATION ( ELSEVIER SCIENCE INC ) 24 ( 5 ) 538 - 543 2005.05

Background: FK 506 inhalant was recently developed for localized administration. We investigated its effects on acute lung allograft rejection and compared its efficacy with that of intramuscular administration of FK 506. Methods: Rats (n = 123) with orthotopic left lung transplantation were divided into 9 groups. Six groups inhaled FK 506 (5 puffs, 10 puffs or 20 puffs per day), or were given intramuscular administration of FK 506 (0.05, 0.1 or 1.0 mg/kg/day). The other groups included rats receiving an isograft, rats with an untreated allograft, and a placebo group. All groups (n = 6 each) were monitored for 14 days post-operatively as an end-point and graft survival time was determined. The remaining animals were killed 4 days after transplantation. The histologic grade of rejection was determined for all groups (n = 6 each). With both (n = 3 each) inhalation therapy and intramuscular administration of FK 506, which showed similar degrees of effectiveness, both blood FK 506 concentration and cytokine expression in the graft and spleen were evaluated. Results: FK 506 inhalation therapy extended allograft survival time and reduced histologic rejection on Day 4 in all groups. Graft survival time and histologic rejection scores at a dose of 10 puffs/day were comparable to those with 0.1 mg/kg/day of intramuscular FK 506. Trough concentrations of FK 506 in blood were detectable with 0.1 mg/kg/day of intramuscular FK 506, but not with 10 puffs/day. The messenger RNA expression levels of interferon-gamma in the lung allograft was suppressed significantly at a dose of 10 puffs/day. Conclusions: FK 506 inhalant enhances acute lung allograft survival with lower blood concentrations than when using comparable intramuscular administration. J Heart Lung Transplant 2005;24:538-43. Copyright (c) 2005 by the International Society for Heart and Lung Transplantation.

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A calcium binding protein, S100A4, mediates T cell dependent cytotoxicity as a transformation-associated antigen

N Kondo, S Ichimiya, Y Tamura, A Tonooka, S Koshiba, T Torigoe, K Kamiguchi, K Takenaga, N Sato

MICROBIOLOGY AND IMMUNOLOGY ( CENTER ACADEMIC PUBL JAPAN ) 49 ( 1 ) 49 - 56 2005

The nature of the target molecule of TCRgammadelta T cell-mediated lysis remains to be determined. As we previously reported, #067 monoclonal antibody (mAb) recognizes one of the transformation-associated antigens, designated as #067 antigen. This antigen is expressed on the cell surface of rat fibrosarcoma W31 cells, which are established by transformation of fetal fibroblastic WFB cells with H-ras oncogene. It has been suggested that the #067 antigen is a target molecule for TCRgammadelta T cells since #067 mAb inhibited TCRgammadelta T cell-mediated lysis against #067 positive cells. In this study we attempted to identify the protein sequence of the #067 antigen. By using molecular cloning techniques, we demonstrated that a calcium binding protein, S100A4, was possibly one and the same molecule as the #067 antigen. It was shown that the expression of S100A4 was higher in W31 cells than in WFB cells at transcription and protein level. Flow cytometry and immunocytochemical studies showed that #067 antigen partially co-localized with S100A4 on the cell surface as well as the cytoplasm of W31 cells. Moreover. rabbit anti-S100A4 polyclonal antibodies (pAb) inhibited TCRgammadelta T cell-mediated lysis against #067 positive cells. Our results indicated that S100A4 may play a role as a possible target molecule for TCRgammadelta T cell-mediated lysis although how S100A4 is involved in TCRgammadelta T cell-mediated lysis remains to be determined.

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Expression and function of tight junctions in the crypt epithelium of human palatine tonsils

M Go, T Kojima, K Takano, M Murata, S Ichimiya, H Tsubota, T Himi, N Sawada

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 52 ( 12 ) 1627 - 1638 2004.12

The human palatine tonsils have surface and crypt stratified epithelium and may be initiated via the epithelium to mount immune responses to various presenting antigens. Here we investigated the expression and function of tight junctions in the epithelium of human palatine tonsils from patients with tonsillar hypertrophy or recurrent tonsillitis. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, -7, -8, and -14 mRNAs were detected in tonsillar hypertrophy. Occludin and claudin-14 were expressed in the uppermost layer of the tonsil surface epithelium, whereas ZO-1, JAM-1, and claudin-1, -4, and -7 were found throughout the epithelium. In the crypt epithelium, claudin-4 was preferentially expressed in the upper layers. In freeze-fracture replicas, short fragments of continuous tight junction strands were observed but never formed networks. In the crypt epithelium of recurrent tonsillitis, the tracer was leaked from the surface regions where occludin and claudin-4 disappeared. Occludin, ZO-1, JAM-1, and claudin-1, -3, -4, and -14, but not claudin-7, mRNAs were decreased in recurrent tonsillitis compared with those of tonsillar hypertrophy. These studies suggest unique expression of tight junctions in human palatine tonsillar epithelium, and the crypt epithelium may possess an epithelial barrier different from that of the surface epithelium.

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Crisscross CTL induction by SYT-SSX junction peptide and its HLA-A*2402 anchor substitute

K Ida, S Kawaguchi, Y Sato, T Tsukahara, Y Nabeta, H Sahara, H Ikeda, T Torigoe, S Ichimiya, K Kamiguchi, T Wada, S Nagoya, H Hiraga, A Kawai, T Ishii, N Araki, A Myoui, S Matsumoto, T Ozaki, H Yoshikawa, T Yamashita, N Sato

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 173 ( 2 ) 1436 - 1443 2004.07

To investigate the effects of anchor substitutions in SYT-SSX junction peptide, an HLA-A24 anchor residue (position 9) of the SYT-SSX B peptide (GYDQIMPKK) was substituted to more favorable residues according to the HLA-A24-binding motif. Among four substitutes constructed, a substitute with isoleucine (termed K9I peptide) most apparently enhanced the affinity for HLA-A24 molecule. Subsequent in vitro CTL induction analysis using PBMCs of 15 HLA-A24(+) synovial sarcoma patients revealed that the original B peptide allowed to induce synovial sarcoma-specific CTLs from 7 patients (47%), whereas such CTLs were inducible from 12 patients (80%) with K9I peptide. Moreover, the extent of cytotoxicity against HLA-A24(+) synovial sarcoma cell lines was higher in K9I peptide-induced CTLs than B peptide-induced CTLs. Influence of anchor substitution on peptide/TCR interaction was evaluated by cytotoxicity assays against autologous cells and tetramer analysis., CTLs induced from a synovial sarcoma patient using K9I peptide did not lyse autologous PHA blasts or EBV-infected B cells. In vitro stimulations of PBMCs from 5 HLA-A24(+) synovial sarcoma patients with K9I peptide increased the frequency of T cells reacting with both HLA-A24/K9I peptide tetramer and HLA-A24/B peptide tetramer. In-contrast, the frequency of T cells reacting with HLA/HIV-derived peptide tetramer remained low. These findings support the validity in design of anchor residue substitution in SYT-SSX fusion gene-derived peptide, and provide a potential clue to the current stagnation in vaccination trials of fusion gene-derived natural junction peptides.

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Expression profiles and functional implications of p53-like transcription factors in thymic epithelial cell subtypes

T Kikuchi, S Ichimiya, T Kojima, L Crisa, S Koshiba, A Tonooka, N Kondo, PT van der Saag, S Yokoyama, N Sato

INTERNATIONAL IMMUNOLOGY ( OXFORD UNIV PRESS ) 16 ( 6 ) 831 - 841 2004.06

In this study, we investigated the localization and functional significance of p53 tumor suppressor-like molecules, p63 and p73, in human thymic epithelial cells (TECs). Immunohistochemical studies showed particular distribution profiles of p63 and p73 in thymic epithelium, in which cortical TECs preferentially expressed p63 in their nuclei whereas subcapsular and medullary TECs expressed both p63 and p73 in their nuclei. The wide distribution of p63 in TECs was further suggested by studies using TECs of primary culture. In vitro studies using two human TEC lines demonstrated that p63 was capable of up-regulating intercellular adhesion molecule-1 (ICAM-1) and enhancing the production of IL-6 and IL-8. Moreover, in vitro studies also indicated that p73, but not p63, had the capacity to induce granulocyte macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF) in the TEC lines. These findings suggest that p63 would regulate the cell adhesive property through ICAM-1/LFA-1 interaction and the production of IL-6 and IL-8, probably in all TEC subtypes. p73 in subcapslar and medullary TECs was suggested to play a role in the regulation of the production of GM-CSF and G-CSF, which might stimulate other stromal cells such as dendritic cells, macrophages and endothelial cells around these regions.

DOI PubMed CiNii

Tumor-derived heat shock protein 70-pulsed dendritic cells elicit tumor-specific cytotoxic T lymphocytes (CTLs) and tumor immunity

G Ueda, Y Tamura, Hirai, I, K Kamiguchi, S Ichimiya, T Torigoe, H Hiratsuka, H Sunakawa, N Sato

CANCER SCIENCE ( JAPANESE CANCER ASSOC ) 95 ( 3 ) 248 - 253 2004.03

Vaccination with autologous tumor-derived heat shock proteins (Hsp), such as Hsp70, Hsp90 and gp96, has been demonstrated to elicit specific immune responses against the tumor from which the Hsps were isolated. The effect of Hsp immunization is wholly dependent on the presence of functional antigen-presenting cells (APCs) in the immunized host, and Hsp receptors on APCs have recently been identified. Here we show that bone marrow-derived dendritic cells (DCs) are able to internalize HSP-peptide complex and that peptides are re-presented by DCs via the major histocompatibility complex (MHC) class I presentation pathway. In addition, immunization with tumor-derived HSP-pulsed IDCs induces strong cytotoxic T cell (CTL) responses against multiple antigenic peptides in a transporter-associated antigen processing (TAP)-dependent manner. The results of the present study provide strong evidence of an efficient cross-priming activity of Hsp70, which could be exploited in the development of new and more effective immunotherapeutic strategies for cancer patients.

DOI PubMed CiNii

Polymorphism of Clara cell 10-kD protein gene of sarcoidosis

T Ohchi, N Shijubo, Kawabata, I, S Ichimiya, S Inomata, A Yamaguchi, Y Umemori, Y Itoh, S Abe, Y Hiraga, N Sato

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE ( AMER THORACIC SOC ) 169 ( 2 ) 180 - 186 2004.01

Clara cell 10-kD protein (CC10) exhibits potent antiinflammatory properties. G38A polymorphism was found in the CC10 gene. We investigated the genetic influence of the allele on the development of sarcoiclosis using case control analysis in a Japanese population (265 sarcoiclosis cases and 258 control subjects). The A allele frequency in sarcoiclosis cases (45.1%) was significantly higher than healthy control subjects (34.9%, p = 0.0002). According to outcomes, we divided 223 patients with follow-up periods of 3 years or more into two subgroups (55 progressive and 168 regressive disease). The A allele frequency in patients with progressive disease was significantly higher than control subjects (odds ratio = 4.55; 95% confidence interval, 2.97-6.97; p < 0.0001), whereas that of regressive disease was not. The A/A genotypes had significantly lower bronchoalveolar lavage fluid CC10 levels than the G/G (nonsmokers, p = 0.0054, and smokers, p = 0.0045) and G/A genotypes (nonsmokers, p = 0.0022, and smokers, p = 0.0402). The reporter gene assay showed significantly lower reporter activities in the presence of interferon-gamma for the 38A construct than the 38G construct (p = 0.0177). The G38A polymorphism in the CC10 gene may influence protein expression and be associated with the development of progressive sarcoidosis.

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Molecules regulating the function of thymic epithelial cells

Clinical Immunology 2004

Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes

Takashi Kojima, Toshinobu Yamamoto, Mengdong Lan, Masaki Murata, Ken-Ichi Takano, Mitsuru Go, Shingo Ichimiya, Hideki Chiba, Norimasa Sawada

Medical Electron Microscopy ( Springer Japan ) 37 ( 2 ) 101 - 113 2004

The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.

DOI PubMed

胸腺上皮細胞機能を制御する分子群

臨床免疫 2004

Chymase-positive mast cells in small sized adenocarcinoma of the lung

M Nagata, N Shijubo, AF Walls, S Ichimiya, S Abe, N Sato

VIRCHOWS ARCHIV ( SPRINGER-VERLAG ) 443 ( 4 ) 565 - 573 2003.10

Mast cells accumulate in angiogenesis-dependent situations of lung adenocarcinoma. Human mast cells are divided into two major subsets: MCT (mast cells with immunoreactivity for tryptase but not chymase) and MCTC (reactive for tryptase and chymase). Chymase is an important mediator of tissue remodeling, but research into chymase-containing mast cell subpopulations has been hampered by the lack of reagents suitable for use with formalin-fixed tissue. We stained chymase using CC1 antibody in 66 cases of small sized lung adenocarcinoma as well as CD34 and tryptase. There were significant positive correlations of microvessel counts with MCT-type and MCTC-type mast cell counts in lung adenocarcinomas. When analyzed according to Noguchi's classification, MCT-type and MCTC-type mast cells were significantly increased in Noguchi type-C tumors [localized bronchioloalveolar carcinoma (LBAC) with active fibroblastic proliferation] compared with in Noguchi type-A (LBAC) plus type-B tumors (LBAC with alveolar collapse). Members in the high-count group of MCTC-type but not MCT-type mast cells showed a significantly worse outcome than those in the low-count group in LBACs. Counting chymase-positive (MCTC-type) mast cells in tumor stroma may be a good prognosis predictor for LBACs, especially Noguchi type-C tumors.

DOI

Histone H2AX sensitizes glioma cells to genotoxic stimuli by recruiting DNA double-strand break repair proteins

H Momota, S Ichimiya, N Kondo, T Kikuchi, T Torigoe, T Yamaki, K Houkin, N Sato

INTERNATIONAL JOURNAL OF ONCOLOGY ( PROFESSOR D A SPANDIDOS ) 23 ( 2 ) 311 - 315 2003.08

Glioblastoma is a life-threatening tumor in the human brain despite the fact that radio-chemotherapy inducing DNA damage has been improved in the last decade. Various studies focusing on the enhancement of the susceptibility of glioblastoma cells to DNA damage have been reported, which are aimed at more efficient treatment for the tumor. In this study, we show that radioresistant T98G glioblastoma cells can develop sensitivity to DNA damage induced by irradiation and etoposide as a result of the introduction of a DNA repair-associated historic, H2AX. Interestingly, when H2AX-transformed T98G cells were irradiated, Brca1 and Nbs1 were readily recruited in DNA double-strand break (DSB) foci and showed the G2/M-phase arrest of the cell cycle. Moreover, up-regulation of Brcal was observed in H2AX-T98G cells after exposure to irradiation. Together with the evidence that H2AX transfection does not affect growth activities of non-tumor cells under genotoxic stimuli, this suggests that H2AX gene transfer would provide a new modality for radio-chemotherapy for glioblastomas, probably through overcoming the instability of the genome, and that Brca1 and Nbs1 might be crucial in this methodology.

Role of the p38 MAP-kinase signaling pathway for Cx32 and claudin-1 in the rat liver

T Kojima, T Yamamoto, M Murata, M Lan, KI Takano, M Go, S Ichimiya, H Chiba, N Sawada

CELL COMMUNICATION AND ADHESION ( TAYLOR & FRANCIS LTD ) 10 ( 4-6 ) 437 - 443 2003.07

Liver regeneration and cholestasis are associated with adaptive changes in expression of gap and tight junctions through signal transduction. The roles of stress responsitive MAP-kinase, p38 MAP-kinase, in the signaling pathway for gap junction protein, Cx32, and tight junction protein, claudin-1, were examined in rat liver in vivo and in vitro , including regeneration following partial hepatectomy and cholestasis after common bile duct ligation. Changes in the expression and function of Cx32 and claudin-1 in hepatocytes in vivo were studied using the p38 MAP-kinase inhibitor SB203580. Following partial hepatectomy and common bile duct ligation, down-regulation of Cx32 protein was inhibited by SB203580 treatment. Up-regulation of claudin-1 protein was enhanced by SB203580 treatment after partial hepatectomy but not common bile duct ligation. However, no change of the Ki-67 labeling index (which is a marker for cell proliferation) in the livers treated with SB203580, was observed compared to that without SB203580 treatment. In primary cultures of rat hepatocytes, however, treatment with a p38 MAP-kinase activator, anisomycin, decreased Cx32 and claudin-1 protein levels. p38 MAP-kinase may be an important signaling pathway for regulation of gap and tight junctions in hepatocytes. Changes of gap and tight junctions during liver regeneration and cholestasis are shown to be in part controlled via the p38 MAP-kinase signaling pathway and are independent of cell growth.

DOI

Frequent beta-catenin alteration in gallbladder carcinomas

Y Kimura, T Furuhata, M Mukaiya, C Kihara, M Kawakami, K Okita, Y Yanai, H Zenbutsu, M Satoh, S Ichimiya, K Hirata

JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH ( APSIT ASSOC PROM STUD IMMUNOL TUMOR ) 22 ( 2 ) 321 - 328 2003.06

To investigate the contribution of beta-catenin to the development of gallbladder carcinoma, genetic alteration in beta-catenin gene, ctnnb-1 and subcellular localization of beta-catenin protein were searched. Mutational analysis of exon 3 in ctnnb-1, which encodes the serine/threonine residues for GSK3beta phosphorylation sites, was performed for 21 gallbladder carcinomas affected with/without the pancreaticobiliary malunion, PBM, and 6 non-cancerous tissues affected with PBM. We also analyzed subcellular localization of beta-catenin protein in all cases immunohistochemically. Nucleotide sequencing analysis revealed that none of them carried mutations that altered amino acid residues in the potential GSK3beta phophorylation sites, but one nucleotide substitution was found. We also analyzed subcellular localization of beta-catenin protein in all cases immunohistochemically, and confirmed its accumulation in both the nucleus and cytoplasm in 10 out of 21 cancer tissues, while the non-cancerous tissues which were affected with PBM and histologically diagnosed as hyperplasia or dysplasia displayed intense membranous staining. A significant correlation between cytoplasmic or nuclear beta-catenin immunoreactivity and clinicopathological status of gallbladder carcinomas was found, especially in the poorer histological differentiation grade(p<0.05). In conclusion our results suggested that β-catenin alteration might be a minor contributor to the development of gallbladder carcinomas through abnormal Wnt-wingless signalling, however, decreased membranous expression of β-catenin might be correlated to carcinoma progression through loss of cell adhesive function in E-cadherin-catenin fashion.

Rapidly growing early gastric cancer with microsatellite instability

K Yamashita, N Kato, H Takahashi, H Shimizu, H Suzuki, S Motoya, Y Arimura, T Endo, H Ura, S Ichimiya, K Imai

JOURNAL OF GASTROENTEROLOGY ( SPRINGER-VERLAG TOKYO ) 38 ( 2 ) 170 - 174 2003.02

A 77-year-old man underwent gastrointestinal endoscopy that revealed a small flat elevated lesion in the gastric antrum. Endoscopically, we assessed the tumor as intramucosal cancer or adenoma, and biopsy specimens were diagnosed as showing borderline malignancy. At the second examination, performed 6 months later, the elevated lesion had grown, showing remarkable changes in its appearance, and a diagnosis of adenocarcinoma was confirmed pathologically. The patient underwent distal gastrectomy about 8 months after the first examination, and the tumor in the resected specimens showed a moderately differentiated adenocarcinoma invading through the entire submucosal layer, with metastasis to a regional lymph node. Microsatellite analysis of this early gastric cancer disclosed mutations in all six markers examined, indicating high-level microsatellite instability (NISI-H). The tumor also showed frameshift mutations of TGFbetaRII and BAX genes, as well as hypermethylation of the hMLH1 promoter. These results suggest that a deficiency of the mismatch repair system played a critical role in the progression of this cancer.

DOI PubMed

T-cell receptor variable gamma chain gene expression in the interaction between rat gamma delta-type T cells and heat-shock protein 70-like molecule

T Ichinohe, S Ichimiya, A Kishi, Y Tamura, N Kondo, G Ueda, T Torigoe, A Yamaguchi, H Hiratsuka, Hirai, I, G Kohama, N Sato

MICROBIOLOGY AND IMMUNOLOGY ( CENTER ACADEMIC PUBL JAPAN ) 47 ( 5 ) 351 - 357 2003

We previously reported that rat T-cell receptor (TCR) Vdelta6 of T-cell hybridomas was preferentially involved in recognition of the cell surface-expressed 70 kDa rat heat-shock cognate (hsc70, a constitutively expressed member of the hsp 70 family) protein-like molecule (#067 molecule). In the present study, we analyzed usage of the TCR Vgamma family of #067-restricted T-cell hybridomas. Our data indicated that most of these hybridomas expressed transcripts of TCR Vgamma1 and/or Vgamma2. However, some of the Vgamma2 transcripts were out-of-frame, suggesting that the TCR Vgamma1 family may be important for the recognition of #067-defined molecules. TCR Vgamma1 transcripts were detected in not only #067-restricted T-cell hybridomas, but #067-non restricted ones as well. However, V-J nucleotide sequences of #067-restricted and #067-non restricted T-cell hybridomas suggested that #067-restricted T-cell hybridomas showed limited insertion of nucleotide stretch as compared with #067-non restricted ones. In terms of amino acids, only one amino acid was added in #067-restricted T-cell hybridomas, whereas two or three amino acids were added in #067-non restricted ones. These data suggest that the heterodimer of the TCR relatively short stretch form of Vgamma1 molecule and TCR Vdelta6 may participate in recognition of the #067 molecule.

Immunohistochemical analysis of the p53 family members in human craniopharyngiomas

Hiroyuki Momota, Shingo Ichimiya, Tatsuru Ikeda, Toshiaki Yamaki, Tomoki Kikuchi, Kiyohiro Houkin, Noriyuki Sato

Brain Tumor Pathology 20 ( 2 ) 73 - 77 2003

Craniopharyngiomas are intracranial tumors that usually arise in the site around the sella turcica. They are composed of distinctive sheets of epithelial cells showing adamantinomatous or squamous-papillary histologic types. Because little is known about the tumorigenesis of craniopharyngiomas, we retrieved samples from 15 tumor cases to investigate the functional significance of the p53 family of transcription factors, which are known to be expressed in various human epithelia. Immunohistochemical analysis of these cases demonstrated similar expression profiles of p53 family members in the two histologic types of the tumor i.e., strong nuclear expression of p63 was observed in all cell layers, and moderate to intense nuclear expression of p73 was observed in the basal cell layers. In contrast to p63 and p73, the reactivity of an archetypal tumor suppressor, p53, was occasional and weak in the two histologic types. Because p63 was widely expressed in the tumors, reverse transcription-polymerase chain reaction (RT-PCR) analysis was conducted to elucidate which spliced variant of p63 was expressed. The results showed that ANp63, lacking a terminal transactivation domain of p63, was the dominant isoform. Together with the reported evidence that the ANp63 isoform is highly expressed in human squamous-cell carcinomas, these data suggest that the cellular architecture characteristic of the expression of p53 family members may be required for the histogenesis of craniopharyngiomas, where ANp63 has a possible role in maintaining proliferative activity of the tumor cells, like squamous-cell carcinomas in other tissues.

DOI PubMed CiNii

Improved generation of HLA class I/peptide tetramers

Y Sato, H Sahara, T Tsukahara, M Kondo, Y Hirohashi, Y Nabeta, S Kawaguchi, H Ikeda, T Torigoe, S Ichimiya, Y Tamura, T Wada, T Yamashita, M Goto, H Takasu, N Sato

JOURNAL OF IMMUNOLOGICAL METHODS ( ELSEVIER SCIENCE BV ) 271 ( 1-2 ) 177 - 184 2002.12

As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL. (C) 2002 Elsevier Science B.V. All rights reserved.

DOI PubMed CiNii

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

Y Sato, Y Nabeta, T Tsukahara, Y Hirohashi, R Syunsui, A Maeda, H Sahara, H Ikeda, T Torigoe, S Ichimiya, T Wada, T Yamashita, H Hiraga, A Kawai, T Ishii, N Araki, A Myoui, S Matsumoto, T Umeda, S Ishii, S Kawaguchi, N Sato

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 169 ( 3 ) 1611 - 1618 2002.08

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYD QIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.

DOI

A gene encoding human gastric signet ring cell carcinoma antigen recognized by HLA-A31-restricted cytotoxic T lymphocytes

H Sahara, Y Nabeta, T Torigoe, Y Hirohashi, S Ichimiya, Y Wada, N Takahashi, K Jimbow, T Yajima, N Watanabe, K Kikuchi, N Sato

JOURNAL OF IMMUNOTHERAPY ( LIPPINCOTT WILLIAMS & WILKINS ) 25 ( 3 ) 235 - 242 2002.05

We previously reported acid-extracted natural antigenic peptide (F4.2[YSWMDISCWI]) of a gastric signet ring cell carcinoma HST-2 cells, recognized by HLA-A*31012-restricted autologous cytotoxic T lymphocytes, TcHST-2 line. In this study, the full-length cDNA (1101 bp), termed c98, predicting a protein composed of 170 amino acids was obtained. Because TcHST-2 cells could lyse the HLA-A31 antigen (+) allogeneic tumor cells that were introduced with c98 gene, this gene was suggested to possess antigenicity. Beginning at N-terminal 61 amino acid, the N-terminal six amino acid sequence that is completely identical to F4.2 was present in c98; however, a sequence of four amino acids in C-terminal was not found. Nevertheless, this peptide, c98(61-70), seemed to be immunogenic, because cells pulsed with c98(61-70) peptide were lysed in a dose-dependent manner by TcHST-2 cells. The c98 gene was expressed ubiquitously in tumor cells as well as in normal tissues. However, some tumor cells, including HST-2 cells, expressed this antigen in a high content, and such cells were lysed by TcHST-2 cells in the context of HLA-A31 antigen. However, TcHST-2 cells did not lyse cells that expressed lower amounts of c98 than HST-2 cells. These data suggested that c98-gene product and/or C98(61-70) peptides could be used as a candidate for tumor vaccines in cancer immunotherapy.

DOI

p73 is expressed in human thymic epithelial cells

S Ichimiya, T Kojima, H Momota, N Kondo, T Ozaki, A Nakagawara, ML Toribio, M Imamura, N Sato

JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY ( HISTOCHEMICAL SOC INC ) 50 ( 4 ) 455 - 462 2002.04

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.

Examination of the Fixation Applicable to Frozen Section

浅沼広子, 東恭悟, 一宮慎吾, 佐藤昌明

臨床検査 ( 株式会社医学書院 ) 46 ( 1 ) 77 - 81 2002

DOI

Reguratory mechanism of thymic epithelial cell function by p53 related transcription factors

Annual Review 2003 2002

[Genetic analysis of microdissected human tissues]

ICHIMIYA Shingo, SATO Noriyuki

Hokkaido Igaku Zasshi ( Hokkaido University ) 77 ( 2 ) 139 - 41 2002

CiNii

p53関連遺伝子による胸腺上皮細胞の機能調節

Annual Review免疫2003 2002

Improved fixation methods for frozen section diagnosis

Rinshokensa 2002

Medicine in Postgenome Era. 1.Genetic analysis of microdissected human tissues

Ichimiya Shingo, Satou Noriyuki

The Hokkaido journal of medical science ( Hokkaido University ) 77 ( 2 ) 139 - 41 2002

CiNii

レーザーマイクロダイセクションを利用した免疫組織の新しい解析法

一宮慎吾

Annual Review 免疫2002 ( 内外医学社 ) 2002

CiNii

The cell surface-expressed HSC70-like molecule preferentially reacts with the rat T-cell receptor V delta 6 family

A Kishi, T Ichinohe, Hirai, I, K Kamiguchi, Y Tamura, M Kinebuchi, T Torigoe, S Ichimiya, N Kondo, K Ishitani, T Yoshikawa, M Kondo, A Matsuura, N Sato

IMMUNOGENETICS ( SPRINGER-VERLAG ) 53 ( 5 ) 401 - 409 2001.07

We previously showed that the cell surface-expressed M-r 70,000 heat shock cognate (hsc70, a constitutively expressed member of the hsp70 family) protein-like molecule (#067 molecule) interacts with rat CD3(+), CD4(-), CD8(-), T-cell receptor (TCR)alpha beta (-), natural killer recetor-Pl(-) T cells. This 70hsc-like molecule was also suggested to present cellular peptide antigens to these T cells. In the present study, we identified the genetic structure of the TCR by establishing T-cell hybridomas between these T cells and mouse BW5147 cells. Our data indicated that these T cells preferentially used TCRs with the V delta6 family. Analysis of the nucleotide sequence of the CDR3 junctional portion showed that there are substantial diversities, with insertion of seven to nine amino acid residues. These data provide indirect evidences for our hypothesis that an hsc70-like molecule could be presented together with cellular peptide antigens to particular T cells with TCR gamma delta chains. Since the expression of this hsc70-like #067 antigen on the cell surface is usually induced along with cell transformation by activated oncogenes, T cells with the TCR V delta6 family are likely to contribute to host resistance to tumor cells.

DOI

A case of inflammatory fibrosarcoma of the retroperitoneum

Atsumori Ueno, Itaru Ozeki, Shigeru Sasaki, Sumio Yamaguchi, Norikazu Iwata, Hiroyuki Kaneto, Fumio Itoh, Takao Endo, Shingo Ichimiya, Masaaki Satoh, Noriyuki Otsuka, Michio Shimizu, Michiaki Matsushita, Satoru Todo, Kohzoh Imai

Japanese Journal of Gastroenterology ( The Japanese Society of Gastroenterology ) 98 ( 4 ) 436 - 441 2001.04

DOI PubMed CiNii

Genetic analysis of p73 localized at chromosome 1p36.3 in primary neuroblastomas

S Ichimiya, Y Nimura, H Kageyama, N Takada, M Sunahara, T Shishikura, Y Nakamura, S Sakiyama, N Seki, M Ohira, Y Kaneko, F McKeon, D Caput, A Nakagawara

MEDICAL AND PEDIATRIC ONCOLOGY ( WILEY-LISS ) 36 ( 1 ) 42 - 44 2001.01

Background. Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers, p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. Procedure. To lest the hypothesis that p73 is a NBL suppressor gene, we examined expression, alleto-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which eve found in intron 9 of the p73 gene. Results. p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P < 0.001), MYCN amplification (P < 0.001), and advanced stages (P < 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. Conclusion. Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage rumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner. Med. Pediatr. Oncol. 36:42-44, 2001. (C) 2001 Wiley-Liss. Inc.

DOI

Downregulation of hASH1 is associated with the retinoic acid-induced differentiation of human neuroblastoma cell lines

S Ichimiya, Y Nimura, N Seki, T Ozaki, T Nagase, A Nakagawara

MEDICAL AND PEDIATRIC ONCOLOGY ( WILEY-LISS ) 36 ( 1 ) 132 - 134 2001.01

Background. MASH1, a transcription factor with basic helix-loop-helix domain, has a pivotal function to promote differentiation of neural crest cells into autonomic neurons. Procedure. To investigate the functional significance of human MASH1 (hASH1) in the pathogenesis of neuroblastoma, which is originated from autonomic precursor cells, we studied hASH1 gene expression in primary neuroblastomas and human neuroblastoma cell lines. Results. The following results were obtained: (i) hASH1 was expressed in 40 out of 61 (66%) primary neuroblastomas, (ii) hASH1 transcripts were downregulated in several cell lines prior to differentiation induced by all-trans retinoic acid (RA), (iii) a neuroblastoma cell line without expression of endogenous hASH1 did not respond to RA at all, and (iv) the analysis of the hASH1 genomic DNA revealed two possible transcription initiation sites, which may correspond to 3.0 kb and 3.5 kb transcripts. Conclusions. Our observations suggest that, although hASH1 may not preserve the growth capacity of neuroblastomas, downregulation of hASH1 may be necessary to promote neuronal differentiation of neuroblastoma. Med. Pediatr. Oncol. 36:132-134, 2001. (C) 2001 Wiley-Liss. Inc.

DOI

H2 gene complex

Dictionary for Immunology, second edition 2001

H2遺伝子複合体、H2ハプロタイプ、Ia、抗Ia抗体、I-A亜領域、I-E亜領域、I-J亜領域

免疫学辞典第2版 2001

Aberrant Wnt-wingless signaling in gallbladder carcinomas

KIMURA Yasutoshi, FURUHATA Tomohisa, MUKAIYA Mitsuhiro, YANAI Yoshiyuki, KAWAKAMI Masayo, SATOH Masaaki, ICHIMIYA Shingo, HIRAMA Toshinori, HIRATA Koichi

Tando ( Japan Biliary Association ) 14 ( 4 ) 339 - 346 2000.10

To investigate the contribution of β-catenin to the development of gallbladder carcinoma, we searched for genetic alterations of the β-cetenin gene, ctnnb-1. Mutational analysis of exon 3 in ctnnb-1, which encodes the serine/threonine residues for GSK-3β phosphorylation sites was performed for 5 gallbladder cancers with pancreaticobiliary malunion, PMB and 8 noncancerous tissues with PBM. PCR-SSCP analysis showed super-shifted bands in the electrophoretic display, but no mutation was detected through nucleotide sequencing analysis near potential GSK-3βphosphorylation sites. We also performed immunohistochemical analysis of β-catenin protein in all cases, and confirmed accumulation in both the nucleus and cytoplasm in three of 5 cancer tissues, while neighboring noncancerous tissue and the biliary tissue with PBM alone displayed membrane staining. Our results indicated that abnormal Wnt-wingless signaling, resulting in β-catenin accumulations, might be involved in the malignant transformation rather than the development of gallbladder carcinomas.

DOI CiNii

275 Salivary duct carcinomaの3例

東恭悟, 浅沼広子, 太田由紀子, 佐々木雅博, 近藤啓, 遠藤明美, 木村幸子, 一宮慎吾, 池田健, 佐藤昌明

日本臨床細胞学会雑誌 ( 特定非営利活動法人日本臨床細胞学会 ) 39 ( 2 ) 494 - 494 2000.09

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178 乳腺小葉癌の穿刺吸引細胞診像の検討

太田由紀子, 東恭悟, 浅沼広子, 佐々木雅博, 岸誼博, 木村幸子, 一宮慎吾, 池田健, 佐藤昌明, 成松英明

日本臨床細胞学会雑誌 ( 特定非営利活動法人日本臨床細胞学会 ) 39 ( 2 ) 445 - 445 2000.09

CiNii

肝原発カルチノイド腫ようの1剖検例

小関至, 佐々木茂, 安井寛, 岩田徳和, 三原真美, 金戸宏行, 一宮慎吾, 佐藤昌明, 今井浩三

日本消化器病学会雑誌 97 A676 2000.09

J-GLOBAL

NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain

T Ozaki, T Nagase, S Ichimiya, N Seki, M Ohiri, N Nomura, N Takada, S Sakiyama, BL Weber, A Nakagawara

DNA AND CELL BIOLOGY ( MARY ANN LIEBERT INC PUBL ) 19 ( 8 ) 475 - 485 2000.08

The BRCT (<(BR)under bar>ECA1 (C) under bar-(t) under bar erminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control. The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089), This gene product has been described as one of the BRCT superfamily proteins. However, its biological significance has been unclarified, Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal. In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity. The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells. Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation. These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

DOI

p73: Structure and function

S Ichimiya, A Nakagawara, Y Sakuma, S Kimura, T Ikeda, M Satoh, N Takahashi, N Sato, M Mori

PATHOLOGY INTERNATIONAL ( BLACKWELL SCIENCE ASIA ) 50 ( 8 ) 589 - 593 2000.08

Book review, literature introduction, etc.

Alteration of the p53 tumor suppressor gene is a common, if not general, observation in human malignant tumors. p73 Is a novel member of the p53 family at chromosome 1p36.3, at which locus frequent defects are seen in many tumors including neuroblastoma. Besides structural similarities, the fact that p73 functions in the regulation of the cell cycle and apoptosis promotes the expansion of the research field concerning p53-associated tumor progression. In this paper, we review the structure and function of p73 as well as the mutational status in various human tumors. In addition, possibilities for new therapeutic applications with p73 for cancer cell control are discussed.

DOI PubMed

A Case of Bizarre parosteal Osteochondromatous Proliferation of the Femur

KAWAGUCHI S

日本整形外科學會雜誌 74 ( 6 ) S1123 2000.06

CiNii

Identification of the full-length KIAA0591 gene encoding a novel kinesin-related protein which is mapped to the neuroblastoma suppressor gene locus at 1p36.2

M Nagai, S Ichimiya, T Ozaki, N Seki, M Mihara, S Furuta, M Ohira, N Tomioka, N Nomura, S Sakiyama, S Kubo, K Takakura, T Hori, A Nakagawara

INTERNATIONAL JOURNAL OF ONCOLOGY ( PROFESSOR D A SPANDIDOS ) 16 ( 5 ) 907 - 916 2000.05

The distal region of a short arm of chromosome Ip is frequently deleted in many human cancers including neuroblastoma (NBL), in which it has been narrowed down to the smallest region of overlap between D1S244 and D1S214 (similar to 7 cM). During the search for the candidate tumor suppressor genes mapped within the region, we found the KIAA0591 gene which encoded a new human kinesin-related protein with a homology to human axonal transporter of synaptic vesicles (ATSV). The kinesin is an intracellular motor protein and often associated with neuronal differentiation and survival. Here we identified a complete open reading frame of the KIAA0591 gene by screening a cDNA library derived from human substantia nigra. The KIAA0591 protein contains a possible pleckstrin homology (PH) domain at its carboxy-terminus. However, it did not possess a force-generating motor domain which is well conserved among kinesin superfamily members (KIFs). Northern blot analysis demonstrated that KIAA0591 mRNA was preferentially expressed in both adult and fetal brains, kidney, skeletal muscle and pancreas. KIAA0591 was expressed in favorable NBLs at higher levels than in unfavorable NBLs, although RT-PCR SSCP analysis showed no mutation within the coding region of the KIAA0591 gene, when 8 neuroblastoma tissues and 15 neuroblastoma-derived cell lines were examined. Thus, the full-length KIAA0591 gene may be a novel member of human KIF superfamily which lacks motor domain and might function as a tumor suppressor in an epigenetic but not a classic Knudson's manner.

山本直敬, 飯島崇利, 砂原正男, 一宮慎吾, 影山肇, 木村秀樹, 中川原章

日本癌学会総会記事 58th 528 1999.08

J-GLOBAL

A case of solitary fibrous tumor of the orbit

中村靖, 大塚賢二, 橋本雅人, 一宮慎吾, 佐藤昌明

臨床眼科 ( 株式会社医学書院 ) 53 ( 6 ) 1381 - 1384 1999.06

DOI

Mutational analysis of the p73 gene in human breast cancers

T Shishikura, S Ichimiya, T Ozaki, Y Nimura, H Kageyama, Y Nakamura, S Sakiyama, M Miyauchi, N Yamamoto, M Suzuki, N Nakajima, A Nakagawara

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 84 ( 3 ) 321 - 325 1999.06

In primary breast cancer, mutations of the p53 tumor suppressor gene lead to loss of growth-suppressive properties and poor outcome. Recently, a p53-related gene, termed p73, has been cloned and its gene product possesses a function similar to p53. p73 has been mapped at chromosome 1p36.3, a region frequently deleted in breast cancer, neuroblastoma and other malignancies. To elucidate the functional significance of p73 in the oncogenesis of breast cancer, we have studied genetic alterations of p73 in tissue specimens obtained from 87 patients with primary breast cancer. Thirteen percent of informative cases showed loss of heterozygosity (LOH) at the p73 gene. However, there was no correlation between the p73 LOH and clinical features such as histopathological types, metastatic behavior or expression of estrogen or progesterone receptor. The levels of p73 transcript in primary breast cancer were not significantly different from those in normal breast tissue. Moreover, PCR-SSCP analysis failed to detect any missense or frameshift mutations in the p73 gene. Our observations suggest that allelic loss, expression levels and mutations of the p73 gene may not contribute to oncogenesis of primary breast cancers. Int. J. Cancer (Pred. Oncol.) 84:321-325, 1999. (C) 1999 Wiley-Liss, Inc.

DOI PubMed CiNii

Identification of a transactivation activity in the COOH-terminal region of p73 which is impaired in the naturally occurring mutants found in human neuroblastomas

N Takada, T Ozaki, S Ichimiya, S Todo, A Nakagawara

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 59 ( 12 ) 2810 - 2814 1999.06

p73 is a recently cloned tumor suppressor gene that is highly homologous to p53, and the products of both possess similar functions in inhibiting cell growth and inducing apoptosis. Interestingly, the COOH-terminal region of p53 displays no significant homology with that of p73, Moreover, p73 has an additional segment at its COOII terminus. Recently, we have found two mutations of p73 with amino acid substitution (P405R and P425L) in primary neuroblastomas, Because the region (amino acid residues 382-491) contains a glutamine- and proline-rich domain, we hypothesized that it has a transactivation function, and the mutations found in tumors result in loss of function. To test it, we used the yeast GAL4 DNA-binding fusion system. Yeast transformants expressing a GAL4-p73(1-112) or a GAL4-p73 alpha(380-513) fusion protein were grown in SD medium lacking histidine and tryptophan and exhibited a significant induction of P-galactosidase activity, Transient transfection experiments revealed that both of fusion proteins could induce the chloramphenicol acetyltransferase activity in mammalian cells, indicating that the COOH-terminaI as well as NH2-terminal regions of p73 had significantly high levels of transactivation activity. Furthermore, the former activity was severely impaired in two naturally occurring mutant forms found in neuroblastomas. These suggest that, unlike p53, p73 has two domains with transactivation function, one in the NH2-terminal region and the other in the COOH-terminal region. Loss of function mutation in the latter might he involved in tumorigenesis and/or tumor progression.

Multiple Orbital Neurofibromas Not Associated with von Recklinghausen's Disease

NAKAMURA Yasushi, OHTSUKA Kenji, HASHIMOTO Masato, ICHIMIYA Singo, SATOH Masaaki

あたらしい眼科 = Journal of the eye 16 ( 2 ) 283 - 285 1999.02

CiNii

O-334 p53 co-activator, p33^<ING1>の神経芽腫発生における役割 : 発現および変異とp53, mdm2との関連について

高橋將人, 一宮慎吾, 藤堂省

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 100 ( 0 ) 191 - 191 1999.02

CiNii

P-804 肺癌における、p53関連遺伝子p73の変異および欠失と、その臨床的意義

山本直敬, 飯島崇利, 一宮慎吾, 影山肇, 木村秀樹, 中川原章

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 100 ( 0 ) 512 - 512 1999.02

CiNii

Absence of mutation of the p73 gene localized at chromosome 1p36.3 in hepatocellular carcinoma

M Mihara, Y Nimura, S Ichimiya, S Sakiyama, S Kajikawa, W Adachi, J Amano, A Nakagawara

BRITISH JOURNAL OF CANCER ( CHURCHILL LIVINGSTONE ) 79 ( 1 ) 164 - 167 1999.01

Accumulating evidence has demonstrated that aberration of the p53 tumour-suppressor gene is one of the pivotal genetic events in hepatocellular carcinogenesis. Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1 p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined.

DOI PubMed CiNii

p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent

S Ichimiya, Y Nimura, H Kageyama, N Takada, M Sunahara, T Shishikura, Y Nakamura, S Sakiyama, N Seki, M Ohira, Y Kaneko, F McKeon, D Caput, A Nakagawara

ONCOGENE ( STOCKTON PRESS ) 18 ( 4 ) 1061 - 1066 1999.01

p73, a novel p53 family member, is a recently identified candidate neuroblastoma (NBL) suppressor gene mapped at chromosome 1p36.33 and was found to inhibit growth and induce apoptosis in cell lines. To test the hypothesis that p73 is a NBL suppressor gene, we analysed the p73 gene in primary human NBLs, Loss of heterozygosity (LOH) for p73 was observed in 19% (28/151) of informative cases which included 92 mass-screening (MS) tumors. The high frequency of p73 LOH was significantly associated with sporadic NBLs (9% vs 34%, P < 0.001), N-myc amplification (10% vs 71%, P < 0.001), and advanced stage (14% vs 28%, P < 0.05), Both p73 alpha and p73 beta transcripts were detectable in only 46 of 134 (34%) NBLs at low levels by RT-PCR methods, while they were easily detectable in most breast cancers and colorectal cancers under the same conditions. They found no correlation between p73 LOH and its expression levels (P > 0.1), We found two mutations out of 140 NBLs, one somatic and one germline, which result in amino acid substitutions in the C-terminal region of p73 which may affect transactivation functions, though, in the same tumor samples, no mutation of the p53 gene was observed as reported previously. These results suggest that allelic loss of the p73 gene may be a later event in NBL tumorigenesis. However, p73 is infrequently mutated in primary NBLs and may hardly function as a tumor suppressor in a classic Knudson's manner.

DOI PubMed CiNii

p73, a gene related to p53, is not mutated in esophageal carcinomas

Y Nimura, M Mihara, S Ichimiya, S Sakiyama, N Seki, M Ohira, N Nomura, M Fujimori, W Adachi, J Amano, M He, YM Ping, A Nakagawara

INTERNATIONAL JOURNAL OF CANCER ( WILEY-LISS ) 78 ( 4 ) 437 - 440 1998.11

A novel gene, termed p73, encodes a protein with a significant homology to p53 and has been mapped at chromosome 1p36.3, which is a locus of multiple suppressor genes for tumors including neuroblastoma and other cancers. Since the 1p36 locus is reported to be deleted and p53 is frequently mutated in esophageal carcinomas, we examined loss of heterozygosity (LOH) and mutation of the p73 gene in 48 untreated esophageal tumors, as well as mRNA expression in 8 tumors. We screened the pi genomic library to obtain a pi clone containing the p73 gene and found a polymorphic short tandem CT repeat site at intron 9, Intragenic sequences for 14 PCR primer sets and a primer pair flanking the repeat were also determined for the analysis of PCR single-strand conformation polymorphism (SSCP) and LOH studies, respectively, Expression of p73 mRNA was detectable but at low levels in all 8 tumor tissues by reverse transcriptase PCR, We did not find any type of mutation other than polymorphisms in the 48 esophageal carcinomas, though aberration of the p53 gene on the PCR-SSCP gels was observed in 15 of 38 (39%) tumors of the same set. In addition, LOH for p73 was found in only 2 of 25 (8%) tumors. These results suggest that, at least in esophageal carcinomas, allelic loss or mutation of p73 may not be a main genetic event for the tumorigenesis as it is with P53. (C) 1998 Wiley-Liss, Inc.

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Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas

M Sunahara, S Ichimiya, Y Nimura, N Takada, S Sakiyama, Y Sato, S Todo, W Adachi, J Amano, A Nakagawara

INTERNATIONAL JOURNAL OF ONCOLOGY ( SPANDIDOS PUBL LTD ) 13 ( 2 ) 319 - 323 1998.08

Genetic alteration of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of human colorectal carcinoma (CRC). Recently, p73, a novel family member of p53, has been identified and found, like p53, to activate p21(Wafl/Cipl) and to induce apoptosis. The p73 gene was mapped at chromosome 1p36.3 which is a region frequently deleted in CRCs and other cancers including neuroblastoma. To assess whether or not p73 is a tumor suppressor gene of CRC, we performed mutational analysis of p73 in 82 colorectal tumor tissues paired with constitutional DNA. Using a microsatellite marker for p73, the loss of heterozygosity (LOH) study was performed and allelic loss of p73 was found in 17% of the CRCs. RT-PCR single strand conformation polymorphism analysis showed no mutation except three polymorphisms in the p73 coding region. In addition, p73 was expressed at higher levels in the CRC tissues than in the normal mucosa or neuroblastoma tissues, though the transcripts were detectable only by the RT-PCR method. Our results suggest that, in CRCs, p73 may not play a role as a tumor suppressor, at least not in a classic Knudson manner.

Mutation Analysis of p73 Gene in Bone & Soft Tissue Tumor

HANAOKA E

日本整形外科學會雜誌 = The Journal of the Japanese Orthopaedic Association 72 ( 6 ) S1022 1998.06

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Mutation, allelotyping, and transcription analyses of the p73 gene in prostatic carcinoma

H Takahashi, S Ichimiya, Y Nimura, M Watanabe, M Furusato, S Wakui, R Yatani, S Aizawa, A Nakagawara

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 58 ( 10 ) 2076 - 2077 1998.05

A novel gene,p73, encoding a protein with significant homology to p53, was recently identified at 1p36, To investigate penetrance of p73 in prostatic carcinogenesis, mutation, allelotyping, and transcription analyses of p73 were performed in prostatic carcinoma. No types of mutation causing amino acid substitutions or frameshifts were found in 106 cases examined. Loss of heterozygosity in the gene was found in 2 of 38 cases (5.3%), Various expression levels of p73 alpha variant were observed in tumor compared with those in normal tissue. These data suggest that the p73 gene is not playing an essential role, but expression of p73 may associate with tumor growth in prostatic carcinogenesis.

Glial cell line-derived neurotrophic factor neurturin-induced differentiation and its enhancement by retinoic acid in primary human neuroblastomas expressing c-Ret, GFR alpha-1, and GFR alpha-2

T Hishiki, Y Nimura, E Isogai, K Kondo, S Ichimiya, Y Nakamura, T Ozaki, S Sakiyama, M Hirose, N Seki, H Takahashi, N Ohnuma, M Tanabe, A Nakagawara

CANCER RESEARCH ( AMER ASSOC CANCER RESEARCH ) 58 ( 10 ) 2158 - 2165 1998.05

Neuroblastomas often undergo spontaneous differentiation and/or regression in vivo, which is at least partly regulated by the signals through neurotrophins and their receptors, Recently, glial cell line-derived neurotrophic factor (GDNF) and a second family member, neurturin (NTN), have been found to mediate their signals by binding to a heterotetrameric complex of c-Ret tyrosine kinase receptors and glycosylphosphatidylinusitol-linked proteins, GFR alpha-1 (GDNFR-alpha) or GFR alpha-2 (TrnR2/GDNFR-beta/ NTNR-alpha/RETL2). Here, we studied the effect of GDNF and NTN on human neuroblastomas in the short-term primary culture system, as well as the expression of c-Ret, GFR alpha-1, GFR alpha-2, GDNF, and NTN. GDNF (1-100 ng/ml) induced morphological differentiation in 34 of 38 primary neuroblastomas and an accompanying increase in c-Fos induction. These effects were markedly enhanced by treatment with 5 mu M all-bans-retinoic acid. Although GDNF alone induced a rather weak differentiation independent of the disease stages, the enhancement of neurite outgrowth induced by treatment with both GDNF and all-trans-retinoic acid was significantly correlated with younger age (less than 1 year; P = 0.0039), non-stage 4 diseases (P = 0.0023), a single copy of N-myc (P = 0.027), and high levels of TRK-A expression (P = 0.0062). To examine the expression levels of GFR alpha-1, we cloned a short form of the human GFR alpha-1 gene with a 15-bp deletion by screening a human adult substantia nigra cDNA library. Many primary neuroblastomas expressed c-Ret, GFR alpha-1, and GFR alpha-2 as well as their ligands, GDNF and NTN, suggesting the presence of a paracrine or autocrine signaling system within the tumor tissue. The effect of NTN on primary culture cells of neuroblastoma was similar to that of GDNF, These imply that the GDNF(NTN)/c-Ret/GFR alpha-1(GFR alpha-2) signaling may have an important role in regulating the growth, differentiation, and cell death of neuroblastomas.

食道癌における新規癌抑制遺伝子p73の検討

二村好憲, 一宮慎吾, 砂原正男, 穴倉朋胤, 崎山樹, 関直彦, 大平美紀, 藤森実, 安達亙, 天野純, 中川原章

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 99 ( 0 ) 378 - 378 1998.03

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大腸癌におけるp73(p53ホモログ)の遺伝子変異

砂原正男, 一宮慎吾, 宍倉朋胤, 二村好憲, 高田尚幸, 安達亙, 佐藤裕二, 天野純, 藤堂省, 崎山樹, 中川原章

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 99 ( 0 ) 498 - 498 1998.03

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乳癌における新規p53関連遺伝子p73の検討

穴倉朋嵐, 一宮慎吾, 砂原正男, 二村好憲, 宮内充, 山本尚人, 鈴木正人, 中嶋伸之, 崎山樹, 中川原章

日本外科学会雑誌 ( 一般社団法人日本外科学会 ) 99 ( 0 ) 339 - 339 1998.03

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Ectopic expression of DAN enhances the retinoic acid-induced neuronal differentiation in human neuroblastoma cell lines

Y Nakamura, T Ozaki, S Ichimiya, A Nakagawara, S Sakiyama

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC ) 243 ( 3 ) 722 - 726 1998.02

Retinoic acid (RA) plays a major role in neuronal cell differentiation. Neuroblastoma cells differentiate in vitro by extending neurites and forming ganglion-like aggregates in response to Rk In the present study, we have examined a biological role(s) of-DAN in the regulation of RA-mediated cellular differentiation in neuroblastoma cells. RTBM1 and SH-SY5Y cells undergo marked morphological changes associated with a remarkable induction of DAN gene expression when exposed to Rk By transfecting an expression vector harboring a rat DAN cDNA into SH-SY5Y sells, we have obtained two independent transfectants which express a large amount of DAN. The forced expression of DAN gene enhanced the neurite extension in the presence of RA, suggesting that DAN gene product might contain some regulatory role(s) in the RA-induced cellular differentiation in neuroblastoma cells. (C) 1998 Academic Press.

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RT1.P, rat class Ib genes related to mouse TL: evidence that CD1 molecules but not authentic TL antigens are expressed by rat thymus

A Matsuura, S Takayama, M Kinebuchi, Y Hashimoto, K Kasai, D Kozutsumi, S Ichimiya, R Honda, T Natori, K Kikuchi

IMMUNOGENETICS ( SPRINGER VERLAG ) 46 ( 4 ) 293 - 306 1997.08

CD1 and TL were once thought to be genetic homologues because of their thymus-specific expression. We investigated their equivalents in the rat to clarify whether their structure and pattern of expression are conserved in rodents. Two rat class Ib genes, containing 3' sequences very similar to mouse TL, were identified and designated RT1.P. Neither of them, however, can encode ordinary class I molecules due to the accumulation of harmful mutations in the 5' regions that are unique to RT1.P, while the 3' TL-like regions still retain protein-coding capacity. Comparison of the structural organization of three types of TL family genes, which include mouse T3/T18-encoding TL antigens, mouse T1/T16, and rat RT1.P1/P2 pseudogenes, revealed the presence of a clear demarcation between the type-specific and TL-specific sequences at intron 3. This finding suggests that recombination plays an important role in creating the TL family genes in rodents. Characteristic features of TL, such as a low level of polymorphism and linkage to the major histocompatibility complex, were also observed in the rat. On the other hand, rat CD1 molecules were expressed at a high level on the surface of thymocytes. Absence of authentic TL antigens and thymic expression of CD id molecules in the rat suggest the plasticity and conservation of class Ib genes in rodent evolution. Functions of TL may be substituted with CD1 or other class Ib molecules expressed by rat thymus.

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Localization of rat CD1 transcripts and protein in rat tissues - An analysis of rat CD1 expression by in situ hybridization and immunohistochemistry

K Kasai, A Matsuura, K Kikuchi, Y Hashimoto, S Ichimiya

CLINICAL AND EXPERIMENTAL IMMUNOLOGY ( BLACKWELL SCIENCE LTD ) 109 ( 2 ) 317 - 322 1997.08

CD1 molecules are cell surface glycoproteins non-covalently associated with Pz-microglobulin. Recently, functional features of the CD1 family such as a target ligand and an antigen-presenting structure for T cells have been reported. In the current study, tissue distribution of rat CD1 was analysed by in situ hybridization (ISH) in conjunction with immunohistochemistry to clarify the precise localization of both transcripts and proteins. CD1 transcripts were detected by ISH in a variety of organs: spleen, thymus, liver, lung, heart, kidney, small intestine and skin. In most organs, CD1 immunoreactivity paralleled the amount of CD1 mRNA expression and was localized in the same regions as its gene transcripts. However, there was a clear difference between the level of rat CD1 transcription and protein expression in the small intestine. CD1 mRNA was detected in the enterocytes of crypts of Lieberkuhn, but not in those of the intestinal villi, while immunoreactivity of CD1 protein was observed in the cells of the intestinal villi but not in those of crypts of Lieberkuhn. This suggests that CD1 gene transcription occurs in the enterocytes of intestinal crypts, and that as the cells of intestinal crypts migrate from the crypts to the intestinal villi, CD1 proteins are synthesized and accumulated in the intestinal villi. Such CD1 expression in the enterocytes appears to be consistent with migration-associated differentiation, and suggests that rat CD1 may take part in mucosal immunity as a first line of defence. In addition, clear cell membrane CD1 immunoreactivity on lymphoid cells raises the possibility that intercellular interaction via rat CD1 and T cell receptors may be involved in both lymphoid cell differentiation and immunoregulation.

Rat CD1 antigen: Structure, expression and function

A Matsuura, Y Hashimoto, M Kinebuchi, K Kasai, S Ichimiya, S Katabami, H Chen, T Shimizu, K Kikuchi

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC ) 29 ( 3 ) 1705 - 1706 1997.05

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G-9 GDNFによる神経芽腫の分化誘導およびRet受容体を介するシグナル伝達の解析(神経芽腫(1))

菱木知郎, 高橋英世, 大沼直躬, 田沼政裕, 一宮慎吾, 近藤和博, 二村好憲, 尾崎俊文, 中川原章

日本小児外科学会雑誌 ( 特定非営利活動法人日本小児外科学会 ) 33 ( 3 ) 542 - 542 1997.05

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Identification and chromosome assignment of a human gene encoding a novel phosphatidylinositol-3 kinase

Naohiko Seki, Yoshinori Nimura, Miki Ohira, Toshiyuki Saito, Shingo Ichimiya, Nobuo Nomura, Akira Nakagawara

DNA Research 4 355 - 358 1997.01

We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

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Veno-occlusive disease of the liver following bone marrow transplantation: A clinical-pathological study of autopsy cases

K Watanabe, H Iwaki, M Satoh, T Ikeda, S Ichimiya, N Suzuki, T Kudoh, T Oda, A Matsuura, M Mori, K Kikuchi

ARTIFICIAL ORGANS ( BLACKWELL SCIENCE INC ) 20 ( 10 ) 1145 - 1150 1996.10

A total of 28 patients with leukemia underwent intensive therapy with megadose chemotherapy and stem cell transplantation. Of 16 patients who had busulfan therapy, 2 developed the clinical syndrome of veno-occlusive disease of the liver (VOD) characterized by progressive abnormalities in liver function, abdominal pain, and ascites. Postmortem findings showed VOD of the central and sublobular hepatic vein. Immunohistochemical analysis using anti-actin and CD34 mAb was employed in an attempt to clarify the process of fibrosis. Actin positive cells had accumulated around the central vein. Ito cell activation might be responsible for secondary fibrosis following endothelial injury. We report autopsy findings and immunohistochemical results.

Glial cell-derived neurotrophic factor (GDNF) による神経芽細胞腫の分化誘導とc-Retチロシンキナーゼのシグナル伝達について

菱木知郎, 近藤和博, 二村好憲, 一宮慎吾, 崎山樹, 中川原章

神経化学 35 ( 3 ) 494 - 495 1996.09

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Thioredoxin dependent peroxidase(TPx)のcDNAクローニングとその生物学的意義

一宮慎吾, GREENE Mark I, 中川原章, 崎山樹

日本分子生物学会年会プログラム・講演要旨集 19 489 - 489 1996.08

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IDENTIFICATION OF THE FUNCTIONAL CLASS-I GENES OF THE RAT BY CDNA CLONING

R HONDA, A MATSUURA, S TAKAYAMA, S ICHIMIYA, Y ITOH, K KIKUCHI

TRANSPLANTATION PROCEEDINGS ( APPLETON & LANGE ) 27 ( 2 ) 1515 - 1515 1995.04

A MONOCLONAL-ANTIBODY REACTS WITH T-CELLS AND NOVEL SUBPOPULATION OF RAT BONE-MARROW CELLS

A MATSUURA, M KINEBUCHI, S ICHIMIYA, M SHEN, Y HASHIMOTO, S KATABAMI, K KIKUCHI

TRANSPLANTATION PROCEEDINGS ( APPLETON & LANGE ) 27 ( 2 ) 1509 - 1510 1995.04

STRUCTURAL-ANALYSIS OF THE RAT HOMOLOG OF CD1 - EVIDENCE FOR EVOLUTIONARY CONSERVATION OF THE CD1D CLASS AND WIDESPREAD TRANSCRIPTION BY RAT-CELLS

S ICHIMIYA, K KIKUCHI, A MATSUURA

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 153 ( 3 ) 1112 - 1123 1994.08

The cDNA encoding the rat homologue of CD1 was isolated and the complete nucleotide sequence was determined. It contained an open reading frame of 1008 bp that was capable of encoding a polypeptide with 336 amino acids composed of hydrophobic leader and transmembrane sequences, three extracellular domains, and 5' and 3' untranslated sequences. Comparison of the amino acid sequence of rat CD1 with those of other species revealed that it showed the highest similarity to mouse CD1, which belongs to the CD1D class of the CD1 system and is distinct from the classic CD1 class including CD1a, CD1b, and CD1c expressed primarily on human thymocytes and some dendritic cells. Widespread transcription of rat CD1 was readily detected by Northern blot analysis in nonlymphoid organs, including the liver, kidney, and heart, as well as in lymphoid organs, including the thymus, lymph node, and spleen. Intestinal expression was also demonstrated by the more sensitive reverse transcription-PCR method. Immunoprecipitation with a rabbit anti-rat CD1 Ab showed that rat CD1 was expressed on the cell surface as a beta(2)-microglobulin-associated heterodimer. Southern blot analysis of inbred rat strains suggested that rat CD1 shows limited polymorphism and that only one CD1 gene is detectable in the F344 rat genome. These results provide evidence for the conservation of CD1D class through mammalian evolution and an apparent lack of the classic CD1 class genes in rodents. Functional similarity of rodent CD1 is implied.

STRUCTURAL-ANALYSIS OF THE CD3-ZETA-ETA LOCUS OF THE RAT - EXPRESSION OF ZETA BUT NOT ETA-TRANSCRIPTS BY RAT T-CELLS

Y ITOH, A MATSUURA, M KINEBUCHI, R HONDA, S TAKAYAMA, S ICHIMIYA, S KON, K KIKUCHI

JOURNAL OF IMMUNOLOGY ( AMER ASSOC IMMUNOLOGISTS ) 151 ( 9 ) 4705 - 4717 1993.11

We analyzed the structure and pattern of expression of rat TCR zeta- and eta-chains to investigate if these components function in activation and development of rat T cells. The rat zeta cDNA contained the complete open reading frame coding for a polypeptide of 164 amino acids and the 5' and 3' noncoding sequences. Comparison of the amino acid sequence to those of mouse and human counterparts revealed a high degree of similarity, more than 85% homology among all three species except for the signal peptide, which was especially high in the cytoplasmic domain including the nucleotide binding site and the possible tyrosine phosphorylation sites. Furthermore, we determined the nucleotide sequences of a rat genomic eta-like sequence located in the 3' region of the rat zeta-gene. Although it showed a high level of nucleotide similarity to mouse and human counterparts, 90.4 and 78.9%, respectively, the deduced polypeptide was very short (only 28 residues) and markedly divergent from the mouse and human eta-specific polypeptides due to frameshift mutations. Transcription of rat zeta was shown to be highly restricted to T cells; abundantly in thymocytes and scarcely in peripheral T cells. Surprisingly, the rat eta transcript could not be detected in any rat tissues so far tested by Northern blot analysis and even by the sensitive reverse transcription-polymerase chain reaction method, whereas it was readily detected in mouse thymus. These findings suggest that the zeta-chain has conserved roles in TCR assembly and TCR-mediated signaling. However, the eta-chain seems not to be indispensable because of its structural diversity among these three species characterized to date and the apparent lack of eta expression in the rat.

MOLECULAR-CLONING OF RAT CLASS-I GENES RELATED TO THE MOUSE TL GENE SUBFAMILY OF THE MAJOR HISTOCOMPATIBILITY COMPLEX

A MATSUURA, S TAKAYAMA, D KOZUTSUMI, S ICHIMIYA, R HONDA, M SHEN, T NATORI, K KIKUCHI

TRANSPLANTATION PROCEEDINGS ( ELSEVIER SCIENCE INC ) 25 ( 5 ) 2754 - 2755 1993.10

Characterization of Rat CD1 Antigen with a Polyclonal Antibody Against a Recombinant Protein Produced by the Prokaryotic Expression System

一宮慎吾, 松浦晃洋

札幌医学雑誌 = The Sapporo medical journal ( 札幌医科大学 ) 62 ( 4 ) 225 - 234 1993.08

CD1, the first cluster of differentiation antigens, is a family of cell surface glycoproteins which are noncovalently associated with β2-microglobulin. Although the structure of CD1 is similar to that of major histocompatibility complex class I molecules and cytolytic T cell lines specific for CD1 molecules have been established, which suggest that CD1 antigens may play a role as a presenting molecule, the actual function of CD1 is presently unknown. We have analyzed and identified the rat homologue of CD1 in attempt to confirm the function of CD1 and whether it has been conserved in evolution. The rat CD1 gene was categorized into the CD1D class. In this report, we produced a specific rabbit antiserum to rat CD1 by the prokaryotic expression system and investigated the expression of its antigen. According to FACS and immunohistochemical analyses with the polyclonal antibody, rat CD1 antigen was expressed on the surface of thymocytes and spleen cells, and was detected in hepatic tissue. The expression level of CD1D class in lymphoid organs varied among species. The functional significance of these results is discussed.

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ESTABLISHMENT OF APOPTOSIS-INDUCING MONOCLONAL-ANTIBODY 2D1 AND 2D1-RESISTANT VARIANTS OF HUMAN T-CELL LINES

S TAKAHASHI, N SATO, S TAKAYAMA, S ICHIMIYA, M SATOH, N HYAKUMACHI, K KIKUCHI

EUROPEAN JOURNAL OF IMMUNOLOGY ( WILEY ) 23 ( 8 ) 1935 - 1941 1993.08

A monoclonal antibody (mAb), 2D1 (IgM), was identified for its anti-proliferative effect on human T leukemia cell line, SUP-T13. The cells bound with 2D1 showed DNA ladder patterns of oligonucleosomes, demonstrating apoptosis. Peripheral mononuclear cells activated by phytohemagglutinin or OKT3 induced expression of 2D1 antigen and were growth-inhibited by the antibodies. Among the cell lines tested, T cell lines tended to be growth-inhibited by the antibodies. Epstein-Barr virus-transformed B cells were reactive with 2D1, but were not growth-inhibited by the antibodies. We established stable 2D1-resistant variants LAC2D1R and JKT2D1R from the original SUP-T13 and Jurkat T cell lines, respectively.These variant cells demonstrated phenotypes identical to the original cells, including reactivity to 2D1 and expression of cytoplasmic Bc1-2 protein. The 2D1-resistant cells were as sensitive as the original cells to the other apoptosis-inducing stimuli, such as gamma-irradiation or calcium ionophore A23187. However, the 2D1-resistant variants were also insensitive to anti-Fas, another apoptosis-inducing mAb. Binding of 2D1 was blocked by anti-Fas mAb, suggesting that 2D1 reacts with an epitope of human Fas molecules. The present results demonstrate that a 2D1-reactive, but not 2D1-sensitive, population may exist in highly 2D1-sensitive human leukemia T cells and that pairs of 2D1-sensitive and 2D1-resistant cells are useful in the biochemical analysis of Fas-mediated apoptosis in human T cells.

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