 | 桒原 一彦 （クワハラ カズヒコ）
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Last Updated :2024/04/25
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ヒト発癌機構を病理学的に解析する。
研究者情報
学位
ホームページURL
J-Global ID
研究キーワード
- 抗体医薬 転写共役型DNA傷害 乳癌
現在の研究分野（キーワード）
- ヒト発癌機構を病理学的に解析する。
研究分野
経歴
- 2022年04月 - 現在 近畿大学近畿大学病院病理診断科講師
- 2021年04月 - 2022年03月 藤田医科大学 医学部病理診断学講師
- 2018年04月 - 2021年03月 藤田医科大学 医学部病理診断学助教
- 2016年08月 - 2018年03月 新潟大学 大学院医歯学総合研究科分子細胞病理学分野助教、講師
- 2016年02月 - 2016年07月 弘前大学医学部附属病院病理部医員
- 2014年04月 - 2016年01月 愛知県がんセンター研究所腫瘍免疫学部室長
- 1999年04月 - 2014年03月 熊本大学 大学院生命科学研究部 感染・免疫学講座免疫学分野助手、講師、准教授
- 2001年12月 - 2005年03月 科学技術振興機構 さきがけ研究21「認識と形成」領域兼任研究者
- 1996年01月 - 1998年12月 日本学術振興会特別研究員（PD）
- 1994年04月 - 1995年12月 鳥取大学医学部生命科学科免疫学講座助手
- 1989年06月 - 1990年03月 佐賀医科大学 医学部附属病院外科学研修医
学歴
所属学協会
研究活動情報
論文
- Yasuhiro Sakai; Kazuhiko KuwaharaPathology International 2024年03月
- Kazuhiko KuwaharaInternational Journal of Molecular Sciences 2023年11月
- Kazuhiko KuwaharaInternational Journal of Molecular Sciences 2023年11月
- Shu Kato; Yasuhiro Sakai; Asako Okabe; Yoshiaki Kawashima; Kazuhiko Kuwahara; Kazuya Shiogama; Masato Abe; Hiroyasu Ito; Shin'ichiro MorimotoJournal of clinical medicine 11 1 2022年01月 [査読有り]
Sarcoidosis is a rare disease of isolated or diffuse granulomatous inflammation. Although any organs can be affected by sarcoidosis, cardiac sarcoidosis is a fatal disorder, and it is crucial to accurately diagnose it to prevent sudden death due to dysrhythmia. Although endomyocardial biopsy is invasive and has limited sensitivity for identifying granulomas, it is the only modality that yields a definitive diagnosis of cardiac sarcoidosis. It is imperative to develop novel pathological approaches for the precise diagnosis of cardiac sarcoidosis. Here, we aimed to discuss commonly used diagnostic criteria for cardiac sarcoidosis and to summarize useful and novel histopathologic criteria of cardiac sarcoidosis. While classical histologic observations including noncaseating granulomas and multinucleated giant cells (typically Langhans type) are the most important findings, others such as microgranulomas, CD68+ CD163- pro-inflammatory (M1) macrophage accumulation, CD4/CD8 T-cell ratio, Cutibacterium acnes components, lymphangiogenesis, confluent fibrosis, and fatty infiltration may help to improve the sensitivity of endomyocardial biopsy for detecting cardiac sarcoidosis. These novel histologic findings are based on the pathology of cardiac sarcoidosis. We also discussed the principal histologic differential diagnoses of cardiac sarcoidosis, such as tuberculosis myocarditis, fungal myocarditis, giant cell myocarditis, and dilated cardiomyopathy. - Naomi Gondo; Yasuhiro Sakai; Zhenhuan Zhang; Yukari Hato; Kiyotaka Kuzushima; Suchada Phimsen; Yoshiaki Kawashima; Makoto Kuroda; Motoshi Suzuki; Seiji Okada; Hiroji Iwata; Tatsuya Toyama; Andri Rezano; Kazuhiko KuwaharaLaboratory investigation; a journal of technical methods and pathology 101 8 1048 - 1059 2021年08月 [査読有り]
Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations. - Yasuhiro Sakai; Suchada Phimsen; Seiji Okada; Kazuhiko KuwaharaExperimental hematology 2020年08月 [査読有り]
Germinal center-associated nuclear protein (GANP) is a unique and multifunctional protein that plays a critical role in cell biology, neurodegenerative disorders, immunohematology, and oncogenesis. GANP is an orthologue of Saccharomyces Sac3, one of the components of the transcription export 2 (TREX-2) complex and a messenger RNA (mRNA) nuclear export factor. GANP is widely conserved in all mammals, including humans. Although GANP was originally discovered as a molecule upregulated in the germinal centers of secondary lymphoid follicles in peripheral lymphoid organs, it is expressed ubiquitously in many tissues. It serves numerous functions, including making up part of the mammalian TREX-2 complex; mRNA nuclear export via nuclear pores; prevention of R-loop formation, genomic instability, and hyper-recombination; and B-cell affinity maturation. In this review, we first overview the extensive analyses that have revealed the basic functions of GANP and its ancestor molecule Sac3, including mRNA nuclear export and regulation of R-loop formation. We then describe how aberrant expression of GANP is significantly associated with cancer development. Moreover, we discuss a crucial role for GANP in B-cell development, especially affinity maturation in germinal centers. Finally, we illustrate that overexpression of GANP in B cells leads to lymphomagenesis resembling Hodgkin lymphoma derived from germinal center B cells, and that GANP may be involved in transdifferentiation of B cells to macrophages, which strongly affects Hodgkin lymphomagenesis. - Qidi Wang; Lianfeng Zhang; Kazuhiko Kuwahara; Li Li; Zijie Liu; Taisheng Li; Hua Zhu; Jiangning Liu; Yanfeng Xu; Jing Xie; Hiroshi Morioka; Nobuo Sakaguchi; Chuan Qin; Gang LiuACS infectious diseases 6 5 1284 - 1285 2020年05月 [査読有り]
- Yasuhiro Sakai; Andri Rezano; Seiji Okada; Takahiro Ohtsuki; Yoshiaki Kawashima; Tetsuya Tsukamoto; Motoshi Suzuki; Michinori Kohara; Motohiro Takeya; Nobuo Sakaguchi; Kazuhiko KuwaharaCancers 12 1 2020年01月 [査読有り]
Hodgkin lymphoma (HL) is one of the most difficult neoplasms in terms of cytopathological research owing to the lack of established cytological murine models. Although HL is believed to be of lymphoid germinal center B-cell origin, HL cells exhibit unique biphenotypic characteristics of B cells and macrophages. B-cell/macrophage biphenotypic cells have also been identified in the spleen of Lyn-deficient mice. Moreover, Lyn-targeting germinal center-associated nuclear protein (GANP)-transgenic mice (Ig-ganpTg mice) spontaneously develop a lymphoid tumor. We aimed to investigate whether the lymphoid tumor developed in Ig-ganpTg mice exhibit biphenotypic characteristics of B cells/macrophages that correspond to human HL. Here, we demonstrated GANP overexpression in human HL cells and found that it may regulate transdifferentiation between B cells and macrophages. We also demonstrated that tumors were comparable with B-cell/macrophage biphenotypic Hodgkinoid lymphomas. The tumor cells expressed macrophage-related F4/80, CD68, and CD204 as well as cytoplasmic B220 and µ-/κ-chains; in addition, these cells exhibited phagocytic activity. These cells also expressed transcripts of CD30; c-fms; and the cytokines monocyte chemoattractant protein (MCP)-1, MCP-5, RANTES, tumor necrosis factor-α and thrombopoietin associated with macrophages as well as granulocyte/macrophage colony-stimulating factor, interleukin (IL)-4, IL-10, IL-12, and IL-13. Ig-ganpTg mice represent a novel cytological model for the study of cytopathological etiology and oncogenesis of HL. - Investigation of GANP and the pathogenesis of breast cancers.Sakai, Y; Kuwahara, K2019 2019 19 - 21 2019年04月 [招待有り]
- Vaeteewoottacharn K; Kariya R; Pothipan P; Fujikawa S; Pairojkul C; Waraasawapati S; Kuwahara K; Wongkham C; Wongkham S; Okada STranslational oncology 12 2 217 - 225 2019年02月 [査読有り]
The involvement of chronic inflammation in cholangiocarcinoma (CCA) progression is well established. Cluster of differentiation 47 (CD47) is mutually expressed in various cancers and serves as a protective signal for phagocytic elimination. CD47 signaling blockage is a recent treatment strategy; however, little is known regarding CD47 in CCA. Therefore, the potential use of CD47 targeting in CCA was focused. CD47 was highly expressed in CCA compared to hepatocellular carcinoma (HCC). Disturbance of CD47-signal regulatory protein-α (SIRPα) interaction by blocking antibodies promoted the macrophage phagocytosis. The therapeutic potential of anti-CD47 therapy was demonstrated in liver metastatic model; alleviation of cancer colonization together with dense macrophage infiltrations was observed. The usefulness of anti-CD47 was emphasized by its universal facilitating macrophage activities. Moreover, increased production of inflammatory cytokines, such as IL-6 and IL-10, in macrophage exposed to CCA-conditioned media suggested that CCA alters macrophages toward cancer promotion. Taken together, interfering of CD47-SIRPα interaction promotes macrophage phagocytosis in all macrophage subtypes and consequently suppresses CCA growth and metastasis. The unique overexpression of CD47 in CCA but not HCC offers an exceptional opportunity for a targeted therapy. CD47 is therefore a novel target for CCA treatment. - Kotani H; Ito H; Kuwahara K; Kuzushima K; Iwata H; Tsunoda N; Nagino M; Matsuo KBreast cancer (Tokyo, Japan) 2019年02月 [査読有り]
- Kulthida Vaeteewoottacharn; Ryusho Kariya; Phattarin Pothipan; Sawako Fujikawa; Chawalit Pairojkul; Sakda Waraasawapati; Kazuhiko Kuwahara; Chaisiri Wongkham; Sopit Wongkham; Seiji OkadaTranslational oncology 12 2 217 - 225 2019年02月 [査読有り]
The involvement of chronic inflammation in cholangiocarcinoma (CCA) progression is well established. Cluster of differentiation 47 (CD47) is mutually expressed in various cancers and serves as a protective signal for phagocytic elimination. CD47 signaling blockage is a recent treatment strategy; however, little is known regarding CD47 in CCA. Therefore, the potential use of CD47 targeting in CCA was focused. CD47 was highly expressed in CCA compared to hepatocellular carcinoma (HCC). Disturbance of CD47-signal regulatory protein-α (SIRPα) interaction by blocking antibodies promoted the macrophage phagocytosis. The therapeutic potential of anti-CD47 therapy was demonstrated in liver metastatic model; alleviation of cancer colonization together with dense macrophage infiltrations was observed. The usefulness of anti-CD47 was emphasized by its universal facilitating macrophage activities. Moreover, increased production of inflammatory cytokines, such as IL-6 and IL-10, in macrophage exposed to CCA-conditioned media suggested that CCA alters macrophages toward cancer promotion. Taken together, interfering of CD47-SIRPα interaction promotes macrophage phagocytosis in all macrophage subtypes and consequently suppresses CCA growth and metastasis. The unique overexpression of CD47 in CCA but not HCC offers an exceptional opportunity for a targeted therapy. CD47 is therefore a novel target for CCA treatment. - Classic Hodgkin lymphoma with osseous involvement mimicking Langerhans cell histiocytosis in a childKazuhiko Kuwahara; Ko Kudo; Akiko Yashima-Abo; Kosuke Katayama; Keiko Kojima; Kiyoshi Tone; Etsuro Ito; Atsuko Nakazawa; Hideto Iwafuchi; Akira KuroseHuman Pathology 77 147 - 151 2018年07月 [査読有り]
Hodgkin lymphoma (HL) commonly presents superficial lymphadenopathy. In addition, HL cells can arise in various organs including the liver and spleen as an extranodal lymphoma. HL in bone is unusual at the initial diagnosis, although some cases show late-stage localization of lymphoma cells to bone. We report the rare case of a young patient with cranial bone classic HL, presumably originating from the skull without any involvement of lymph nodes. As the main clinical manifestation was only tumor mass in the skull without osteoscopic pain, the tentative diagnosis of Langerhans cell histiocytosis was histologically confirmed by an excisional biopsy. Before the final pathological diagnosis as classic HL, we noticed several small lesions in extranodal regions through systemic surveys, suggesting that the cranial lesion appeared antecedent to those lesions. This is a rare and instructive case of cranial bone HL for which a histological diagnosis has been meticulously made. - Qidi Wang; Lianfeng Zhang; Kazuhiko Kuwahara; Li Li; Zijie Liu; Taisheng Li; Hua Zhu; Jiangning Liu; Yanfeng Xu; Jing Xie; Hiroshi Morioka; Nobuo Sakaguchi; Chuan Qjn; Gang LiuACS INFECTIOUS DISEASES 2 5 361 - 376 2016年05月 [査読有り]
Severe acute respiratory syndrome (SARS) is caused by a coronavirus (SARS-CoV) and has the potential to threaten global public health and socioeconomic stability. Evidence of antibody-dependent enhancement (AIDE) of SARS-CoV infection in vitro and in non-human primates clouds the prospects for a safe vaccine. Using antibodies from SARS patients, we identified and characterized SARS-CoV B-cell peptide epitopes with disparate functions. In rhesus macaques, the spike glycoprotein peptides S471-503, S604-625, and S1164-1191 elicited antibodies that efficiently prevented infection in non-human primates. In contrast, peptide S597-603 induced antibodies that enhanced infection both in vitro and in non-human primates by using an epitope sequence-dependent (ESD) mechanism. This peptide exhibited a high level of serological reactivity (64%), which resulted from the additive responses of two tandem epitopes (S597-603 and S604-625) and a long-term human B-cell memory response with antisera from convalescent SARS patients. Thus, peptide-based vaccines against SARS-CoV could be engineered to avoid ADE via elimination of the S597-603 epitope. We provide herein an alternative strategy to prepare a safe and effective vaccine for ADE of viral infection by identifying and eliminating epitope sequence-dependent enhancement of viral infection. - Kazuhiko Kuwahara; Mutsuko Yamamoto-Ibusuki; Zhenhuan Zhang; Suchada Phimsen; Naomi Gondo; Hiroko Yamashita; Toru Takeo; Naomi Nakagata; Daisuke Yamashita; Yoshimi Fukushima; Yutaka Yamamoto; Hiroji Iwata; Hideyuki Saya; Eisaku Kondo; Keitaro Matsuo; Motohiro Takeya; Hirotaka Iwase; Nobuo SakaguchiCANCER SCIENCE 107 4 469 - 477 2016年04月 [査読有り]
Human chromosome 21 is known to be associated with the high risk of hematological malignancy but with resistance to breast cancer in the study of Down syndrome. In human cancers, we previously observed the significant alterations of the protein expression encoded by the ganp/MCM3AP gene on human chromosome 21q22.3. Here, we investigated GANP protein alterations in human breast cancer samples (416 cases) at various stages by immunohistochemical analysis. This cohort study clearly showed that expression of GANP is significantly decreased in human breast cancer cases with poor prognosis as an independent risk factor (relapse-free survival, hazard ratio = 2.37, 95% confidence interval, 1.27-4.42, P = 0.007 [univariate analysis]; hazard ratio = 2.70, 95% confidence interval, 1.42-5.13, P = 0.002 [multivariate analysis]). To investigate whether the altered GANP expression is associated with mammary tumorigenesis, we created mutant mice that were conditionally deficient in the ganp/MCM3AP gene using wap-cre recombinase transgenic mice. Mammary gland tumors occurred at a very high incidence in female mammary gland-specific GANP-deficient mice after severe impairment of mammary gland development during pregnancy. Moreover, tumor development also occurred in female post parous GANP-heterodeficient mice. GANP has a significant role in the suppression of DNA damage caused by estrogen in human breast cancer cell lines. These results indicated that the GANP protein is associated with breast cancer resistance. - Masahiro Kitabatake; Miho Soma; Tianli Zhang; Kazuhiko Kuwahara; Yoshimi Fukushima; Takuya Nojima; Daisuke Kitamura; Nobuo SakaguchiJOURNAL OF IMMUNOLOGY 194 4 1480 - 1488 2015年02月 [査読有り]
Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black 3 New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody-secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells. - Hiroki Goto; Yuki Kojima; Kouki Matsuda; Ryusho Kariya; Manabu Taura; Kazuhiko Kuwahara; Hirokazu Nagai; Harutaka Katano; Seiji OkadaEUROPEAN JOURNAL OF CANCER 50 10 1836 - 1846 2014年07月 [査読有り]
Background: Recently, the critical role of CD47 on the surface of resistant cancer cells has been proposed in their evasion of immunosurveillance. Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion in body cavities, especially in advanced acquired immunodeficiency syndrome (AIDS). PEL is resistant to conventional chemotherapy and has a poor prognosis. In this study, we evaluated the effect of anti-CD47 antibody (Ab) on PEL in vitro and in vivo. Methods: Surface CD47 of PEL cell lines was examined by flow cytometry. Efficacy of knocking down CD47 or anti-CD47 Ab-mediated phagocytosis against PEL was evaluated using mouse peritoneal macrophages and human macrophages in vitro. Primary PEL cells were injected intraperitoneally into NOD/Rag-2/Jak3 double-deficient (NRJ) mice to establish a direct xenograft mouse model. Results: Surface CD47 of PEL cell lines was highly expressed. Knocking down CD47 and anti-CD47 Ab promoted phagocytic activities of macrophages in a CD47 expression-dependent manner in vitro. Treatment with anti-CD47 Ab inhibited ascite formation and organ invasion completely in vivo compared with control IgG-treated mice. Conclusion: CD47 plays the pivotal role in the immune evasion of PEL cells in body cavities. Therapeutic antibody targeting of CD47 could be an effective therapy for PEL. (C) 2014 Elsevier Ltd. All rights reserved. - Buqing Ye; Zhonghua Dai; Benyu Liu; Rui Wang; Chong Li; Guanling Huang; Shuo Wang; Pengyan Xia; Xuan Yang; Kazuhiko Kuwahara; Nobuo Sakaguchi; Zusen FanSTEM CELLS 32 3 623 - 635 2014年03月 [査読有り]
Self-renewal and differentiation are the hallmarks of embryonic stem cells (ESCs). However, it is largely unknown about how the pluripotency is regulated. Here we demonstrate that Pcid2 is required for the maintenance of self-renewal both in mouse and human ESCs. Pcid2 plays a critical role in suppression of ESC differentiation. Pcid2 deficiency causes early embryonic lethality before the blastocyst stage. Pcid2 associates with EID1 and is present in the CBP/p300-EID1 complex in the ESCs. We show that MDM2 is an E3 ligase for K48-linked EID1 ubiquitination for its degradation. For the maintenance of self-renewal, Pcid2 binds to EID1 to impede the association with MDM2. Then EID1 is not degraded to sustain its stability to block the HAT activity of CBP/p300, leading to suppression of the developmental gene expression. Collectively, Pcid2 is present in the CBP/p300-EID1 complex to control the switch balance of mouse and human ESCs through modulation of EID1 degradation. Stem Cells 2014;32:623-635 - Haru Ogiwara; Fumihiko Yasui; Keisuke Munekata; Asako Takagi-Kamiya; Tsubasa Munakata; Namiko Nomura; Futoshi Shibasaki; Kazuhiko Kuwahara; Nobuo Sakaguchi; Yoshihiro Sakoda; Hiroshi Kida; Michinori KoharaAMERICAN JOURNAL OF PATHOLOGY 184 1 171 - 183 2014年01月 [査読有り]
Patients infected with highly pathogenic avian influenza A H5N1 viruses (H5N1 HPAIV) show diffuse alveolar damage. However, the temporal progression of tissue damage and repair after viral infection remains poorly defined. Therefore, we assessed the sequential histopathological characteristics of mouse lung after intranasal infection with H5N1 HPAIV or H1N1 2009 pandemic influenza virus (H1N1 pdm). We determined the amount and localization of virus in the Lung through IHC staining and in situ hybridization. IHC used antibodies raised against the virus protein and antibodies specific for macrophages, type II pneumocytes, or proliferating cell nuclear antigen. In situ hybridization used RNA probes against both viral RNA and mRNA encoding the nucleoprotein and the hemagglutinin protein. H5N1 HPAIV infection and replication were observed in multiple lung cell types and might result in rapid progression of lung injury. Both type II pneumocytes and macrophages proliferated after H5N1 HPAIV infection. However, the abundant macrophages failed to block the viral attack, and proliferation of type II pneumocytes failed to restore the damaged alveoli. In contrast, mice infected with H1N1 pdm exhibited modest proliferation of type II pneumocytes and macrophages and slight alveolar damage. These results suggest that the virulence of H5N1 HPAIV results from the wide range of cell tropism of the virus, excessive virus replication, and rapid development of diffuse alveolar damage. - Andri Rezano; Kazuhiko Kuwahara; Mutsuko Yamamoto-Ibusuki; Masahiro Kitabatake; Penpak Moolthiya; Suchada Phimsen; Taiji Suda; Shigenobu Tone; Yutaka Yamamoto; Hirotaka Iwase; Nobuo SakaguchiBMC CANCER 13 562 2013年12月 [査読有り]
Background: Genetic BRCA2 insufficiency is associated with breast cancer development; however, in sporadic breast cancer cases, high BRCA2 expression is paradoxically correlated with poor prognosis. Because DSS1, a mammalian component of the transcription/RNA export complex, is known to stabilize BRCA2, we investigated how the expression of DSS1 is associated with clinical parameters in breast cancers. Methods: DSS1 mRNA and p53 protein were examined by RT-PCR and immunohistochemical staining of breast cancer specimens to classify DSS1(high) and DSS1(low) or p53(high) and p53(low) groups. Patient survival was compared using Kaplan-Meier method. DSS1(high) or DSS1(low) breast cancer cells were prepared by retroviral cDNA transfection or DSS1 siRNA on proliferation, cell cycle progression, and survival by flow cytometric analyses with or without anti-cancer drugs. Results: In comparison to patients with low levels of DSS1, high-DSS1 patients showed a poorer prognosis, with respect to relapse-free survival period. The effect of DSS1 was examined in breast cancer cells in vitro. DSS1 high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs, as observed in cell cycle and apoptosis analyses. DSS1 knockdown, however, increased the susceptibility to the DNA-damaging drugs camptothecin and etoposide and caused early apoptosis in p53 wild type MCF7 and p53-insufficient MDA-MB-231 cells. DSS1 knockdown suppresses the proliferation of drug-resistant MDA-MB-231 breast cancer cells, particularly effectively in combination with DNA-damaging agents. Conclusion: Breast cancers with high DSS1 expression have worse prognosis and shorter relapse-free survival times. DSS1 is necessary to rescue cells from DNA damage, but high DSS1 expression increases drug resistance. We suggest that DSS1 expression could be a useful marker for drug resistance in breast cancers, and DSS1 knockdown can induce tumor apoptosis when used in combination with DNA-damaging drugs. - Akira Sakurai; Katsuyoshi Takayama; Namiko Nomura; Tsubasa Munakata; Naoki Yamamoto; Tsuruki Tamura; Jitsuho Yamada; Masako Hashimoto; Kazuhiko Kuwahara; Yoshihiro Sakoda; Yoshihiko Suda; Yukuharu Kobayashi; Nobuo Sakaguchi; Hiroshi Kida; Michinori Kohara; Futoshi ShibasakiPLoS ONE 8 11 e76753 2013年11月 [査読有り]
Immunochromatography (IC) is an antigen-detection assay that plays an important role in the rapid diagnosis of influenza virus because the protocol is short time and easy to use. Despite the usability of IC, the sensitivity is approximately 103 pfu per reaction. In addition, antigen-antibody interaction-based method cannot be used for the detection of influenza viruses with major antigenic change. In this study, we established the use of fluorescent immunochromatography (FLIC) to detect a broad spectrum of H5 subtype influenza A viruses. This method has improved sensitivity 10-100 fold higher than traditional IC because of the use of fluorescent conjugated beads. Our Type-E FLIC kit detected all of the H5 subtype influenza viruses that were examined, as well as recombinant hemagglutinin (HA) proteins (rHAs) belonging to the Eurasian H5 subtype viruses and the Type-N diagnosed North American H5 subtype influenza A viruses. Thus, this kit has the improved potential to detect H5 subtype influenza viruses of different clades with both Type-E and Type-N FLIC kits. Compared with PCR-based diagnosis, FLIC has a strong advantage in usability, because the sample preparation required for FLIC is only mix-and-drop without any additional steps such as RNA extraction. Our results can provide new strategies against the spread and transmission of HPAI H5N1 viruses in birds and mammals including humans. Copyright: © 2013 Sakurai et al. - Atit Silsirivanit; Norie Araki; Chaisiri Wongkham; Kulthida Vaeteewoottacharn; Chawalit Pairojkul; Kazuhiko Kuwahara; Yoshiki Narimatsu; Hiromichi Sawaki; Hisashi Narimatsu; Seiji Okada; Nobuo Sakaguchi; Sopit WongkhamCancer Science 104 10 1278 - 1284 2013年10月 [査読有り]
Early and specific diagnosis is critical for treatment of cholangiocarcinoma (CCA). In this study, a carbohydrate antigen-S27 (CA-S27) monoclonal antibody (mAb) was established using pooled CCA tissue-extract as immunogen. The epitope recognized by CA-S27-mAb was a new Lewis-a (Lea) associated modification of MUC5AC mucin. A Soybean agglutinin/CA-S27-mAb sandwich ELISA to determine CA-S27 in serum was successfully developed. High level of CA-S27 was detected in serum of CCA patients and could differentiate CCA patients from those of gastro-intestinal cancers, hepatomas, benign hepatobiliary diseases and healthy subjects with high sensitivity (87.5%) and high negative predictive value (90.4%). The level of serum CA-S27 was dramatically reduced after tumor removal, indicating tumor origin of CA-S27. Patients with high serum CA-S27 had significantly shorter survivals than those with low serum CA-S27 regardless of serum MUC5AC levels. Fucosyltransferase-III (FUT3) was shown to be a regulator of CA-S27 expression. Suppression of CA-S27 expression with siRNA-FUT3 or neutralization with CA-S27 mAb significantly reduced growth, adhesion, invasion and migration potentials of CCA cells in vitro. In summary, we demonstrate that serum CA-S27, a novel carbohydrate antigen, has potential as diagnostic and prognostic markers for CCA patients. CA-S27 involves in promoting cell growth, adhesion, migration and invasion of CCA cells. © 2013 Japanese Cancer Association. - 原発性滲出性リンパ腫における悪性滲出液抑制のためのCD47-SIRPA標的化(Targeting CD47-SIRPA for the controlling malignant effusion in primary effusion lymphoma)Goto Hiroki; Kariya Ryusho; Matsuda Kouki; Kudo Eriko; Taura Manabu; Kuwahara Kazuhiko; Katano Harutaka; Okada Seiji臨床血液 54 9 1157 - 1157 2013年09月 [査読有り]
- Ken Saito; Nagio Takigawa; Naoko Ohtani; Hidekazu Iioka; Yuki Tomita; Ryuzo Ueda; Junya Fukuoka; Kazuhiko Kuwahara; Eiki Ichihara; Katsuyuki Kiura; Eisaku KondoMolecular cancer therapeutics 12 8 1616 - 28 2013年08月 [査読有り]
Activation of the epidermal growth factor receptor (EGFR) has been observed in many malignant tumors and its constitutive signal transduction facilitates the proliferation of tumors. EGFR-tyrosine kinase inhibitors, such as gefitinib, are widely used as a molecular-targeting agent for the inactivation of EGFR signaling and show considerable therapeutic effect in non-small cell lung cancers harboring activating EGFR mutations. However, prolonged treatment inevitably produces tumors with additional gefitinib-resistant mutations in EGFR, which is a critical issue for current therapeutics. We aimed to characterize the distinct molecular response to gefitinib between the drug-resistant and drug-sensitive lung adenocarcinoma cells in order to learn about therapeutics based on the molecular information. From the quantitative PCR analysis, we found a specific increase in p14(ARF) expression in gefitinib-sensitive lung adenocarcinoma clones, which was absent in gefitinib-resistant clones. Moreover, mitochondria-targeted p14(ARF) triggered the most augmented apoptosis in both clones. We identified the amino acid residues spanning from 38 to 65 as a functional core of mitochondrial p14(ARF) (p14 38-65 a.a.), which reduced the mitochondrial membrane potential and caused caspase-9 activation. The synthesized peptide covering the p14 38-65 a.a. induced growth suppression of the gefitinib-resistant clones without affecting nonneoplastic cells. Notably, transduction of the minimized dose of the p14 38-65 peptide restored the response to gefitinib like that in the sensitive clones. These findings suggest that the region of p14(ARF) 38-65 a.a. is critical in the pharmacologic action of gefitinib against EGFR-mutated lung adenocarcinoma cells and has potential utility in the therapeutics of gefitinib-resistant cancers. - Silsirivanit A; Araki N; Wongkham C; Vaeteewoottacharn K; Pairojkul C; Kuwahara K; Narimatsu Y; Sawaki H; Narimatsu H; Okada S; Sakaguchi N; Wongkham SCancer Sci 2013年 [査読有り]
- Kazuhiko Kuwahara; Teruo Nakaya; Suchada Phimsen; Teppei Toda; Masahiro Kitabatake; Tomohiro Kaji; Toshitada Takemori; Takeshi Watanabe; Nobuo SakaguchiJOURNAL OF IMMUNOLOGY 189 7 3472 - 3479 2012年10月 [査読有り]
Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenylchicken gamma-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs. The Journal of Immunology, 2012, 189: 3472-3479. - Teppei Toda; Kazuhiko Kuwahara; Naoyuki Kondo; Zene Matsuda; Yosuke Maeda; Kazuhiko Maeda; Nobuo SakaguchiIMMUNOBIOLOGY 217 9 864 - 872 2012年09月 [査読有り]
HIV-1 entry into cells is mediated by interactions between the envelope (Env) gp120 and gp41 proteins with CD4 and chemokine receptors via an intermediate called the viral fusion complex (vFC). Here, mAbs were used to find the dynamic changes in expression of antigenic epitopes during vFC formation. A CD4-specific mAb (R275) and anti-vFC mAbs, designated F12-1, F13-6 and F18-4 that recognize the epitopes only appeared by the co-culture of env-transfected 293FT and CD4-transfected 293 cells, were developed by immunizing ganp-gene transgenic mice with an vFC-like structure formed by the same co-culture. The epitopes recognized by the mAbs appeared at different time points during vFC formation: F18-4 appeared first, followed by F13-6, and finally F12-1. The anti-vFC mAbs had little effect on vFC formation or virus neutralization; however, interestingly F12-1 and F18-4 increased exposure of the OKT4-epitope on the domain 3 in the extracellular region of CD4. R275, which recognizes the epitope closely associated with the OKT4-determinant on the domain 3, showed the marked inhibition of vFC formation and viral neutralization activity. The Ab binding to the epitopes appeared during viral membrane fusion might reinforce the appearance of the target epitopes for effective neutralization activity. (C) 2011 Elsevier GmbH. All rights reserved. - Masahiro Kitabatake; Teppei Toda; Kazuhiko Kuwahara; Hideya Igarashi; Mareki Ohtsuji; Hiromichi Tsurui; Sachiko Hirose; Nobuo SakaguchiJOURNAL OF IMMUNOLOGY 189 3 1193 - 1201 2012年08月 [査読有り]
To investigate signals that control B cell selection, we examined expression of G5PR, a regulatory subunit of the serine/threonine protein phosphatase 2A, which suppresses JNK phosphorylation. G5PR is upregulated in activated B cells, in Ki67-negative centrocytes at germinal centers (GCs), and in purified B220(+)Fas(+)GL7(+) mature GC B cells following Ag immunization. G5PR rescues transformed B cells from BCR-mediated activation-induced cell death by suppression of late-phase JNK activation. In G5PR-transgenic (G5PR(Tg)) mice, G5PR overexpression leads to an augmented generation of GC B cells via an increase in non-Ag-specific B cells and a consequent reduction in the proportion of Ag-specific B cells and high-affinity Ab production after immunization with nitrophenyl-conjugated chicken gamma-globulin. G5PR overexpression impaired the affinity-maturation of Ag-specific B cells, presumably by diluting the numbers of high-affinity B cells. However, aged nonimmunized female G5PR(Tg) mice showed an increase in the numbers of peritoneal B-1a cells and the generation of autoantibodies. G5PR overexpression did not affect the proliferation of B-1a and B-2 cells but rescued B-1a cells from activation-induced cell death in vitro. G5PR might play a pivotal role in B cell selection not only for B-2 cells but also for B-1 cells in peripheral lymphoid organs. The Journal of Immunology, 2012, 189: 1193-1201. - Suchada Phimsen; Kazuhiko Kuwahara; Teruo Nakaya; Kazutaka Ohta; Taiji Suda; Andri Rezano; Masahiro Kitabatake; Kulthida Vaeteewoottacharn; Seiji Okada; Shigenobu Tone; Nobuo SakaguchiAPOPTOSIS 17 7 679 - 690 2012年07月 [査読有り]
Cancer cells often contain p53 abnormalities that impair cell-cycle checkpoint progression and cause resistance to various anti-cancer treatments. DNA damage occurs at actively transcribed genes during G1-phase in yeast cells that have a deficient mRNA export capacity. Here, we show that germinal center-associated nuclear protein (GANP), a homologue of yeast Sac3 that is involved in mRNA export, is indispensable for ensuring the stability of human genomic DNA and that GANP knockdown causes apoptosis and necrosis of p53-insufficient cancer cells. Ganp small interfering RNA (siGanp)-induced DNA damage, accompanied by a decrease in the number of cells in S-phase, caused late apoptosis and necrosis in p53-insufficient cancer cells through both caspase-dependent and -independent mechanisms. siGanp effectively induced DNA damage leading to cell death in p53-insufficient cancer cells in vitro and protect the growth of cancer cells transplanted into immunocompromized mice, suggesting that siGanp has potential as a selective treatment for p53-insufficient cancer cells. - Atit Silsirivanit; Norie Araki; Chaisiri Wongkham; Chawalit Pairojkul; Yoshiki Narimatsu; Kazuhiko Kuwahara; Hisashi Narimatsu; Sopit Wongkham; Nobuo SakaguchiCANCER 117 15 3393 - 3403 2011年08月 [査読有り]
BACKGROUND: The incidence of cholangiocarcinoma (CCA) is increasing globally. Currently, there is no powerful marker for the diagnosis of CCA, which has led to late diagnosis and poor patient outcome. This study was designed to establish a new monoclonal antibody (MoAb) for detecting a serum marker associated with CCA. METHODS: Pooled CCA tissue extracts were immunized to germinal center associated nuclear protein (GANP)-transgenic mice. The antibody-producing hybridomas were prepared and initially screened by using an indirect enzyme-linked immunosorbent assay (ELISA). A positive clone that reacted strongly with CCA serum or tumor tissue extract and failed to react with normal human serum and liver extract was selected. RESULTS: An S121 immunoglobulin M MoAb that recognized a novel glycan epitope was obtained. Immunohistochemistry of CCA tissues revealed that the MoAb reacted strongly with hyperplastic/dysplastic and neoplastic bile ducts but not with normal bile ducts. In addition, experiments demonstrated that mucin 5AC (MUC5AC) is a core glycoprotein for the S121 epitope. A sandwich ELISA using soybean agglutinin and an S121 MoAb was developed for detecting S121 reactive antigen in patient sera. The level of serum S121 from patients with CCA was reduced significantly after tumor removal, indicating the tumor origin of this antigen. The test was able to distinguish patients with CCA from healthy individuals, active Opisthorchis viverrini-infected individuals and patients with various gastrointestinal cancers, hepatoma, and benign hepatobiliary diseases with 87.63% sensitivity, 89.58% specificity, an 80.95% positive predictive value, and a 93.47% negative predictive value. Moreover, high serum S121 levels were related to a poor patient outcome. CONCLUSIONS: The sugar antigen recognized by S121 MoAb is a new serum marker for the diagnosis and prognosis of CCA. Cancer 2011;117:3393-403. (C) 2011 American Cancer Society - N. Sakaguchi; K. Maeda; K. KuwaharaSCANDINAVIAN JOURNAL OF IMMUNOLOGY 73 6 520 - 526 2011年06月 [査読有り]
The immune system produces specific antibodies (Ab) against any antigens (Ag) of exogenous and endogenous origins with a diverse repertoire of V-region specificities. The primary V-region repertoire is created by the rearrangement of immunoglobulin (Ig) V-region, D- and J-segments with the insertion of N- and P-sequences during early B cell differentiation. Recent studies revealed that secondary diversification of the IgV-region generated in the peripheral lymphoid organs plays a critical role in the generation of effective Ab production for protection from various pathogens. Naive B cells that react with Ags initiate proliferation and differentiation in the follicular region and create the germinal centres (GCs), where activation-induced cytidine deaminase (AID)-dependent IgV-region somatic hypermutation (SHM) and class-switch recombination generate high-affinity and class-switched mature Ag-specific B cells. Our studies have discovered a 210-kDa nuclear protein, named GC-associated nuclear protein (GANP) that is up-regulated in GC B cells during the T cell-dependent (TD) immune responses. By studying mice with mutant forms of the ganp gene, we demonstrated that GANP is essential for the generation of high-affinity B cells against TD-Ag by affecting SHM at the IgV-regions. GANP is associated with AID in the cytoplasm and the GANP/AID complex is recruited to the nucleus, specifically, the chromatin, and targeted selectively to the IgV-region gene in B cells. GANP augments the access of AID towards IgV-regions in B cells. Here, we review the role of GANP in acquired immunity through the detailed analysis of the molecular mechanism generating SHM specifically at IgV-regions in B cells. - Yuri Kasama; Satoshi Sekiguchi; Makoto Saito; Kousuke Tanaka; Masaaki Satoh; Kazuhiko Kuwahara; Nobuo Sakaguchi; Motohiro Takeya; Yoichi Hiasa; Michinori Kohara; Kyoko Tsukiyama-KoharaBLOOD 116 23 4926 - 4933 2010年12月 [査読有り]
Extrahepatic manifestations of hepatitis C virus (HCV) infection occur in 40%-70% of HCV-infected patients. B-cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with HCV infection. The mechanism by which HCV infection of B cells leads to lymphoma remains unclear. Here we established HCV transgenic mice that express the full HCV genome in B cells (RzCD19Cre mice) and observed a 25.0% incidence of diffuse large B-cell non-Hodgkin lymphomas (22.2% in males and 29.6% in females) within 600 days after birth. Expression levels of aspartate aminotransferase and alanine aminotransferase, as well as 32 different cytokines, chemokines and growth factors, were examined. The incidence of B-cell lymphoma was significantly correlated with only the level of soluble interleukin-2 receptor alpha subunit (sIL-2R alpha) in RzCD19Cre mouse serum. All RzCD19Cre mice with substantially elevated serum sIL-2R alpha levels (> 1000 pg/mL) developed B-cell lymphomas. Moreover, compared with tissues from control animals, the B-cell lymphoma tissues of RzCD19Cre mice expressed significantly higher levels of IL-2R alpha. We show that the expression of HCV in B cells promotes non-Hodgkin-type diffuse B-cell lymphoma, and therefore, the RzCD19Cre mouse is a powerful model to study the mechanisms related to the development of HCV-associated B-cell lymphoma. (Blood. 2010;116(23):4926-4933) - Teruo Nakaya; Kazuhiko Kuwahara; Kazutaka Ohta; Masahiro Kitabatake; Teppei Toda; Naoki Takeda; Tokio Tani; Eisaku Kondo; Nobuo SakaguchiJOURNAL OF IMMUNOLOGY 185 9 5180 - 5187 2010年11月 [査読有り]
The mitotic checkpoint is essential for maintaining genomic stability in differentiating B cells undergoing genetic alterations of the Ig gene. In this study, using real-time RT-PCR and in situ RNA hybridization, we demonstrated that MAD2 mRNA export is selectively regulated by Pcid2/Thp1. Pcid2 small interfering RNA induced a cell-cycle abnormality with increased apoptosis and polyploidy, as previously observed in MAD2-knockdown cells. Pcid2 small interfering RNA reduced MAD2 expression, but not the expression of other cell-cycle checkpoint proteins, such as MAD1 and BUBR1, or the cell-cycle-associated proteins, cyclin A, cyclin B1, and cyclin-dependent kinase 1. In mouse B lineage cells, Pcid2 transcripts appeared in a stage-dependent manner at high levels in bone marrow pre-B and immature B cells, and in spleen transitional 1 and follicular B cells, but at lower levels in pro-B, transitional 2, and marginal zone B cells, suggesting a stage-dependent requirement for MAD2 regulation. Cd19-cre-derived targeting of the Pcid2 gene induced a mature B cell deficiency in mice. These findings indicate that Pcid2 is essential for B cell survival through the regulation of MAD2 expression during B cell differentiation. The Journal of Immunology, 2010, 185: 5180-5187. - Nobukazu Okamoto; Kazuhiko Kuwahara; Kazutaka Ohta; Masahiro Kitabatake; Katsumasa Takagi; Hiroshi Mizuta; Eisaku Kondo; Nobuo SakaguchiGENES TO CELLS 15 5 471 - 483 2010年05月 [査読有り]
Germinal center-associated nuclear protein (GANP) is a 210-kDa protein that is upregulated in rapidly proliferating B cells. GANP contains regions for RNA-primase and minichromosome maintenance 3 (MCM3)-associated activities, as well as a Sac3-homology region, which is associated with mRNA export in yeast. Here, we examined the role of GANP in mRNA export and cell proliferation in mammalian cells. The ganp small interfering RNA (siRNA) induced cell-cycle arrest at the G2/M-phase, but increased abnormal chromosome alignment of metaphase chromosomes and cell apoptosis in HeLa cells. These changes were not associated with either the abnormality of the spindle assembly checkpoint or the expression level of cohesin. ganp siRNA disrupted the assembly and localization of cohesin at the centromeres in metaphase cells, which is a quite similar phenotype caused by Shugoshin-1 (Sgo1) siRNA-treatment, which was reported previously. ganp siRNA did induce a selective decrease in Sgo1 transcript levels in the cytoplasm, resulting in a lack of cohesin at the centromeres in metaphase and premature separation of the sister chromatids at mitosis. GANP lacking the Sac3-homology region caused the dominant-negative effect with similar abnormalities and impaired mRNA export. Thus, human GANP is critically involved in cell proliferation at the mitotic phase through its selective support of Sgo1 mRNA export. - Kazutaka Ohta; Kazuhiko Kuwahara; Zhenhuan Zhang; Keishi Makino; Yoshihiro Komohara; Hideo Nakamura; Jun-ichi Kuratsu; Nobuo SakaguchiCANCER SCIENCE 100 11 2069 - 2076 2009年11月 [査読有り]
Malignant glioma (MG) is highly proliferative and invasive, with the malignant characteristics associated with aneuploidy and chromosomal instability (CIN). Here, we found that the level of germinal center-associated nuclear protein (GANP), a mammalian homologue of yeast Sac3, was markedly decreased in MGs with a poor prognosis; and thus we explored the effect of its decrease on cell-cycle progression of MG cell lines. Glioblastomas showed a significantly lower level of ganp mRNA than anaplastic astrocytomas, as measured by real-time reverse transcription-PCR, in 101 cases of adult MG. MGs of ganpLow expression displayed more malignant characteristics, with loss of heterozygosity on chromosome 10, epidermal growth factor receptor gene amplification, and significantly poorer prognosis than the ganpHigh group. Human diploid fibroblasts depleted of ganp mRNA by the RNA interference (RNAi) method showed a decreased percentage of S-phase cells and a cellular-senescence phenotype. MG cell lines harboring abnormalities of various cell-cycle checkpoint molecules displayed slippage of mitotic checkpoints and an increased proportion of hyperploid cells after ganp RNAi-treatment. These results suggest that GANP protects cells from cellular senescence caused by DNA damage and that a significant decrease in GANP expression leads to malignancy by generating hyperploidy and CIN. (Cancer Sci 2009); 00: 000-000). - Waraporn Chan-On; Kazuhiko Kuwahara; Naoya Kobayashi; Kazutaka Ohta; Tatsuya Shimasaki; Banchob Sripa; Chanvit Leelayuwat; Nobuo SakaguchiINTERNATIONAL JOURNAL OF ONCOLOGY 35 2 287 - 295 2009年08月 [査読有り]
Cholangiocarcinoma (CCA) represents a model of tumor development after long-term inflammation which causes DNA damage or impairs DNA repair mechanism. AID and GANP, both appearing in antigen-driven B cells, are involved in affinity maturation of the immunoglobulin V-region with increased somatic mutation. A normal cholangiocyte line showed the induction of AID transcripts after stimulation with TNF-alpha, whereas ganp transcripts appeared constitutively in this cell line. Next, we examined the expression of AID and GANP in clinical CCA specimens to obtain information whether their expression levels are associated with the malignant grade of CCA. AID expression was similarly detected in the clinical cases of both well-differentiated and poorly-differentiated CCAs. On the contrary, GANP expression was detected in CCA cells at a higher level in the nucleus of poorly-differentiated CCAs with shorter survivals than in that of well-differentiated CCAs. The high and low cases of nuclear GANP expression showed no change in the frequency of the TP53 mutations, however, further investigation by in vitro experiment demonstrated that the high GANP expression caused the increased number of gamma H2AX foci after DNA damage by ionizing-irradiation. These results suggest that GANP is involved in regulation of DNA repair mechanism and the abnormal over-expression of GANP together with AID might be associated with rigorous DNA damage, potentially causing the malignant development of CCAs during long-term inflammation. - Hideya Igarashi; Kazuhiko Kuwahara; Mikoto Yoshida; Yan Xing; Kazuhiko Maeda; Koichi Nakajima; Nobuo SakaguchiMOLECULAR IMMUNOLOGY 46 6 1031 - 1041 2009年03月 [査読有り]
Antigen (Ag)-driven B cells undergo antibody (Ab) affinity maturation and class switching in germinal center (GC) B cells. GANP is one of the molecules required for Ab affinity maturation. We herein found an increase of IgE in B cell ganp-deficient mice and studied the signal transduction pathway regulated by GANP. GANP suppresses the STAT-mediated transcription activity in GC B cells with the regulation of arginine methyltransferase activity by the interaction with JAK-binding protein arginine methyltransferase (PRMT) 5 and JAK1/JAK3 that are responsible for STAT6 activation. The prmt5 mRNA was up-regulated in B cells after stimulation in vitro and in vivo in GC B cells. The loss of GANP caused up-regulation of phosphorylation and arginine dimethylation of STAT6 in B cells after stimulation with LIPS and IL-4 in vitro. On the contrary, GANP over-expressed B cells in ganp gene-transgenic mice showed a low STAT6 phosphorylation after stimulation. The over-expression of PRMT5 caused the up-regulation of STAT6-mediated gene transcription, which was also suppressed by the co-transfection of GANP, in luciferase reporter assay. GANP down-regulates JAK1/JAK3 to STAT6-signaling with regulation of arginine methylation activity, which might be responsible for the B cell endogenous suppressive mechanism of hyper-IgE. (C) 2008 Elsevier Ltd. All rights reserved. - Nobuo Sakaguchi; Teppei Toda; Teruo Nakaya; Masataka Kitabatake; Kazuhiko Maeda; Kazuhiko KuwaharaRecent Patents on DNA and Gene Sequences 3 2 88 - 95 2009年 [査読有り]
The most critical issue for the application of high affinity monoclonal antibodies is their creation. Here, we summarize the cellular and molecular mechanisms by which high affinity antibodies are generated, and then review the attempts of many investigators to create high affinity monoclonal antibodies against various target molecules. High affinity monoclonal antibodies are generated by one or a combination of the following three major methods. (1) The improvement of antibody affinity by introducing mutations in the immunoglobulin V-region genes by in vitro mutagenesis. (2) Screening many clones from a random combinatory repertoire of IgV-region genes using a phage library established in yeast or bacteria. (3) Attempting to introduce many somatic hypermutation of IgV-region genes. We summarize the advantages and applications of each of these methods including recent patents to facilitate informed individual choice. We also extend our review to the current creation of antibodies for HIV research. © 2009 Bentham Science Publishers Ltd. - Satoru Fujimura; Takeshi Matsui; Kazuhiko Kuwahara; Kazuhiko Maeda; Nobuo SakaguchiMOLECULAR IMMUNOLOGY 45 6 1712 - 1719 2008年03月 [査読有り]
T-cell-dependent antigen induces differentiation of germinal center (GC) B-cell in peripheral lymphoid follicles. We studied whether GC B-cell differentiation is associated with DNA methylation status by examining regulatory regions of mouse AID transcription that are essential for B-cell maturation. AID-negative cell lines of pre-B cells, immature B cells, mature B cells, plasmacytomas or T cells showed various hypermethylation profiles in the 5'-promoter and intronic regions. In contrast, AID-positive GC-type B cells were hypomethylated in these regions. Stimulation of splenic B cells with lipopolysaccharide and interleukin-4 caused DNA hypornethylation in the 5'-promoter and intronic CpG sites proportional to the increase in AID transcription. Mature GL7(+)Fas(+) GC B cells were hypomethylated at these CpG sites, especially near the Pax5-consensus site and an intronic site. However, Syndecan-1(+) plasma cells showed DNA hypermethylation, as seen in plasmacytomas. Methylation status of the transcriptional regulatory region might contribute to stage-dependent activation of AID transcription during GC B-cell differentiation. (c) 2007 Elsevier Ltd. All rights reserved. - Mikoto Yoshida; Kazuhiko Kuwahara; Tatsuya Shimasaki; Naomi Nakagata; Masao Matsuoka; Nobuo SakaguchiGENES TO CELLS 12 10 1205 - 1213 2007年10月 [査読有り]
Immunoglobulin V-region somatic hypermutation and C-region class-switch recombination are initiated by activation-induced cytidine deaminase (AID) in B-cells. AID-induced DNA damage at the immunoglobulin S-region is known to be repaired by non-homologous end-joining, but repair mechanisms at the V-region remain to be elucidated. In Saccharomyces cerevisiae, DNA homologous recombination is regulated by the expression of Sac3, involved in actin assembly, cell cycle transition and mRNA metabolism. Here, we demonstrate that the Sac3-homologue GANP suppresses DNA recombination in a direct-repeat beta-galactosidase gene construct in mammalian cells. Homozygous ganp gene knockout is embryonic lethal in mice. Embryonic fibroblasts immortalized from hetero-deficient ganp(+/-) mice showed more DNA recombination than wild-type. In contrast, over-expression of GANP suppressed either spontaneous DNA recombination or that caused by the introduction of aid cDNA into NIH3T3 cells (susceptible to I-sceI restriction enzyme cleavage but not to RAG-mediated immunoglobulin gene recombination). GANP suppresses the DNA recombination not only on the extrachromosomal DNA construct but also on the integrated DNA. The Sac3-homology portion is necessary for the suppressive activity, but the truncated carboxyl terminal MCM3-binding/acetylating region adversely augmented DNA recombination, acting as a dominant negative form. Expression of full-length GANP is critical for suppression of DNA hyper-recombination in mammalian cells. - Taichi Ezaki; Kazuhiko Kuwahara; Shunichi Morikawa; Kazuhiko Shimizu; Nobuo Sakaguchi; Kouji Matsushima; Kenjiro MatsunoANATOMY AND EMBRYOLOGY 211 5 379 - 393 2006年10月 [査読有り]
We produced two novel rat monoclonal antibodies (LA102 and LA5) to identify mouse lymphatic vessels and blood vessels, respectively. We characterized the two antibodies as to the morphological and functional specificities of endothelial cells of both types of vessels. The antibodies were produced by a rapid differential immunization of DA rats with collagenase- and neuraminidase-treated mouse lymphangioma tissues. LA102 specifically reacted with mouse lymphatic vessels except the thoracic duct and the marginal sinus of lymph nodes, but not with any blood vessels. In contrast, LA5 reacted with most mouse blood vessels with a few exceptions, but not with lymphatics. LA102 recognized a protein of 25-27 kDa, whereas LA5 recognized a molecule of 12-13 kDa. Neither antibody recognized any currently identified lymphatic or vascular endothelial cell antigens. Immunoelectron microscopy revealed that the antigens recognized by LA102 and LA5 were localized on both luminal and abluminal endothelial cell membranes of each vessel type. Interestingly, LA102 immunoreactivity was strongly expressed on pinocytic or transport vesicle membrane in the cytoplasm of lymphatic endothelium. Besides endothelial cells, both antibodies also recognized some types of lymphoid cells. Since, the LA102 antigen molecule is expressed on some lymphoid cells, it may play important roles in the migration of lymphoid cells and in some transport mechanisms through lymphatic endothelial cells. - T Kageshita; K Kuwahara; M Oka; DL Ma; T Ono; N SakaguchiJOURNAL OF DERMATOLOGICAL SCIENCE 42 1 55 - 63 2006年04月 [査読有り]
Background: Germinal center-associated nuclear protein (GANP) is a newly cloned molecule that is up-regulated in the germinal center B cells. Although GANP functions in the regulation of DNA repair during replication and survival of B cells, little is known about its expression in melanocytic cells. Objectives: To investigate whether GANP and phosphorylated-GANP (P-GANP) are expressed in cultured human melanocytes and melanoma cells and in benign and malignant melanocytic lesions. In addition, we aim to determine whether GANP and P-GANP are associated with malignant transformation of melanocytic lineage. Methods: GANP and P-GANP expression in cultured melanocytic cells was analyzed by immunostaining and in vitro kinase assay. GANP and P-GANP expression in melanocytic lesions was analyzed by immunohistochemistry. Results: GANP and P-GANP were up-regulated in cultured melanoma cells compared to melanocytes. GANP and P-GANP were restricted to nucleus of melanocytes but co-expressed in cytoplasm of melanoma cells. On the other hand, GANP and P-GANP were widely expressed at various levels in melanocytic nevi and melanoma lesions with nuclear and cytoplasmic staining pattern. Melanoma cells showed a stronger intensity of GANP and P-GANP than melanocytic nevus cells, however the staining intensity in primary melanoma lesions was not associated with any clinicopathological variables. Cytoplasmic GANP and P-GANP expression was associated with MCM3 and Ki67 expression. Conclusions: These data suggest, for the first time, that GANP and P-GANP are upregulated in cultured melanoma cells compared to melanocytes and also they are widely expressed in benign and malignant melanocytic tumor cells. (c) 2005 Japanese Society for Investigative Dermatotogy. Published by Elsevier Ireland Ltd. All rights reserved. - Y Kawatani; H Igarashi; T Matsui; K Kuwahara; S Fujimura; N Okamoto; K Takagi; N SakaguchiJOURNAL OF IMMUNOLOGY 175 9 5615 - 5618 2005年11月 [査読有り]
Double-stranded DNA breaks (DSBs) at the JgV region (JgV) genes might be involved in somatic hypermutation and affinity-maturation of the B cell receptor in response to T cell-dependent Ag. By ligation-mediated PCR, we studied IgV DSBs that occurred in mature germinal center B cells in response to nitrophenyl-chicken gamma-globulin in a RAG1-independent, Ag-dependent, and IgV-selective manner. We quantified their levels in GANP-deficient B cells that have impaired generation of high-affinity Ab. GANP(-/-) B cells showed a decreased level of DSBs with blunt ends than control B cells and, on the contrary, the ganp gene transgenic (GANP(Tg)) B cells showed an increased level. These results suggested that the level of JgV DSBs in germinal center B cells is associated with GANP expression, which is presumably required for B cell receptor affinity maturation. - S Fujimura; Y Xing; M Takeya; Y Yamashita; K Ohshima; K Kuwahara; N SakaguchiCANCER RESEARCH 65 13 5925 - 5934 2005年07月 [査読有り]
Lymphomas arise containing abnormalities of various differentiation stage-specific molecules. In the study reported here, we have shown abnormal up-regulation of germinal center B cell-associated GANT in various human lymphomas including mantle cell, diffuse large B cell, and Hodgkin lymphoma, by immunohistochemical analysis. To study the role of GANP in lymphomagenesis, we generated mutant mice (ganp-Tg) that express the transgenic ganp gene under immunoglobulin enhancer and promoter control. Ganp-Tg mice showed a high incidence of lymphomagenesis (29.5%) after aging with a non-B/non-T cell surface phenotype having slight CD45R/B220 expression and Ig transcripts of rearranged VH-DH-JH IgH loci. Lymphomas generated in ganp-Tg mice displayed similar pathologic characteristics to mouse reticulum cell neoplasm or Hodgkin lymphoma-like lesions. The VH sequences of individual mice showed that the tumors proliferated from a single clone or oligoclones, as is found in human diffuse large B-cell lymphomas and Hodgkin lymphoma. These results suggest that GANP overexpression is a causative factor in the generation of B lymphomas. - N Sakaguchi; T Kimura; S Matsushita; S Fujimura; J Shibata; M Araki; T Sakamoto; C Minoda; K KuwaharaJOURNAL OF IMMUNOLOGY 174 8 4485 - 4494 2005年04月 [査読有り]
Generation of high-affinity Ab is impaired in mice lacking germinal center-associated DNA primase (GANP) in B cells. In this study, we examined the effect of its overexpression in ganp transgenic C57BL/6 mice (Ganp(Tg)). Ganp(Tg) displayed normal phenotype in B cell development, serum Ig levels, and responses against T cell-independent Ag; however, it generated the Ab with much higher affinity against nitrophenyl-chicken gammaglobulin in comparison with C57BL/6. To further examine the affinity increase, we established hybridomas producing high-affinity mAbs and compared their affinities using BIAcore. C57BL/6 generated high-affinity anti-nitrophenyl mAbs (K-D similar to 2.50 X 10(-7) M) of IgG1/lambda 1 and contained the V(H)186.2 region with W33L mutation. GanpTg generated much higher affinity (K-D > 1.57 X 10(-9) M) by usage of V(H)186.2 as well as noncanonical V(H)7183 regions. Ganp(Tg) also generated exceptionally high-affinity anti-HIV-1 (V3 peptide) mAbs (K, > 9.90 x 10-11 m) with neutralizing activity. These results demonstrated that GANP is involved in V region alteration generating high-affinity Ab. - Sefat-e-Khuda; M Yoshida; Y Xing; T Shimasaki; M Takeya; K Kuwahara; N SakaguchiJOURNAL OF BIOLOGICAL CHEMISTRY 279 44 46182 - 46190 2004年10月 [査読有り]
Saccharomyces Sac3 required for actin assembly was shown to be involved in DNA replication. Here, we studied the function of a mammalian homologue SHD1 in cell cycle progression. SHD1 is localized on centrosomes at interphase and at spindle poles and mitotic spindles, similar to alpha-tubulin, at M phase. RNA interference suppression of endogenous shd1 caused defects in centrosome duplication and spindle formation displaying cells with a single apparent centrosome and down-regulated Mad2 expression, generating increased micronuclei. Conversely, increased expression of SHD1 by DNA transfection with shd1-green fluorescent protein (gfp) vector for a fusion protein of SHD1 and GFP caused abnormalities in centrosome duplication displaying cells with multiple centrosomes and deregulated spindle assembly with up-regulated Mad2 expression until anaphase, generating polyploidy cells. These results demonstrated that shd1 is involved in cell cycle progression, in particular centrosome duplication and a spindle assembly checkpoint function. - ZK Mirnics; E Caudell; Y Gao; K Kuwahara; N Sakaguchi; T Kurosaki; J Burnside; K Mirnics; SJ CoreyJOURNAL OF IMMUNOLOGY 172 7 4133 - 4141 2004年04月 [査読有り]
Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal, organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-kappaB, elongation factor 1alpha, CD79b, octamer binding factor 1, Ig H chain, stathmin, and gamma-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn-->PU.1-->GANP. - N Sakaguchi; Y Takahashi; T Takemori; K KuwaharaIMMUNOLOGY 2004: GENOMIC ISSUES, IMMUNE SYSTEM ACTIVATION AND ALLERGY 379 - 384 2004年 [査読有り]
Acquired immunity is dependent on generation of high-affinity B cells against the T cell dependent (TD)-antigens (Ags) during the proliferation and differentiation in germinal center (GC) of peripheral lymphoid organs. The primary repertoire of virgin B cells provides a necessary but limited source of Ag-reactive B cells, but they further differentiate into high-affinity B cells during immune responses. To elucidate the molecular mechanism of generation of high-affinity B cells, we studied GC-associated nuclear protein (GANP) that appeared up-regulated in GC-B cells upon immunization with TD-Ags. Conditional targeting of ganp in B cells displayed impairment of generation of high-affinity B cells upon immunization with nitrophenyl chicken gamma-globulin (NP-CG). The low-affinity in the ganp-deficient B cells is manifested in lack of the mutation at W-33 to L of VH186.2 with lambda1 light chain. The data suggested the new notion regarding mechanism in Ig diversification in peripheral lymphoid tissues. - K Kuwahara; S Fujimura; Y Takashi; N Nakagata; T Takemori; S Aizawa; N SakaguchiPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 4 1010 - 1015 2004年01月 [査読有り]
Acquired immunity depends on proliferation and differentiation of antigen (Ag)-specific B cells in germinal centers (GCs) of lymphoid follicles in response to T cell-dependent Ags. Here, we studied the function of GC-associated nuclear protein that is selectively up-regulated in GC-B cells. B cell-specific ganp-deficient mice were compromised in affinity maturation of hapten-specific antibodies against T cell-dependent Ags with retarded development of GCs. B cell numbers and development, serum Ig levels, mitogen-induced B cell proliferation in vitro, and responses to T cell-independent Ag were nearly normal; however, the mutant B cells showed a decrease in anti-CD40-induced proliferation and an increased susceptibility to B cell apoptosis in vitro and in vivo. B cell-specific ganp-deficient mice showed a decreased frequency of variable-region somatic mutations, especially of the high-affinity type (W-33 --> L) in the V(H)186.2 region against nitrophenyl-chicken gamma globulin, whereas the class switching was normal. We conclude that GC-associated nuclear protein is necessary for generation or maintenance of 13 cells with high-affinity B cell Ag receptors during the maturation in GCs. - Shinjiro Tomiyasu; Kazuhiko Kuwahara; Nobuo Sakaguchi; Michio OgawaInternational Congress Series 1255 C 283 - 288 2003年08月 [査読有り]
GC-associated DNA primase (GANP) contains two functionally important regions, the N-terminal RNA/DNA primase and C-terminal MCM3-associated/import domains, which might be involved in DNA replication. Here, we investigated the functions of these regions by transfection studies. In addition to the C-terminal MCM3-binding (Gmap80) region, the RNA/DNA primase region (Gp) possesses an additional MCM3-interaction activity that mediates its translocation between the nucleus and the cytoplasm. Gp with an N-terminal nuclear localization signal sequence (NLS) is present in the nucleus, but Gmap80 with a C-terminal NLS appeared in the cytoplasm. The whole GANP (Ganp) protein is present in both nuclear and cytoplasmic compartments, in a Crm1-dependent manner. Gp associated with MCM3 through the NLS, and MCM3 induced nuclear localization of Ganp. NLS-negative MCM3 does not have such activity. These results suggest that the NLS of MCM3 is involved in nuclear translocation of Ganp. The nuclear-cytoplasm MCM3 translocation was demonstrated in mammalian cells, and over-expression of Ganp, but not of Gp or Gmap80, facilitated this translocation of MCM3 in co-transfectants. Moreover, introduction of Ganp induced DNA synthesis in the transfectants but dominant-negative mutants did not. It is concluded that GANP is translocated from the nucleus to the cytoplasm in association with MCM3 and is involved in DNA synthesis. © 2003, Elsevier Inc. All rights reserved. - Nobuo Sakaguchi; Satoru Fujimura; Kazuhiko KuwaharaDevelopmental Immunology 9 3 169 - 172 2002年09月 [査読有り]
Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist. - Y Kono; K Maeda; K Kuwahara; H Yamamoto; E Miyamoto; K Yonezawa; K Takagi; N SakaguchiGENES TO CELLS 7 8 821 - 834 2002年08月 [査読有り]
Background: GANP, carrying DNA-primase and MCM3-binding domains, is up-regulated in germinal centre B cells. To understand the regulatory function of GANP upon MCM complex, we searched for GANP-associated molecules by yeast two-hybrid screening. Results: Using the 1 kb fragment (G5) of the ganp cDNA, we identified a clone named G5PR that is structurally homologous to known regulatory subunits of protein phosphatases (PPases) and determined the association of G5PR with GANP in vivo in the DNA transfectant. G5PR is associated with protein phosphatase 5 (PP5) through its tetratricopeptide-repeat (TPR) domain. Pull-down assays demonstrated that G5PR is also associated with protein phosphatase 2A (PP2A), the complex of A subunit (PR65) and the catalytic (C) subunit (PP2Ac), similar to the B'' subunit. The G5PR-associated complex had phosphatase activity on casein, histone H1 and MCM3 in vitro , but the addition of G5PR did not stimulate or inhibit the phosphatase activities of PP5 and PP2A. The cellular localization of G5PR in transfected cells varies during cell cycling, appearing in the nucleus during prophase, in the peri-chromatin during mitotic phase, and in the cytoplasm after cell division. Conclusion: G5PR is capable of recruiting two kinds of PPases, PP5 and PP2A, into the GANP/MCM3 complex, which might regulate its phosphorylation state during cell cycle progression. - Mohamed A. El-Gazzar; Kazuhiko Maeda; Hisayuki Nomiyama; Mitsuyoshi Nakao; Kazuhiko Kuwahara; Nobuo SakaguchiJournal of Biological Chemistry 276 48000 - 48008 2001年12月Germinal center-associated DNA primase (GANP) associated with MCM3 of the DNA replication complex is up-regulated selectively in germinal center B cells. We studied promoter activity of the 5′ region involved in the developmental stage-dependent expression in B lineage cells by luciferase reporter assay. Selective regulation of ganp expression was observed in the -737-bp promoter region in B and plasma cell lines but was significantly low in pre-B and T cell lines. The deletion constructs displayed a gap decrease after shortening the region from -134 to -108 bp. Further narrowing suggested the involvement of the PU.1 consensus sequence at -126 bp by electrophoretic mobility shift assay. The protein component PU.1 complex is not inhibited with mutated probes at the consensus site but is inhibited with the known PU.1 probe of CD72 and with anti-PU.1 antibody. Moreover, introduction of PU.1 cDNA enhanced the reporter gene activity in a dose-dependent manner in B cells, whereas the reporter construct with the mutated PU.1 site did not respond. Anti-CD40 stimulation induced the reporter activity with a 100% increase, which is not observed with the PU.1-mutated reporter construct. These results demonstrate that the germinal center-associated DNA primase expression is partly regulated by the transcription factor PU.1 expressed in B lineage cells.
- Eiji Abe; Kazuhiko Kuwahara; Mikoto Yoshida; Mikio Suzuki; Hidenori Terasaki; Yoshinobu Matsuo; Ei-Ichi Takahashi; Nobuo SakaguchiGene 255 2 219 - 227 2000年09月 [査読有り]
A 210 kDa protein named GANP is upregulated in germinal center (GC)-B cells in the spleen of antigen-immunized mouse. We studied a human ganp gene (hganp) encoding a putative polypeptide of 1980 amino acids. The carboxyl-terminal 721-amino-acid sequence of hGANP is identical to Map80, that is presumably generated by alternative splicing of hganp/Map80 gene. The genomic segment carrying hganp and Map80 genes was isolated, and the chromosomal location was determined on 21q22.3. Northern blot analysis with RNAs from various organs demonstrated a single band of 7 kb hganp mRNA, which suggests a preferential transcription of hganp gene from the hganp/Map80 locus. The hGANP expression was upregulated in GCs of the tonsil, as demonstrated by in-situ RNA hybridization and immunohistochemical analyses. The hGANP, with the domain (Map-box) capable of binding to MCM3 in B cells, might be involved in regulation of cell-cycle progression and DNA replication of GC-B cells. (C) 2000 Elsevier Science B.V. All rights reserved. - A Sakata; K Kuwahara; T Ohmura; S Inui; N SakaguchiIMMUNOLOGY LETTERS 68 2-3 301 - 309 1999年06月 [査読有り]
Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgM(high)/IgD(low) B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-KB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells. (C) 1999 Elsevier Science B.V. All rights reserved. - Sachiko Yamanouchi; Kazuhiko Kuwahara; Atsuko Sakata; Taichi Ezaki; Shuji Matsuoka; Jun-Ichi Miyazaki; Sachiko Hirose; Toshiki Tamura; Hideo Nariuchi; Nobuo SakaguchiEuropean Journal of Immunology 28 2 696 - 707 1998年02月 [査読有り]
A T cell activation antigen, Ly6C, is considered to be involved in the autoimmunity of some autoimmune-prone mice however, the function of Ly6C remains largely unknown. We prepared a rat anti-mouse Ly6C monoclonal antibody (mAb) (S14) that inhibits the proliferation of peripheral T cells stimulated with anti-CD3 mAb in vitro. S14 mAb, the specificity of which is confirmed by a cDNA transfectant, recognizes Ly6C antigen preferentially expressed on a part of CD8+ T cells in peripheral lymphoid organs. The immunohistochemical analysis demonstrates that Ly6C appears on CD8+ T cells in the conventional T cell-associated area of BALB/c but not of nonobese diabetic (NOD) mice, confirming the absence of Ly6C+ T cells in NOD mice. Addition of soluble S14 mAb to the culture does not influence the proliferation of T cells in vitro, however, the S14 mAb coated on the plate clearly inhibits the proliferation and IL-2 production of anti-CD3-stimulated peripheral T cells. The T cells are arrested at the transitional stage from G0/G1 to S+G2/M phases, but they are not induced to undergo apoptotic changes in vitro. This inhibitory signal provided through the Ly6C molecule inhibited IL-2 secretion in a subpopulation of the activated CD4+ T cells. Ly6C is expressed on T cell clones of both Th1 and Th2 cells, but the cytokine secretion from Th1 clones is preferentially inhibited. These results suggest that Ly6C mediates an inhibitory signal for secretion of cytokines from Th1 CD4+ T cells, potentially causing the inhibition of immune response in peripheral lymphoid tissues. - Y. Matsuo; A. Sugimoto; K. Kuwahara; Y. Watanabe; A. Sakata; N. Sakaguchi; K. Sagawa; K. OritaTissue Antigens 52 5 422 - 429 1998年 [査読有り]
To analyze the cellular antigens of human B-cell lineage, a monoclonal antibody, NU-B1, was raised using the acute lymphoblastic leukemia (ALL) cell line NALM-16 as the immunogen. NU-B1 reacted with 7.7 ± 3.9% of the healthy adult peripheral blood mononuclear cells but not with neutrophils, monocytes, red blood cells or thymocytes. In order to distinguish the reaction specificity of NU-B1, two-color immunofluorescence staining using tonsillar cells was performed, and it was demonstrated that NU-B1-positive cells coexpressed CD20, which is a representative B-cell antigen. The expression of NU-B1 was highly restricted to cells of B-cell lineage when a panel of hematopoietic cell lines was examined. In a pathoimmunohistological study using human lymph node tissue, NU-B1-positive cells were localized in the mantle and marginal zones. In a clinical study, NU-B1 reacted specifically with leukemias/lymphomas of B-cell lineage: all 43 cases of ALL including common ALL and biphenotypic leukemia, all 4 cases of B-cell ALL, 6/7 B-cell type malignant lymphomas and 2/4 B-cell chronic lymphocytic leukemias. NU-B1 did not react with multiple myeloma, T-cell or myeloid leukemias/lymphomas. Immunoprecipitation of NU-B1 revealed two clear bands at 50 kDa and 42 kDa under either reducing or nonreducing conditions. Although anti-IgM treatment induced dramatic down modulation of CD79b, the NU-B1 antigen was also down modulated, but only slightly. However, crosslinking of NU-B1 did not induce tyrosine phosphorylation of intracellular proteins or the mobilization of calcium in NALM-16. The present results revealed that the antigenic determinant recognized by NU-B1 is not surface immunoglobulin chains, HLA-DR, a receptor for C3, Fc for immunoglobulin chains or any known CD molecule. We conclude that monoclonal antibody NU-B1 recognizes a novel human B-cell restricted antigen distinct from known CD molecules, and that it is a useful antibody in the immunophenotyping and classification of leukemias/lymphomas. - Y. Matsuo; R. A.F. MacLeod; K. Kojima; K. Kuwahara; A. Sakata; H. G. Drexler; C. Nishizaki; S. Fukuda; Y. Inoue; T. Sezaki; N. Sakaguchi; K. OritaLeukemia 11 12 2168 - 2174 1997年 [査読有り]
A human acute lymphoblastic leukemia (ALL) cell line, BALM-16, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) in relapse. As with the original leukemia cells, the established line was negative for both cell surface and cytoplasmic immunoglobulin (Ig) chains. Absence of Ig expression was confirmed by Western blotting. Southern blot analysis demonstrated homozygous deletion of the Cκ gene, germ line configuration of the Cλ and rearrangement of IgJH genes. Cytogenetic analysis of both leukemic bone marrow and BALM-16 cells showed the t(8 22)(q24 q11) abnormality which is specifically associated with ALL-L3 and Burkitt lymphoma. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which showed parathyroid hormone-related peptide (PTHrP) mRNA detected by reverse-transcriptase polymerase chain reaction. However, PTHrP production was not detected in the culture supernatant. The established cell line, BALM-16, could provide a useful material for analyzing the lack of Ig expression and of clarifying the pathogenesis of this type of B cell malignancy. - Y. Matsuo; S. Nakamura; T. Ariyasu; R. Terao; K. Imajyo; T. Tsubota; K. Kuwahara; N. SakaguchiLeukemia 10 4 700 - 706 1996年 [査読有り]
A human acute lymphoblastic leukemia (ALL) cell line, BALM-9, was established from the peripheral blood specimen of a patient with B cell ALL (L3 type). In contrast to the original immunoglobulin (Ig) phenotype, the established BALM-9 cell line expressed both κ and λ light (L) chains simultaneously in a range of 30-80%. Two-color flow cytometric analysis demonstrated that there was a distinct population of κλ double positive cells as well as κ single, A single and double negative populations. Therefore, subclones were obtained from each population by limiting dilution and were designated BALM-9KL (κ+λ+), BALM-9K (κ+λ-), BALM-9L (κ-λ+) and BALM-9N (κ-λ-). Western blotting confirmed the results of the immunofluorescence test at the protein level. In BALM-9N, L chains were absent even in the cytoplasm as demonstrated by Western blotting. Evidence that the subclones have the same ancestry was provided both by cytogenetic analysis and by Southern blotting, which revealed the 14q32 chromosomal rearrangement as a common abnormality and the same IgH gene rearrangement among the subclones. The existence of a κλ positive B cell population suggests a transient stage of normal B cell maturation. These subclones might represent such a stage and thus provide a useful means of analyzing the mechanism of this double light chain expression. - Inui S; Kuwahara K; Mizutani J; Maeda K; Kawai T; Nakayasu H; Sakaguchi NThe Journal of Immunology 154 6 2714 - 2723 1995年03月 [査読有り]
- Kuwahara K; Igarashi H; Kawai T; Ichigi Y; Muraguchi A; Mason DY; Kimoto M; Inui S; Sakaguchi NBiochemical and Biophysical Research Communications 197 3 1563 - 1569 1993年12月 [査読有り]
- Matsuo T; Nomura J; Kuwahara K; Igarashi H; Inui S; Hamaguchi M; Kimoto M; Sakaguchi NJournal of immunology (Baltimore, Md. : 1950) 150 9 3766 - 3775 1993年05月 [査読有り]
講演・口頭発表等
- The role of mammalian TREX2 complex in sporadic breast cancers. [招待講演]Kazuhiko KuwaharaThe 3rd Bandung International Biomolecular Medicine Conference (BIBMC) 2014年 口頭発表（招待・特別）
- DNA傷害の新たな視点 -mRNA輸送分子GANP欠損による転写共役型DNA損傷と発癌の実証- [招待講演]桑原 一彦佐賀大学分子生命科学セミナー 2012年 公開講演，セミナー，チュートリアル，講習，講義等
- New Insights of transcription-coupled DNA damage in cell development and differentiation. [招待講演]Kazuhiko KuwaharaSpecial seminar in Department of Biochemistry, Faculty of Medicine, Khon Kaen University 2011年 公開講演，セミナー，チュートリアル，講習，講義等
作品等
MISC
- Kazuhiko Kuwahara; Hidemi Ito; Kiyotaka Kuzushima; Hiroji Iwata; Hideo Tanaka; Keitaro Matsuo CANCER RESEARCH 76 2016年07月
- Andri Rezano; Kazuhiko Kuwahara; Mutsuko Yamamoto-Ibusuki; Masahiro Kitabatake; Penpak Moolthiya; Suchada Phimsen; Taiji Suda; Shigenobu Tone; Yutaka Yamamoto; Hirotaka Iwase; Nobuo Sakaguchi INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 34 S114 -S114 2014年
- HIV-1 gp120/CD4&CXCR4膜融合複合体エピトープと感染阻止効果戸田 哲平; 桑原 一彦; 近藤 直幸; 前田 洋助; 松田 善衛; 阪口 薫雄 日本エイズ学会誌 13 (4) 342 -342 2011年11月
- 笠間由里; 関口敏; 齊藤誠; 佐藤正明; 桑原一彦; 竹屋元裕; 阪口薫雄; 小原道法; 小原恭子 生化学 83回・33回 ROMBUNNO.2T11-7 -7 2010年12月
- 笠間由里; 関口敏; 齊藤誠; 佐藤正明; 桑原一彦; 竹屋元裕; 阪口薫雄; 小原道法; 小原恭子 日本ウイルス学会学術集会プログラム・抄録集 58th 268 2010年10月
- 全長HCV遺伝子のB細胞発現マウスにおけるBリンパ腫の発生(Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous B-cell lymphomas)笠間 由里; 関口 敏; 齊藤 誠; 佐藤 正明; 桑原 一彦; 阪口 薫雄; 竹屋 元裕; 日浅 陽一; 小原 道法; 小原 恭子 日本癌学会総会記事 69回 91 -92 2010年08月
- Kazutaka Ohta; Kazuhiko Kuwahara; Keishi Makino; Nobuo Sakaguchi; Jun-ichi Kuratsu NEURO-ONCOLOGY 11 (6) 959 -959 2009年12月
- Nobuo Sakaguchi; Hideya Igarashi; Mikoto Yoshida; Kazuhiko Maeda; Kazuhiko Kuwahara JOURNAL OF IMMUNOLOGY 176 S164 -S164 2006年04月
- Nobuo Sakaguchi; Kazuhiko Kuwahara CELL STRUCTURE AND FUNCTION 29 88 -88 2004年05月
- 胚中心B細胞の親和性成熟に関与するRNAプライマーゼGANP（共著）桑原一彦; 阪口薫雄 羊土社 実験医学増刊号「免疫研究のフロンティア」 22 (5) 88' 2004年
- K Kuwahara; S Fujimura; Y Takashi; N Nakagata; T Takemori; S Aizawa; N Sakaguchi PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 101 (4) 1010 -1015 2004年01月
- S Fujimura; K Kuwahara; T Ezaki; K Tomita; S Hirose; N Sakaguchi JOURNAL OF AUTOIMMUNITY 20 (4) 291 -301 2003年06月
- N Sakaguchi; K Kuwahara; S Fujimura; Y Takahashi; T Takemori FASEB JOURNAL 17 (7) C190 -C190 2003年04月
- 抗原特異的B細胞の活性化とGANP分子の免疫応答における機能（共著）桑原一彦; 阪口薫雄 中山書店 Molecular Medicine臨時増刊号「免疫2004」 40 40' 2003年
- K Maeda; Y Kono; K Kuwahara; N Sakaguchi FASEB JOURNAL 16 (4) A348 -A348 2002年03月
- K Kuwahara; S Tomiyasu; S Fujimura; K Nomura; Y Xing; N Nishiyama; M Ogawa; S Imajoh-Ohmi; S Izuta; N Sakaguchi PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 98 (18) 10279 -10283 2001年08月
- K Kuwahara; M Yoshida; E Kondo; A Sakata; Y Watanabe; E Abe; Y Kouno; S Tomiyasu; S Fujimura; T Tokuhisa; H Kimura; T Ezaki; N Sakaguchi BLOOD 95 (7) 2321 -2328 2000年04月
- Y Matsuo; K Kuwahara; N Sakaguchi; K Orita FASEB JOURNAL 12 (5) A1075 -A1075 1998年03月
- K Kuwahara; T Kawai; S Mitsuyoshi; Y Matsuo; H Kikuchi; S ImajohOhmi; E Hashimoto; S Inui; MD Cooper; N Sakaguchi INTERNATIONAL IMMUNOLOGY 8 (8) 1273 -1285 1996年08月
- B細胞抗原レセプターを介するシグナル伝達河合 太郎; 桑原 一彦; 阪口 薫雄 実験医学 12 (17) 2172 -2178 1994年11月
- H IGARASHI; K KUWAHARA; J NOMURA; A MATSUDA; K KIKUCHI; S INUI; N SAKAGUCHI JOURNAL OF IMMUNOLOGY 153 (6) 2381 -2393 1994年09月
- K KUWAHARA; T MATSUO; J NOMURA; H IGARASHI; M KIMOTO; S INUI; N SAKAGUCHI JOURNAL OF IMMUNOLOGY 152 (6) 2742 -2752 1994年03月
- N SAKAGUCHI; T MATSUO; J NOMURA; K KUWAHARA; H IGARASHI; S INUI ADVANCES IN IMMUNOLOGY, VOL 54 54 337 -392 1993年
産業財産権
- 特願2009-025607:胆管がん特異的糖鎖エピトープを認識するモノクローナル抗体 2009年02月06日阪口薫雄, 桑原一彦, 荒木令江, 坂本珠美
- 特開4426728:GANP蛋白質 1999年02月24日阪口薫雄, 桑原一彦特願平11-047035
- 特願2012-278228:高病原性トリインフルエンザに対する抗体阪口薫雄, 桑原一彦, 小原道法, 芝崎太
受賞
- 2022年11月 日本病理学会 学術研究賞演説（A演説）
受賞者: 桑原 一彦 - 2018年10月 藤田学園医学会 産学連携推進センター長賞
受賞者: 桑原 一彦 - 2015年 愛知健康増進財団 医学研究・健康増進活動等研究助成金
受賞者: 桑原 一彦 - 2015年 第24回日本医学会総会記念医学振興基金 研究助成金
受賞者: 桑原 一彦 - 2014年 愛知県がん研究振興会 がんその他の悪性新生物研究助成金
受賞者: 桑原 一彦 - 2005年 神澤医学研究振興財団研究助成金
JPN受賞者: 桑原 一彦 - 2005年 黒住医学研究振興財団研究助成金
JPN受賞者: 桑原 一彦 - 2004年 ノバルティス研究奨励金
JPN受賞者: 桑原 一彦 - 2004年 東京生化学研究会研究奨励金
JPN受賞者: 桑原 一彦 - 2002年 熊本医学会奨励賞
JPN受賞者: 桑原 一彦 - 2001年 上原記念生命科学財団研究奨励金
JPN受賞者: 桑原 一彦
共同研究・競争的資金等の研究課題
- PARP阻害剤による非遺伝性乳癌に対する新規抗がん治療戦略日本学術振興会：科学研究費助成事業 基盤研究(C)研究期間 : 2020年04月 -2023年03月代表者 : 桑原 一彦
- TREX2複合体機能不全の誘導は抗がん剤感受性亢進の標的になるのか日本学術振興会：科学研究費助成事業 挑戦的萌芽研究研究期間 : 2016年04月 -2018年03月代表者 : 権藤 なおみ
- 非遺伝性散発性乳癌の発症、悪性進展におけるTREX2複合体因子の機能解析日本学術振興会：科学研究費助成事業 基盤研究(C)研究期間 : 2015年04月 -2018年03月代表者 : 桑原 一彦
- 転写に共役したDNA傷害に起因する新規乳癌発症機構の解析日本学術振興会：科学研究費助成事業 基盤研究(C)研究期間 : 2012年04月 -2015年03月代表者 : 桑原 一彦
- 非遺伝性乳癌におけるp53 mRNA核外輸送分子GANPの機能日本学術振興会：科学研究費助成事業 基盤研究(C)研究期間 : 2009年04月 -2012年03月代表者 : 桑原 一彦
- 胚中心における免疫応答の解析科学技術振興機構：戦略的創造研究推進制度（個人研究型） （個人研究推進事業：さきがけ研究２１‐ＰＲＥＳＴＯ）研究期間 : 2001年12月 -2005年03月代表者 : 桑原 一彦
- Analysis of immune response in germinal centerJST Basic Research Programs (Precursory Research for Embryonic Science and Technology :PRESTO)研究期間 : 1988年