研究者総覧

菊池 次郎 (キクチ ジロウ)

  • 幹細胞制御研究部 准教授
Last Updated :2021/12/04

研究者情報

学位

  • 医学博士(自治医科大学)

ホームページURL

J-Global ID

研究キーワード

  • 血液腫瘍   分子標的薬   エピジェネティクス   癌   

研究分野

  • ライフサイエンス / 血液、腫瘍内科学

研究活動情報

論文

  • Naoki Osada, Jiro Kikuchi, Daisuke Koyama, Yoshiaki Kuroda, Hiroshi Yasui, Joel D Leverson, Yusuke Furukawa
    Haematologica 2021年07月 
    Not available.
  • Daisuke Koyama, Jiro Kikuchi, Yoshiaki Kuroda, Masatsugu Ohta, Yusuke Furukawa
    Cancer science 2020年10月 [査読有り]
     
    Chronic myeloid leukemia is driven by the BCR-ABL oncoprotein, a constitutively active protein tyrosine kinase. Although tyrosine kinase inhibitors (TKIs) have greatly improved the prognosis of CML patients, the emergence of TKI resistance is an important clinical problem, which deserves additional treatment options based on unique biological properties to CML cells. In this study, we show that metabolic homeostasis is critical for survival of CML cells, especially when the disease is in advanced stages. The BCR-ABL protein activates AMP-activated protein kinase (AMPK) for ATP production and the mTOR pathway to suppress autophagy. BCR-ABL is detected in the nuclei of advanced-stage CML cells, in which ATP is sufficiently supplied by enhanced glucose metabolism. AMP-activated protein kinase is further activated under energy-deprived conditions and triggers autophagy through ULK1 phosphorylation and mTOR inhibition. In addition, AMPK phosphorylates 14-3-3 and Beclin 1 to facilitate cytoplasmic translocation of nuclear BCR-ABL in a BCR-ABL/14-3-3τ/Beclin1/XPO1 complex. Cytoplasmic BCR-ABL protein undergoes autophagic degradation when intracellular ATP is exhausted by disruption of the energy balance or forced autophagy flux with AMP mimetics, mTOR inhibitors, or arsenic trioxide, leading to apoptotic cell death. This pathway represents a novel therapeutic vulnerability that could be useful for treating TKI-resistant CML.
  • Yoshiaki Kuroda, Akiko Yashima-Abo, Daisuke Koyama, Jiro Kikuchi, Shigehisa Mori, Shigeki Ito, Yusuke Furukawa
    Leukemia 2020年09月 [査読有り]
  • Akiko Nagamachi, Jiro Kikuchi, Akinori Kanai, Yusuke Furukawa, Toshiya Inaba
    Haematologica 105 7 e325-e327  2020年07月 [査読有り]
  • Yusuke Furukawa, Jiro Kikuchi
    International journal of hematology 111 4 496 - 511 2020年04月 [査読有り]
     
    The treatment outcome of multiple myeloma (MM) is worse than expected from the average numbers of non-synonymous mutations, which are roughly correlated with the prognosis of cancer patients. The refractoriness of MM may be ascribed to the complex genomic architecture and clonal behavior of the disease. In MM, disease progression is accomplished by branching patterns of subclonal evolution from reservoir clones with a propagating potential and/or the emergence of minor clones, which already exist at the MGUS stage and outcompete other clones through selective pressure mainly by therapeutic agents. Each subclone harbors novel mutations and distinct phenotypes including drug sensitivities. In general, mature clones are highly sensitive to proteasome inhibitors (PIs), whereas immature clones are resistant to PIs but could be eradicated by immunomodulatory drugs (IMiDs). The branching evolution is a result of the fitness of different clones to microenvironment and their evasion of immune surveillance; therefore, IMiDs are effective for MM with this pattern of evolution. In contrast, ~ 20% of MM evolve neutrally in the context of strong oncogenic drivers, such as high-risk IgH translocations, and are relatively resistant to IMiDs. Further understanding of the genomic landscape and the pattern of clonal evolution may contribute to the development of more effective treatment strategies for MM.
  • Jiro Kikuchi, Mitsuo Hori, Hidekatsu Iha, Noriko Toyama-Sorimachi, Shotaro Hagiwara, Yoshiaki Kuroda, Daisuke Koyama, Tohru Izumi, Hiroshi Yasui, Atsushi Suzuki, Yusuke Furukawa
    Leukemia 34 1 180 - 195 2020年01月 [査読有り][通常論文]
     
    SLAMF7 is expressed mainly on multiple myeloma (MM) cells and considered an ideal target for immunotherapeutic approaches. Indeed, elotuzumab, an anti-SLAMF7 antibody, is used for the treatment of MM in combination with immunomodulatory drugs. SLAMF7 is cleaved via unknown mechanisms and detected as a soluble form (sSLAMF7) exclusively in the serum of MM patients; however, little is known about the role of sSLAMF7 in MM biology. In this study, we found that sSLAMF7 enhanced the growth of MM cells via homophilic interaction with surface SLAMF7 and subsequent activation of the SHP-2 and ERK signaling pathways. Elotuzumab suppressed sSLAMF7-induced MM cell growth both in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the SLAMF7 gene. Pharmacological targeting of Ikaros by lenalidomide and its analog pomalidomide downregulated SLAMF7 expression and ameliorated the response of MM cells to sSLAMF7. Elotuzumab blocked the growth-promoting function of sSLAMF7 when combined with lenalidomide in a murine xenograft model. Neutralization of sSLAMF7 is a novel antimyeloma mechanism of elotuzumab, which is enhanced by immunomodulatory drugs via downregulation of surface SLAMF7 expression on MM cells. These findings may provide important information for the optimal use of elotuzumab in MM treatment.
  • Kikuchi J, Hayashi N, Osada N, Sugitani M, Furukawa Y
    Biochemical and biophysical research communications 518 1 134 - 140 2019年10月 [査読有り][通常論文]
  • Wada T, Kikuchi J, Koyama D, Honda H, Furukawa Y
    Leukemia research 82 29 - 32 2019年07月 [査読有り][通常論文]
  • Saito S, Kikuchi J, Koyama D, Sato S, Koyama H, Osada N, Kuroda Y, Akahane K, Inukai T, Umehara T, Furukawa Y
    Clinical cancer research : an official journal of the American Association for Cancer Research 25 5 1601 - 1611 2019年03月 [査読有り][通常論文]
  • Jiro Kikuchi, Yoshiaki Kuroda, Daisuke Koyama, Naoki Osada, Tohru Izumi, Hiroshi Yasui, Takakazu Kawase, Tatsuo Ichinohe, Yusuke Furukawa
    Cancer Research 78 7 1766 - 1778 2018年04月 [査読有り][通常論文]
     
    Multiple myeloma (MM) cells acquire dormancy and drug resistance via interaction with bone marrow stroma cells (BMSC) in a hypoxic microenvironment. Elucidating the mechanisms underlying the regrowth of dormant clones may contribute to further improvement of the prognosis of MM patients. In this study, we find that the CD180/MD-1 complex, a noncanonical lipopolysaccharide (LPS) receptor, is expressed on MM cells but not on normal counterparts, and its abundance is markedly upregulated under adherent and hypoxic conditions. Bacterial LPS and anti-CD180 antibody, but not other Toll-like receptor ligands, enhanced the growth of MM cells via activation of MAP kinases ERK and JNK in positive correlation with expression levels of CD180. Administration of LPS significantly increased the number of CD180/CD138 double-positive cells in a murine xenograft model when MM cells were inoculated with direct attachment to BMSC. Knockdown of CD180 canceled the LPS response in vitro and in vivo. Promoter analyses identified IKZF1 (Ikaros) as a pivotal transcriptional activator of the CD180 gene. Both cell adhesion and hypoxia activated transcription of the CD180 gene by increasing Ikaros expression and its binding to the promoter region. Pharmacological targeting of Ikaros by the immunomodulatory drug lenalidomide ameliorated the response of MM cells to LPS in a CD180-dependent manner in vitro and in vivo. Thus, the CD180/MD-1 pathway may represent a novel mechanism of growth regulation of MM cells in a BM milieu and may be a therapeutic target of preventing the regrowth of dormant MM cells. Significance: This study describes a novel mechanism by which myeloma cells are regulated in the bone marrow, where drug resistance and dormancy can evolve after treatment, with potential therapeutic implications for treating this often untreatable blood cancer.
  • Jiro Kikuchi, Yoshiaki Kuroda, Daisuke Koyama, Yusuke Furukawa
    International Journal of Hematology 107 3 383 - 385 2018年03月 [査読有り][通常論文]
  • Naoki Osada, Jiro Kikuchi, Takashi Umehara, Shin Sato, Masashi Urabe, Tomoyuki Abe, Nakanobu Hayashi, Masahiko Sugitani, Yutaka Hanazono, Yusuke Furukawa
    Oncotarget 9 5 6450 - 6462 2018年 [査読有り][通常論文]
     
    Human induced pluripotent stem cells (hiPSCs) are creating great expectations for regenerative medicine. However, safety strategies must be put in place to guard against teratoma formation after transplantation of hiPSC-derived cells into patients. Recent studies indicate that epigenetic regulators act at the initial step of tumorigenesis. Using gain-of-function and loss-of-function approaches, we show here that the expression and function of lysine-specific demethylase 1 (LSD1) are tightly regulated in hiPSCs, and their deregulation underlies the development of teratomas. Consistent with these results, we demonstrate that an LSD1 inhibitor, S2157, prevented teratoma formation from hiPSCs transplanted into immunodeficient mice. This novel action of LSD1 and the effects of its inhibition potentially allow for the development of new clinical applications and therapeutic strategies using hiPSCs.
  • Furukawa Y, Kuroda Y, Kikuchi J
    [Rinsho ketsueki] The Japanese journal of clinical hematology 59 8 1048 - 1057 2018年 [査読有り][通常論文]
  • Kazuya Takahashi, Takeshi Inukai, Toshihiko Imamura, Mio Yano, Chihiro Tomoyasu, David M. Lucas, Atsushi Nemoto, Hiroki Sato, Meixian Huang, Masako Abe, Keiko Kagami, Tamao Shinohara, Atsushi Watanabe, Shinpei Somazu, Hiroko Oshiro, Koshi Akahane, Kumiko Goi, Jiro Kikuchi, Yusuke Furukawa, Hiroaki Goto, Masayoshi Minegishi, Shotaro Iwamoto, Kanji Sugita
    PLOS ONE 12 12 e0188680  2017年12月 [査読有り][通常論文]
     
    Prognosis of childhood acute lymphoblastic leukemia (ALL) has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ), a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+) ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin) in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ), a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17; 19) ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17; 19) ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17; 19) ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good sensitivity to CFZ and BTZ, and that CFZ combination chemotherapy may be a new therapeutic option with higher anti-leukemic activity for refractory ALL that contain P-glycoprotein-negative leukemia cells.
  • 黒田 芳明, 菊池 次郎, 古川 雄祐
    電気泳動 61 2 93 - 96 日本電気泳動学会 2017年 

    多発性骨髄腫は形質細胞に由来する難治性の造血器腫瘍である.近年,骨髄腫は進展に伴い枝分かれ的に複数種類のクローンが存在することが明らかになってきたが,これらのクローンの違いは遺伝子変異などgeneticなものだけでなく,後天的に獲得された細胞環境因子によるものもあると考えられている.その後天的獲得因子の一つとして細菌感染が骨髄腫増悪に関わっている可能性は,骨髄腫患者の臨床経過から推測可能であり我々の後方視的解析においても細菌感染後に骨髄腫が産生する異常蛋白(M蛋白)が一過性に増加する症例も認められた.そこで我々は細菌感染が骨髄腫細胞の増殖・悪性化に関わる機序を解明すべく基礎研究を行った.結果として,多発性骨髄腫細胞は,骨髄微小環境内においてCD180分子を細胞表面に発現し,細菌感染時にLPSを介したシグナルを受けて増殖能が亢進し,病態の進展に働く機序が示唆された.

  • Yusuke Furukawa, Jiro Kikuchi
    INTERNATIONAL JOURNAL OF HEMATOLOGY 104 3 281 - 292 2016年09月 [査読有り][通常論文]
     
    Multiple myeloma cells acquire the resistance to anti-cancer drugs through physical and functional interactions with the bone marrow microenvironment via two overlapping mechanisms. First, bone marrow stromal cells (BMSCs) produce soluble factors, such as interleukin-6 and insulin-like growth factor-1, to activate signal transduction pathways leading to drug resistance (soluble factor-mediated drug resistance). Second, BMSCs up-regulate the expression of cell cycle inhibitors, anti-apoptotic members of the Bcl-2 family and ABC drug transporters in myeloma cells upon direct adhesion [cell adhesion-mediated drug resistance (CAM-DR)]. Elucidation of the mechanisms underlying drug resistance may greatly contribute to the advancement of cancer therapies. Recent investigations, including ours, have revealed the involvement of epigenetic alterations in drug resistance especially CAM-DR. For example, we found that class I histone deacetylases (HDACs) determine the sensitivity of proteasome inhibitors and the histone methyltransferase EZH2 regulates the transcription of anti-apoptotic genes during the acquisition of CAM-DR by myeloma cells. In addition, another histone methyltransferase MMSET was shown to confer drug resistance to myeloma cells by facilitating DNA repair. These findings provide a rationale for the inclusion of epigenetic drugs, such as HDAC inhibitors and histone methylation modifiers, in combination chemotherapy for MM patients to increase the therapeutic index.
  • Furukawa Y, Kikuchi J
    [Rinsho ketsueki] The Japanese journal of clinical hematology 57 5 546 - 555 2016年05月 [査読有り][通常論文]
  • Atsushi Nemoto, Satoshi Saida, Itaru Kato, Jiro Kikuchi, Yusuke Furukawa, Yasuhiro Maeda, Koshi Akahane, Hiroko Honna-Oshiro, Kumiko Goi, Keiko Kagami, Shinya Kimura, Yuko Sato, Seiichi Okabe, Akira Niwa, Kenichiro Watanabe, Tatsutoshi Nakahata, Toshio Heike, Kanji Sugita, Takeshi Inukai
    MOLECULAR CANCER THERAPEUTICS 15 1 94 - 105 2016年01月 [査読有り][通常論文]
     
    S-phase progression of the cell cycle is accelerated in tumors through various genetic abnormalities, and, thus, pharmacologic inhibition of altered cell-cycle progression would be an effective strategy to control tumors. In the current study, we analyzed the antileukemic activity of three available small molecules targeting CDK4/CDK6 against lymphoid crisis of chronic myeloid leukemia (CML-LC) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and found that all three molecules showed specific activities against leukemic cell lines derived from CML-LC and Ph+ ALL. In particular, PD0332991 exhibited extremely high antileukemic activity against CML-LC and Ph+ ALL cell lines in the nanomolar range by the induction of G(0)-G(1) arrest and partially cell death through dephosphorylation of pRb and downregulation of the genes that are involved in S-phase transition. As an underlying mechanism for favorable sensitivity to the small molecules targeting CDK4/CDK6, cell-cycle progression of Ph+ lymphoid leukemia cells was regulated by transcriptional and posttranscriptional modulation of CDK4 as well as Cyclin D2 gene expression under the control of BCR-ABL probably through the PI3K pathway. Consistently, the gene expression level of Cyclin D2 in Ph+ lymphoid leukemia cells was significantly higher than that in Ph+ lymphoid leukemia cells. Of note, three Ph+ ALL cell lines having the T315I mutation also showed sensitivity to PD0332991. In a xenograft model, PD0332991, but not imatinib, suppressed dissemination of Ph+ ALL having the T315I mutation and prolonged survival, demonstrating that this reagent would be a new therapeutic modality for relapsed CML-LC and Ph+ ALL patients after treatment with tyrosine kinase inhibitors. (C) 2015 AACR.
  • Jiro Kikuchi, Daisuke Koyama, Taeko Wada, Tohru Izumi, Peter O. Hofgaard, Bjarne Bogen, Yusuke Furukawa
    JOURNAL OF CLINICAL INVESTIGATION 125 12 4375 - 4390 2015年12月 [査読有り][通常論文]
     
    Alterations in chromatin modifications, such as histone methylation, have been suggested as mediating chemotherapy resistance in several cancer types; therefore, elucidation of the epigenetic mechanisms that underlie drug resistance may greatly contribute to the advancement of cancer therapies. In the present study, we identified histone H3-lysine 27 (H3K27) as a critical residue for epigenetic modification in multiple myeloma. We determined that abrogation of drug-induced H3K27 hypermethylation is associated with cell adhesion-mediated drug resistance (CAM-DR), which is the most important form of drug resistance, using a coculture system to evaluate stroma cell adhesion-dependent alterations in multiple myeloma cells. Cell adhesion counteracted anticancer drug-induced hypermethylation of H3K27 via inactivating phosphorylation of the transcription regulator EZH2 at serine 21, leading to the sustained expression of antiapoptotic genes, including IGF1, B cell CLL/Iymphoma 2 (BCL2), and hypoxia inducible factor 1, alpha subunit (H/F1A). Pharmacological and genetic inhibition of the IGF-1R/P13K/AKT pathway reversed CAM-DR by promoting EZH2 dephosphorylation and H3K27 hypermethylation both in vitro and in refractory murine myeloma models. Together, our findings identify and characterize an epigenetic mechanism that underlies CAM-DR and suggest that kinase inhibitors to counteract EZH2 phosphorylation should be included in combination chemotherapy to increase therapeutic index.
  • Taeko Wada, Daisuke Koyama, Jiro Kikuchi, Hiroaki Honda, Yusuke Furukawa
    BLOOD 125 24 3731 - 3746 2015年06月 [査読有り][通常論文]
     
    Recent investigations indicate that epigenetic regulators act at the initial step of myeloid leukemogenesis by forming preleukemic hematopoietic stem cells (HSCs), which possess the increased self-renewal potential but retain multidifferentiation ability, and synergize with genetic abnormalities in later stages to develop full-blown acute myeloid leukemias. However, it is still unknown whether this theory is applicable to other malignancies. In this study, we demonstrate that lysine-specific demethylase 1 (LSD1) overexpression is a founder abnormality for the development of T-cell lymphoblastic leukemia/lymphoma (T-LBL) using LSD1 transgenic mice. LSD1 expression is tightly regulated via alternative splicing and transcriptional repression in HSCs and is altered in most leukemias, especially T-LBL. Overexpression of the shortest isoform of LSD1, which is specifically repressed in quiescent HSCs and demethylates histone H3K9 more efficiently than other isoforms, increases self-renewal potential via upregulation of the HoxAfamily but retains multidifferentiation ability with a skewed differentiation to T-cell lineages at transcriptome levels in HSCs. Transgenic mice overexpressing LSD1 in HSCs did not show obvious abnormalities but developed T-LBL at very high frequency after g-irradiation. LSD1 overexpression appears to be the first hit in T-cell leukemogenesis and provides an insight into novel strategies for early diagnosis and effective treatment of the disease.
  • Yusuke Furukawa, Jiro Kikuchi
    INTERNATIONAL JOURNAL OF CLINICAL ONCOLOGY 20 3 413 - 422 2015年06月 [査読有り][通常論文]
     
    Multiple myeloma (MM), one of the most intractable malignancies, is characterized by the infiltration and growth of plasma cells, the most differentiated cells in the B-cell lineage, in the bone marrow. Despite the introduction of novel therapeutic agents, including proteasome inhibitors and immunomodulatory drugs, the prognosis of patients with MM is still worse than that of most hematological malignancies. A better understanding of the molecular pathogenesis of the disease is essential to achieve any improvement of treatment outcome of MM patients. All MM cases pass through the phase of asymptomatic expansion of clonal plasma cells, referred to as monoclonal gammopathy of undetermined significance (MGUS). It has long been believed that MM evolves linearly from MGUS to terminal phases, such as extramedullary tumors and plasma cell leukemia via the accumulation of novel mutations. However, recent studies using next-generation sequencing have disclosed the complex genomic architecture of the disease. At each step of progression, the acquisition of novel mutations is accompanied by subclonal evolution from reservoir clones with branching patterns. Each subclone may carry novel mutations and distinct phenotypes, including drug sensitivity. In addition, minor clones already exist at the MGUS stage, which could expand later in the clinical course, resulting in relapse and/or leukemic conversion. The ultimate goal of treatment is to eradicate all clones, including subclonal populations with distinct biological characteristics. This goal could be achieved by further improving treatment strategies that reflect the genomic landscape of the disease.
  • K. Tago, M. Funakoshi-Tago, H. Itoh, Y. Furukawa, J. Kikuchi, T. Kato, K. Suzuki, K. Yanagisawa
    ONCOGENE 34 3 310 - 318 2015年01月 [査読有り][通常論文]
     
    Tumor suppressor protein p19(ARF) (Arf; p14(ARF) in humans) functions in both p53-dependent and -independent modes to counteract hyper-proliferative signals caused by proto-oncogene activation, but its p53-independent activities remain poorly understood. Using the tandem affinity purification-tag technique, we purified Arf-containing protein complexes and identified p68 DEAD-box protein (DDX5) as a novel interacting protein of Arf. In this study, we found that DDX5 interacts with c-Myc, and harbors essential roles for c-Myc-mediated transcription and its transforming activity. Furthermore, when c-Myc was forcibly expressed, the expression level of DDX5 protein was drastically increased through the acceleration of protein synthesis of DDX5, suggesting the presence of an oncogenic positive feedback loop including c-Myc and DDX5. Strikingly, Arf blocked the physical interaction between DDX5 and c-Myc, and drove away DDX5 from the promoter of c-Myc target genes. These observations most likely indicate the mechanism by which Arf causes p53-independent tumor-suppressive activity.
  • Kikuchi J, Furukawa Y
    Nihon rinsho. Japanese journal of clinical medicine 73 1 57 - 61 2015年01月 [査読有り][通常論文]
  • D. Koyama, J. Kikuchi, N. Hiraoka, T. Wada, H. Kurosawa, S. Chiba, Y. Furukawa
    LEUKEMIA 28 6 1216 - 1226 2014年06月 [査読有り][通常論文]
     
    The Notch signaling pathway has been recognized as a key factor for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL), because of the high incidence of activating mutations of Notch1. Notch inhibition could serve as a new treatment strategy for T-ALL; however, the attempts to perturb Notch signaling pathways have been unsuccessful so far. In this study, we found that proteasome inhibitors exert cytotoxic effects on T-ALL cells with constitutive activation of Notch1 to a similar extent as myeloma cells. The proteasome inhibitor bortezomib repressed the transcription of Notch1 and downstream effectors including Hes1, GATA3, RUNX3 and nuclear factor-kappa B (NF-kappa B) (p65 and p50), coincided with downregulation of the major transactivator Sp1 and its dissociation from Notch1 promoter. Overexpression of the Notch1 intracellular domain (NICD) significantly ameliorated bortezomib-induced cytotoxicity against T-ALL cells. Drug combination studies revealed that bortezomib showed synergistic or additive effects with key drugs for the treatment of T-ALL such as dexamethasone (DEX), doxorubicin and cyclophosphamide, which were readily abolished by NICD overexpression. The synergy of bortezomib and DEX was confirmed in vivo using a murine xenograft model. Our findings provide a molecular basis and rationale for the inclusion of proteasome inhibitors in treatment strategies for T-ALL.
  • Jiro Kikuchi, Daisuke Koyama, Harumi Y. Mukai, Yusuke Furukawa
    INTERNATIONAL JOURNAL OF HEMATOLOGY 99 6 726 - 736 2014年06月 [査読有り][通常論文]
     
    Several clinical trials have demonstrated the effectiveness of bortezomib in combination with various anti-myeloma agents; however, no definitive information is available regarding drugs best suited for use in combination with bortezomib. Using isobologram analysis, we investigated the combined effects of bortezomib with four key anti-myeloma drugs (melphalan, cyclophosphamide, doxorubicin and lenalidomide), which represent components of major bortezomib-based regimens with corticosteroids, in three myeloma cell lines (U266, RPMI8226 and KMS-12BM) under various conditions. Melphalan showed the best performance with bortezomib under all culture conditions tested (liquid culture, on fibronectin-coated plates, and co-culture with bone marrow stromal cells), whereas cyclophosphamide was antagonistic with bortezomib especially in the presence of stromal cells. Doxorubicin showed additive effects under stroma-free conditions and in contact with fibronectin, but was rather antagonistic in the presence of stromal cells. In contrast, lenalidomide exerted the most favorable effect with bortezomib in contact with stromal cells. Consistent with these results, caspase-3 was activated more strongly by melphalan than by other agents in combination with bortezomib. Moreover, bortezomib-induced up-regulation of CHOP was readily enhanced by lenalidomide in contact with stromal cells. The present findings may provide fundamental information for the selection of bortezomib-based regimens for myeloma patients.
  • Kikuchi J, Furukawa Y
    Nihon rinsho. Japanese journal of clinical medicine 72 6 1136 - 1142 2014年06月 [査読有り][通常論文]
  • Nobuya Hiraoka, Jiro Kikuchi, Takahiro Yamauchi, Daisuke Koyama, Taeko Wada, Mitsuyo Uesawa, Miyuki Akutsu, Shigehisa Mori, Yuichi Nakamura, Takanori Ueda, Yasuhiko Kano, Yusuke Furukawa
    PLOS ONE 9 3 e90675  2014年03月 [査読有り][通常論文]
     
    Bendamustine has shown considerable clinical activity against indolent lymphoid malignancies as a single agent or in combination with rituximab, but combination with additional anti-cancer drugs may be required for refractory and/or relapsed cases as well as other intractable tumors. In this study, we attempted to determine suitable anti-cancer drugs to be combined with bendamustine for the treatment of mantle cell lymphoma, diffuse large B-cell lymphoma, aggressive lymphomas and multiple myeloma, all of which are relatively resistant to this drug, and investigated the mechanisms underlying synergism. Isobologram analysis revealed that bendamustine had synergistic effects with alkylating agents (4-hydroperoxy-cyclophosphamide, chlorambucil and melphalan) and pyrimidine analogues (cytosine arabinoside, gemcitabine and decitabine) in HBL-2, B104, Namalwa and U266 cell lines, which represent the above entities respectively. In cell cycle analysis, bendamustine induced late S-phase arrest, which was enhanced by 4-hydroperoxy-cyclophosphamide, and potentiated early S-phase arrest by cytosine arabinoside (Ara-C), followed by a robust increase in the size of sub-G1 fractions. Bendamustine was able to elicit DNA damage response and subsequent apoptosis faster and with shorter exposure than other alkylating agents due to rapid intracellular incorporation via equilibrative nucleoside transporters (ENTs). Furthermore, bendamustine increased the expression of ENT1 at both mRNA and protein levels and enhanced the uptake of Ara-C and subsequent increase in Ara-C triphosphate (Ara-CTP) in HBL-2 cells to an extent comparable with the purine analog fludarabine. These purine analog-like properties of bendamustine may underlie favorable combinations with other alkylators and pyrimidine analogues. Our findings may provide a theoretical basis for the development of more effective bendamustine-based combination therapies.
  • Piyanuch Sripayap, Tadashi Nagai, Kaoru Hatano, Jiro Kikuchi, Yusuke Furukawa, Keiya Ozawa
    ACTA HAEMATOLOGICA 132 1 1 - 4 2014年 [査読有り][通常論文]
  • N. Hiraoka, J. Kikuchi, D. Koyama, T. Wada, S. Mori, Y. Nakamura, Y. Furukawa
    BLOOD CANCER JOURNAL 3 e169  2013年12月 [査読有り][通常論文]
  • F. J. Calero-Nieto, A. Joshi, N. Bonadies, S. Kinston, W-I Chan, E. Gudgin, C. Pridans, J-R Landry, J. Kikuchi, B. J. Huntly, B. Gottgens
    ONCOGENE 32 48 5471 - 5480 2013年11月 [査読有り][通常論文]
     
    The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2.
  • Jiro Kikuchi, Satoshi Yamada, Daisuke Koyama, Taeko Wada, Masaharu Nobuyoshi, Tohru Izumi, Miyuki Akutsu, Yasuhiko Kano, Yusuke Furukawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 288 35 25593 - 25602 2013年08月 [査読有り][通常論文]
     
    Bortezomib therapy is now indispensable for multiple myeloma, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. The development of orally active proteasome inhibitors with distinct mechanisms of action is therefore eagerly awaited. Previously, we identified homopiperazine derivatives as a novel class of proteasome inhibitors with a different mode of proteasome binding from bortezomib. In this study, we show that K-7174, one of proteasome inhibitory homopiperazine derivatives, exhibits a therapeutic effect, which is stronger when administered orally than intravenously, without obvious side effects in a murine myeloma model. Moreover, K-7174 kills bortezomib-resistant myeloma cells carrying a beta 5-subunit mutation in vivo and primary cells from a patient resistant to bortezomib. K-7174 induces transcriptional repression of class I histone deacetylases (HDAC1, -2, and -3) via caspase-8-dependent degradation of Sp1, the most potent transactivator of class I HDAC genes. HDAC1 overexpression ameliorates the cytotoxic effect of K-7174 and abrogates histone hyperacetylation without affecting the accumulation of ubiquitinated proteins in K-7174-treated myeloma cells. Conversely, HDAC inhibitors enhance the activity of K-7174 with an increase in histone acetylation. These results suggest that class I HDACs are critical targets of K-7174-induced cytotoxicity. It is highly anticipated that K-7174 increases the tolerability and convenience of patients by oral administration and has the clinical utility in overcoming bortezomib resistance as a single agent or in combination with HDAC inhibitors.
  • Furukawa Y, Kikuchi J
    [Rinsho ketsueki] The Japanese journal of clinical hematology 54 513 - 521 6 2013年06月 [査読有り][通常論文]
  • Jiro Kikuchi, Naoya Shibayama, Satoshi Yamada, Taeko Wada, Masaharu Nobuyoshi, Tohru Izumi, Miyuki Akutsu, Yasuhiko Kano, Kanako Sugiyama, Mio Ohki, Sam-Yong Park, Yusuke Furukawa
    PLOS ONE 8 4 e60649  2013年04月 [査読有り][通常論文]
     
    The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (beta 1, beta 2 and beta 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the beta 5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a beta 5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib.
  • I. Kuroda, T. Inukai, X. Zhang, J. Kikuchi, Y. Furukawa, A. Nemoto, K. Akahane, K. Hirose, H. Honna-Oshiro, K. Goi, K. Kagami, H. Yagita, T. Tauchi, Y. Maeda, K. Sugita
    Oncogene 32 13 1670 - 1681 2013年03月 [査読有り][通常論文]
     
    Allogeneic stem cell transplantation (allo-SCT) is a potentially curative therapy for chronic myeloid leukemia and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia, and the graft-vs-leukemia (GVL) effect can eradicate residual leukemia after allo-SCT. Ph(+) leukemia cells frequently express death-inducing receptors (DR4 and DR5) for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is one of the cytotoxic ligands expressed on cytotoxic T cells and natural killer cells mediating the GVL effect. Here we demonstrate that imatinib specifically downregulated DR4 and DR5 expression in cell lines and clinical samples of Ph(+) leukemia. Second-generation tyrosine kinase inhibitors (dasatinib and nilotinib) and short hairpin RNA against bcr-abl also downregulated DR4 and DR5 expression in Ph(+) leukemia cells, and transfection of bcr-abl into a Ph(-) leukemia cell line induced DR4 and DR5 expression, which was abrogated by imatinib treatment. Accordingly, Ph(+) leukemia cells that had been pretreated with imatinib showed resistance to the pro-apoptotic activity of recombinant human soluble TRAIL. These observations demonstrate that BCR-ABL is critically involved in the leukemia-specific expression of DR4 and DR5 and in the susceptibility of Ph(+) leukemia to TRAIL-mediated anti-leukemic activity, providing new insight into the mechanisms of the tumor-specific cytotoxic activities of TRAIL. © 2013 Macmillan Publishers Limited All rights reserved.
  • Masahiro Azuma, Daisuke Koyama, Jiro Kikuchi, Hiromichi Yoshizawa, Dissayabutra Thasinas, Kazuhiro Shiizaki, Makoto Kuro-o, Yusuke Furukawa, Eiji Kusano
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26 10 4264 - 74 2012年10月 [査読有り][通常論文]
     
    The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.
  • Masahiro Azuma, Daisuke Koyama, Jiro Kikuchi, Hiromichi Yoshizawa, Dissayabutra Thasinas, Kazuhiro Shiizaki, Makoto Kuro-O, Yusuke Furukawa, Eiji Kusano
    FASEB Journal 26 10 4264 - 4274 2012年10月 [査読有り][通常論文]
     
    The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to - 1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho downregulation. © FASEB.
  • Kanae Mitsunaga, Jiro Kikuchi, Taeko Wada, Yusuke Furukawa
    JOURNAL OF CELLULAR PHYSIOLOGY 227 3 1138 - 1147 2012年03月 [査読有り][通常論文]
     
    Latexin is the only known carboxypeptidase A inhibitor in mammals and shares structural similarity with cystatin C, suggesting that latexin regulates the abundance of as yet unidentified target proteins. A forward genetic approach revealed that latexin is involved in homeostasis of hematopoietic stem cells (HSCs) in mice; however, little is known about the mechanisms by which latexin negatively affects the numbers of HSCs. In this study, we found that latexin is preferentially expressed in hematopoietic stem/progenitor cells, and is co-localized with the molecules responsible for the interaction of HSCs with a bone marrow niche, such as N-cadherin, Tie2, and Roundabout 4. Latexin-knockout young female mice showed an increase in the numbers of KSL (c-Kit+/Sca-1+/linegae marker-negative) cells, which may be attributable to enhanced self-renewal because latexin-deficient KSL cells formed more colonies than their wild-type counterparts in methylcellulose culture. Proteomic analysis of Sca-1+ bone marrow cells demonstrated that latexin ablation reduced the abundance of multiple cellular proteins, including N-cadherin, Tie2, and Roundabout 4. Finally, we found that latexin expression was lost or greatly reduced in approximately 50% of human leukemia/lymphoma cell lines. These results imply that latexin inhibits the self-renewal of HSCs by facilitating the lodgment of HSCs within a bone marrow niche to maintain HSC homeostasis. J. Cell. Physiol. 227: 11381147, 2012. (C) 2011 Wiley Periodicals, Inc.
  • Taeko Wada, Jiro Kikuchi, Yusuke Furukawa
    EMBO REPORTS 13 2 142 - 149 2012年01月 [査読有り][通常論文]
     
    Relatively little is known about the regulatory mechanisms of the Drosha/DGCR8 complex, which processes miRNAs at the initial step of biogenesis. We found that histone deacetylase 1 (HDAC1) increases the expression levels of mature miRNAs despite repressing the transcription of host genes. HDAC1 is an integral component of the Drosha/DGCR8 complex and enhances miRNA processing by increasing the affinity of DGCR8 to primary miRNA transcripts via deacetylation of critical lysine residues in the RNA-binding domains of DGCR8. This finding suggests that HDACs have two arms for gene silencing: transcriptional repression by promoter histone deacetylation and post-transcriptional inhibition by increasing miRNA abundance.
  • Furukawa Y, Hiraoka N, Wada T, Kikuchi J, Kano Y
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 138 1 26 - 32 1 2011年07月 [査読有り][通常論文]
  • Kinuko Hirose, Takeshi Inukai, Jiro Kikuchi, Yusuke Furukawa, Tomokatsu Ikawa, Hiroshi Kawamoto, S. Helen Oram, Berthold Goettgens, Nobutaka Kiyokawa, Yoshitaka Miyagawa, Hajime Okita, Koshi Akahane, Xiaochun Zhang, Itaru Kuroda, Hiroko Honna, Keiko Kagami, Kumiko Goi, Hidemitsu Kurosawa, A. Thomas Look, Hirotaka Matsui, Toshiya Inaba, Kanji Sugita
    BLOOD 116 6 962 - 970 2010年08月 [査読有り][通常論文]
     
    LMO2, a critical transcription regulator of hematopoiesis, is involved in human T-cell leukemia. The binding site of proline and acidic amino acid-rich protein (PAR) transcription factors in the promoter of the LMO2 gene plays a central role in hematopoietic-specific expression. E2A-HLF fusion derived from t(17;19) in B-precursor acute lymphoblastic leukemia (ALL) has the transactivation domain of E2A and the basic region/leucine zipper domain of HLF, which is a PAR transcription factor, raising the possibility that E2A-HLF aberrantly induces LMO2 expression. We here demonstrate that cell lines and a primary sample of t(17;19)-ALL expressed LMO2 at significantly higher levels than other B-precursor ALLs did. Transfection of E2A-HLF into a non-t(17;19) B-precursor ALL cell line induced LMO2 gene expression that was dependent on the DNA-binding and transactivation activities of E2A-HLF. The PAR site in the LMO2 gene promoter was critical for E2A-HLF-induced LMO2 expression. Gene silencing of LMO2 in a t(17;19)-ALL cell line by short hairpin RNA-induced apoptotic cell death. These observations indicated that E2A-HLF promotes cell survival of t(17;19)-ALL cells by aberrantly up-regulating LMO2 expression. LMO2 could be a target for a new therapeutic modality for extremely chemo-resistant t(17;19)-ALL. (Blood. 2010;116(6):962-970)
  • Jiro Kikuchi, Taeko Wada, Rumi Shimizu, Tohru Izumi, Miyuki Akutsu, Kanae Mitsunaga, Kaoru Noborio-Hatano, Masaharu Nobuyoshi, Keiya Ozawa, Yasuhiko Kano, Yusuke Furukawa
    BLOOD 116 3 406 - 417 2010年07月 [査読有り][通常論文]
     
    Bortezomib is now widely used for the treatment of multiple myeloma (MM); however, its action mechanisms are not fully understood. Despite the initial results, recent investigations have indicated that bortezomib does not inactivate nuclear factor-kappa B activity in MM cells, suggesting the presence of other critical pathways leading to cytotoxicity. In this study, we show that histone deacetylases (HDACs) are critical targets of bortezomib, which specifically down-regulated the expression of class I HDACs (HDAC1, HDAC2, and HDAC3) in MM cell lines and primary MM cells at the transcriptional level, accompanied by reciprocal histone hyperacetylation. Transcriptional repression of HDACs was mediated by caspase-8-dependent degradation of Sp1 protein, the most potent transactivator of class I HDAC genes. Short-interfering RNA-mediated knockdown of HDAC1 enhanced bortezomib-induced apoptosis and histone hyperacetylation, whereas HDAC1 overexpression inhibited them. HDAC1 overexpression conferred resistance to bortezomib in MM cells, and administration of the HDAC inhibitor romidepsin restored sensitivity to bortezomib in HDAC1-overexpressing cells both in vitro and in vivo. These results suggest that bortezomib targets HDACs via distinct mechanisms from conventional HDAC inhibitors. Our findings provide a novel molecular basis and rationale for the use of bortezomib in MM treatment. (Blood. 2010; 116(3): 406-417)
  • T. Odgerel, J. Kikuchi, T. Wada, R. Shimizu, Y. Kano, Y. Furukawa
    LEUKEMIA 24 5 1087 - 1090 2010年05月 [査読有り][通常論文]
  • Mayuko Okuya, Hidemitsu Kurosawa, Jiro Kikuchi, Yusuke Furukawa, Hirotaka Matsui, Daisuke Aki, Takayuki Matsunaga, Takeshi Inukai, Hiroaki Goto, Rachel A. Altura, Kenich Sugita, Osamu Arisaka, A. Thomas Look, Toshiya Inaba
    JOURNAL OF BIOLOGICAL CHEMISTRY 285 3 1850 - 1860 2010年01月 [査読有り][通常論文]
     
    The E2A-HLF fusion transcription factor generated by t(17;19)(q22;p13) translocation is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs) and promotes leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that t(17;19)(+) ALL cells express Survivin at high levels and that a dominant negative mutant of E2A-HLF suppresses Survivin expression. Forced expression of E2A-HLF in t(17;19)(-) leukemia cells up-regulated Survivin expression, suggesting that Survivin is a downstream target of E2A-HLF. Analysis using a counterflow centrifugal elutriator revealed that t(17;19)(+) ALL cells express Survivin throughout the cell cycle. Reporter assays revealed that E2A-HLF induces survivin expression at the transcriptional level likely through indirect down-regulation of a cell cycle-dependent cis element in the promoter region. Down-regulation of Survivin function by a dominant negative mutant of Survivin or reduction of Survivin expression induced massive apoptosis throughout the cell cycle in t(17;19)(+) cells mainly through caspase-independent pathways involving translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. AIF knockdown conferred resistance to apoptosis caused by down-regulation of Survivin function. These data indicated that reversal of AIF translocation by Survivin, which is induced by E2A-HLF throughout the cell cycle, is one of the key mechanisms in the protection of t(17;19)(+) leukemia cells from apoptosis.
  • R. Shimizu, J. Kikuchi, T. Wada, K. Ozawa, Y. Kano, Y. Furukawa
    Leukemia 24 10 1760 - 1768 2010年 [査読有り][通常論文]
     
    Anti-CD20 antibody rituximab is now essential for the treatment of CD20-positive B-cell lymphomas. Decreased expression of CD20 is one of the major mechanisms underlying both innate and acquired resistance to rituximab. In this study, we show that histone deacetylase (HDAC) inhibitors augment the cytotoxic activity of rituximab by enhancing the surface expression of CD20 antigen on lymphoma cells. HDAC inhibitors, valproic acid (VPA) and romidepsin, increased CD20 expression at protein and mRNA levels in B-cell lymphoma cell lines with relatively low CD20 expression levels. The VPA-mediated increase in CD20 expression occurred at 1 m, which is clinically achievable and safe, but insufficient for inducing cell death. Chromatin immunoprecipitation assays revealed that HDAC inhibitors transactivated the CD20 gene through promoter hyperacetylation and Sp1 recruitment. HDAC inhibitors potentiated the activity of rituximab in complement-dependent cytotoxic assays. In mouse lymphoma models, HDAC inhibitors enhanced CD20 expression along with histone hyperacetylation in transplanted cells, and acted synergistically with rituximab to retard their growth. The combination with HDAC inhibitors may serve as an effective strategy to overcome rituximab resistance in B-cell lymphomas. © 2010 Macmillan Publishers Limited All rights reserved.
  • Taeko Wada, Jiro Kikuchi, Noriko Nishimura, Rumi Shimizu, Toshio Kitamura, Yusuke Furukawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 44 30673 - 30683 2009年10月 [査読有り][通常論文]
     
    Histone deacetylases (HDACs) are globally implicated in the growth and differentiation of mammalian cells; however, relatively little is known about their specific roles in hematopoiesis. In this study, we investigated the expression of HDACs in human hematopoietic cells and their functions during hematopoiesis. The expression of HDACs was very low in hematopoietic progenitor cells, which was accompanied by histone hyperacetylation. HDACs were detectable in more differentiated progenitors and erythroid precursors but down-regulated in mature myeloid cells especially granulocytes. In contrast, acute myeloid leukemias showed HDAC overexpression and histone hypoacetylation. Transcription of the HDAC1 gene was repressed by CCAAT/enhancer binding proteins during myeloid differentiation, and activated by GATA-1 during erythromegakaryocytic differentiation. Small interfering RNA-mediated knockdown of HDAC1 enhanced myeloid differentiation in immature hematopoietic cell lines and perturbed erythroid differentiation in progenitor cells. Myeloid but not erythromegakaryocytic differentiation was blocked in mice transplanted with HDAC1-overexpressing hematopoietic progenitor cells. These findings suggest that HDAC is not merely an auxiliary factor of genetic elements but plays a direct role in the cell fate decision of hematopoietic progenitors.
  • Mizuho Maekawa, Jiro Kikuchi, Kazuhiko Kotani, Kohjiro Nagao, Tsogbadrakh Odgerel, Kazumitsu Ueda, Mikihiko Kawano, Yusuke Furukawa, Ikunosuke Sakurabayashi
    ATHEROSCLEROSIS 206 1 216 - 222 2009年09月 [査読有り][通常論文]
     
    Tangier disease (TD) is a hereditary disorder characterized by the severe deficiency or absence of high-density lipoprotein cholesterol (HDL-C). TD is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene, most of which are located in the extracellular loops and nucleotide-binding domains. Here we describe the first case of TD carrying a missense mutation in a transmembrane alpha-helix of ABCA1. A 31-year-old Japanese woman had an extremely low level of HDL-C (1 mg/dl) and yellowish tonsillar swelling, leading to the diagnosis of TD. The proband was homozygous for a point mutation of T4978C in exon 37, which results in the substitution of cysteine-1660 to arginine (C1660R) in the 8th transmembrane segment of ABCA1. Her parents, grandmother, and brother were found to be heterozygous for the same mutation. Both peripheral blood leukocytes from the patient and HEK293 cells transfected with T4978C-mutated ABCA1 normally expressed ABCA1 on the plasma membrane and had normal apolipoprotein A-I-binding ability. However, apolipoprotein A-I-mediated efflux of cholesterol and phospholipids was markedly diminished in HEK293 cells transfected with T4978C-mutated ABCA1. These results suggest that this mutant is normally translated and exists as a stable product with normal localization, yet is functionally defective. Cysteine-1660 appears to be a critical residue for cholesterol transport of ABCA1. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Takahisa Kobayashi, Yusuke Furukawa, Jiro Kikuchi, Chiharu Ito, Yukio Miyata, Shigeaki Muto, Akira Tanaka, Eiji Kusano
    KIDNEY INTERNATIONAL 75 11 1173 - 1183 2009年06月 [査読有り][通常論文]
     
    Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention. Kidney International (2009) 75, 1173-1183; doi:10.1038/ki.2009.44; published online 25 February 2009
  • K. Noborio-Hatano, J. Kikuchi, M. Takatoku, R. Shimizu, T. Wada, M. Ueda, M. Nobuyoshi, I. Oh, K. Sato, T. Suzuki, K. Ozaki, M. Mori, T. Nagai, K. Muroi, Y. Kano, Y. Furukawa, K. Ozawa
    ONCOGENE 28 2 231 - 242 2009年01月 [査読有り][通常論文]
     
    Multiple myeloma ( MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using short hairpin RNA (shRNA) to de. ne the molecule(s) responsible for CAM-DR of MM. Using four bona. de myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226 and U266) and primary myeloma cells, we identified CD29 (beta 1-integrin), CD44, CD49d (alpha 4-integrin, a subunit of VLA-4), CD54 ( intercellular adhesion molecule-1 (ICAM-1)), CD138 (syndecan-1) and CD184 (CXC chemokine receptor-4 (CXCR4)) as major adhesion molecules expressed on MM. shRNA-mediated knockdown of CD49d but not CD44, CD54, CD138 and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically down-regulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.
  • Satoko Oka, Yasuko Nagatsuka, Jiro Kikuchi, Taiji Yokote, Yoshio Hirabayashi, Toshiaki Hanafusa, Keiya Ozawa, Kazuo Muroi
    LEUKEMIA & LYMPHOMA 50 7 1190 - 1197 2009年 [査読有り][通常論文]
     
    Phosphatidylglucoside (PtdGlc), a new type of glycolipid, was recently identified. We examined PtdGlc expression in normal blood cells and leukemic cells using an anti-PtdGlc monoclonal antibody, DIM-21. Neutrophils, monocytes, HL-60 cells and a subset of cord blood (CB) CD34(+) cells, but not erythroblasts, expressed lipid antigen. PtdGlc was preferentially expressed along the neutrophil differentiation pathway of CB CD34(+) cells treated with cytokines and HL-60 cells treated with retinoic acid. PtdGlc expression was not increased in HL-60 cells treated with phorbol ester. CB CD34(+) cells contained a population of PtdGlc(+) cells, and CB CD34(+)PtdGlc(+) cells produced mainly granulocyte-macrophage colonies and a small number of erythroid colonies. A positive correlation between PtdGlc expression and CD15 expression in leukemic cells from patients with acute myeloblastic leukemia was shown. These results indicate that increasing PtdGlc expression is seen with neutrophil maturation.
  • T. Odgerel, J. Kikuchi, T. Wada, R. Shimizu, K. Futaki, Y. Kano, Y. Furukawa
    ONCOGENE 27 22 3102 - 3110 2008年05月 [査読有り][通常論文]
     
    PKC412 is a staurosporine derivative that inhibits several protein kinases including FLT3, and is highly anticipated as a novel therapeutic agent for acute myeloblastic leukemia (AML) carrying FLT3 mutations. In this study, we show that PKC412 exerts differential cell cycle effects on AML cells depending on the presence of FLT3 mutations. PKC412 elicits massive apoptosis without markedly affecting cell cycle patterns in AML cell lines with FLT3 mutations (MV4-11 and MOLM13), whereas it induces G(2) arrest but not apoptosis in AML cell lines without FLT3 mutations (THP-1 and U937). In MV4-11 and MOLM13 cells, PKC412 inactivates Myt-1 and activates CDC25c, leading to the activation of CDC2. Activated CDC2 phosphorylates Bad at serine-128 and facilitates its translocation to the mitochondria, where Bad triggers apoptosis. In contrast, PKC412 inactivates CDC2 by inducing serine-216 phosphorylation and subsequent cytoplasmic sequestration of CDC25c in THP-1 and U937 cells. As a result, cells are arrested in the G2 phase of the cell cycle, but do not undergo apoptosis because Bad is not activated. The FLT3 mutation-dependent differential cell cycle effect of PKC412 is considered an important factor when PKC412 is combined with cell cycle-specific anticancer drugs in the treatment of cancer and leukemia.
  • Chizu Nonomura, Jiro Kikuchi, Nobutaka Kiyokawa, Hidenori Ozaki, Kanae Mitsunaga, Hidenobu Ando, Akiko Kanamori, Reiji Kannagi, Junichiro Fujimoto, Kazuo Muroi, Yusuke Furukawa, Mitsuru Nakamura
    CANCER RESEARCH 68 3 790 - 799 2008年02月 [査読有り][通常論文]
     
    B-cell precursor acute lymphoblastic leukemia (BCP-ALL/B-precursor ALL) is characterized by a high rate of tissue infiltration. The mechanism of BCP-ALL cell extravasation is not fully understood. In the present study, we have investigated the major carrier of carbohydrate selectin ligands in the BCP-ALL cell line NALL-1 and its possible role in the extravascular infiltration of the leukemic cells. B-precursor ALL cell lines and clinical samples from patients with BCP-ALL essentially exhibited positive flow cytometric reactivity with E-selectin, and the reactivity was significantly diminished by O-sialoglycoprotein endopeptidase treatment in NALL-1 cells. B-precursor ALL cell lines adhered well to E-selectin but only very weakly to P-selectin with low-shear-force cell adhesion assay. Although BCP-ALL cell lines did not express the well-known core protein P-selectin glycoprotein ligand-1 (PSGL-1), a major proportion of the carbohydrate selectin ligand was carried by a sialomucin, CD43, in NALL-1 cells. Most clinical samples from patients with BCP-ALL exhibited a PSGL-1(neg/low)/CD43(high) phenotype. NALL-1 cells rolled well on E-selectin, but knockdown of CD43 on NALL-1 cells resulted in reduced rolling activity on E-selectin. In addition, the CD43 knockdown NALL-1 cells showed decreased tissue engraftment compared with the control cells when introduced into gamma-irradiated immunodeficient mice. These results strongly suggest that CD43 but not PSGL-1 plays an important role in the extravascular infiltration of NALL-1 cells and that the degree of tissue engraftment of B-precursor ALL cells may be controlled by manipulating CD43 expression.
  • Jiro Kikuchi, Rumi Shimizu, Taeko Wada, Hidenobu Ando, Mitsuru Nakamura, Keiya Ozawa, Yusuke Furukawa
    STEM CELLS 25 10 2439 - 2447 2007年 [査読有り][通常論文]
     
    E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F- 6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation.
  • J Kikuchi, H Ozaki, C Nonomura, H Shinohara, S Iguchi, H Nojiri, H Hamada, A Kiuchi, M Nakamura
    LEUKEMIA 19 11 1934 - 1940 2005年11月 [査読有り][通常論文]
     
    B-cell precursor (BCP) leukemia cells infiltrate into peripheral organs and the disease often relapses. Inhibition of tissue infiltration may improve the treatment outcome of BCP-leukemia patients. Selectin ligand has been suggested to play an important role in the infiltration of leukemia cells. However, the regulation mechanisms and involvement in tissue infiltration of selectin ligand expression in BCP-leukemia cells are not fully understood. In this study, we report that BCP-leukemia cells express selectin ligand on O-sialoglycoproteins. Core 2 beta 1,6-N-acetylglucosaminyltransferase-1 (C2GnT-1) is mainly expressed in BCP-leukemia cells. Transfection of the antisense C2GnT-1 cDNA resulted in a significant reduction of either selectin ligand expression or selectin-dependent cell adhesion in BCP-leukemia cell line KM3 cells. Migration ability into mouse peripheral organs was reduced significantly in the antisense transfectant. These findings suggest that C2GnT-1 regulates selectin ligand expression. Downregulation of the selectin ligand expression level inhibits tissue infiltration of BCP-leukemia cells. C2GnT-1 may be a candidate of therapeutic target for the inhibition of infiltration of leukemia cells.
  • J Kikuchi, H Shinohara, C Nonomura, H Ando, S Takaku, H Nojiri, ML Nakamura
    GLYCOBIOLOGY 15 3 271 - 280 2005年03月 [査読有り][通常論文]
     
    Sialyl-Lewis x (sLeX), one of the major selectin ligands, is expressed on T and B cells in a differentiation or activation stage-specific manner. We have demonstrated before that sLeX expression and core 2 beta1,6-N-acetylglucosaminyl-transferase (C2GnT) were simultaneously regulated during precursor B (pre-B) cell differentiation. Three C2GnT family genes, designated C2GnT-1, -2, and -3,: were previously identified, but their roles have not, been fully examined. In this study, we have investigated the roles of C2GnTs in the regulation of sLeX expression level during pre-B cell differentiation comparing with alpha1,3fucosyltransferase-VII (FucT-VII) and alpha2,3sialyltransferase-IV (ST3Gal-IV). Overexpression of not FucT-VII and ST3Gal-IV but C2GnT-1 blocked the down-regulation of sLeX expression by differentiation induction. Neither C2GnT-2 nor -3 but C2GnT-1 transcript Was mainly expressed in B lineage cell lines and bone-marrow derived B lineage cells. Significant down-regulation of C2GnT-1 of the three C2GnTs was observed in KM3 cells during differentiation. The expression of C2GnT-1 correlated well to sLeX expression and differentiation stage. Furthermore, introduction of short interfering RNA against C2GnT-1 markedly ' reduced C2GnT-1 expression and resulted in down-regulation of sLeX expression. These results suggest - that not the other. glycosyl-transferases but C2GnT-1 regulates. sLeX expression level during differentiation of pre-B cells, providing, the cells with substrate of sLeX structure biosynthesis.
  • J Kikuchi, J Mimuro, K Ogata, T Tabata, Y Ueda, A Ishiwata, K Kimura, K Takano, S Madoiwa, H Mizukami, Y Hanazono, A Kume, M Hasegawa, K Ozawa, Y Sakata
    JOURNAL OF GENE MEDICINE 6 10 1049 - 1060 2004年10月 [査読有り][通常論文]
     
    Background Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34(+) cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). Methods CD34(+) cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34(+) cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34(+) cells and expression of transgenes were studied. Results We could efficiently transduce CD34(+) cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34(+) cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 106 CD34(+) cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34(+) cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34(+) cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Conclusions Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients. Copyright (C) 2004 John Wiley Sons, Ltd.
  • Sp4は糖鎖遺伝子の転写制御を介してヒトプレB白血病細胞のセレクチンリガンド糖鎖発現を制御している
    菊池 次郎, 野々村 智尋, 安藤 秀信, 藤井 崇, Hur Man-Wook, 古川 雄祐, 高久 静香, 成松 久, 中村 充
    日本血液学会・日本臨床血液学会総会プログラム・抄録集 66回・46回 804 - 804 日本臨床血液学会 2004年09月
  • K Ogata, J Mimuro, J Kikuchi, T Tabata, Y Ueda, M Naito, S Madoiwa, K Takano, M Hasegawa, K Ozawa, Y Sakata
    GENE THERAPY 11 3 253 - 259 2004年02月 [査読有り][通常論文]
     
    We demonstrate that transduction of adipocytes with a simian immunodeficiency virus agm TYO1 ( SIVagm)-based lentiviral vector carrying the human coagulation factor VIII gene (SIVhFVIII) resulted in expression of the human FVIII transgene in vitro and in db/db mice in vivo. Cultured human adipocytes were transduced with the SIVagm vector carrying the GFP gene in a dose-dependent manner and transduction of adipocytes with SIVhFVIII resulted in efficient expression of human coagulation factor VIII (hFVIII; 320 +/- 39.8 ng/10(6) adipocytes/ 24 h) in vitro. Based upon successful transduction of adipocytes by SIV vectors carrying the lacZ gene in vivo in mice, the adipose tissue of db/db mice was transduced with SIVhFVIII. There was a transient appearance of human FVIII in mouse plasma (maximum 1.8 ng/ml) on day 11 after the injection. Transcripts of human FVIII transgene and human FVIII antigen also were detected in the adipose tissue by RT-PCR and immunofluorescence, respectively, on day 14. Emergence of anti-human FVIII antibodies 14 days after the injection of SIVhFVIII may explain the disappearance of human FVIII from the circulation. These results suggest that transduction of the adipocytes with vectors carrying the human FVIII gene may be potentially applicable for gene therapy of hemophilia A.
  • M Naito, J Mimuro, H Endo, S Madoiwa, K Ogata, J Kikuchi, T Sugo, T Yasu, Y Kariya, Y Hoshino, Y Sakata
    CIRCULATION RESEARCH 92 8 865 - 872 2003年05月 [査読有り][通常論文]
     
    Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.
  • J Kikuchi, Y Furukawa, N Hayashi
    MOLECULAR BIOTECHNOLOGY 23 3 203 - 212 2003年03月 [査読有り][通常論文]
     
    Electrospray tandem mass spectrometry (ESI-MS/MS) was combined with biomolecular interaction analysis (BIA) to develop a method of direct protein identification after real-time analysis of protein-protein interactions. Using this,,method, called BIA-MS/MS, we detected multiple p53-interacting proteins in whole tissue extracts from human placenta and liver. Peptide sequencing revealed three proteins whose interaction with p53 had not been previously reported: a cyclin-dependent kinase inhibitor p57/Kip2, a serine/threonine protein phosphatase PPIC, and hemoglobin. Using our system, unambiguous sequence information can be obtained at the femto- to picomole level after repeating the recovery procedure five times. Furthermore, the association and dissociation constants are easily determined by kinetic analysis. This system provides a powerful tool for analyzing complex biological materials in a simple but highly specific and sensitive manner.
  • J Mimuro, S Muramatsu, Y Hakamada, K Mori, J Kikuchi, M Urabe, S Madoiwa, K Ozawa, Y Sakata
    GENE THERAPY 8 22 1690 - 1697 2001年11月 [査読有り][通常論文]
     
    We were able to facilitate plasminogen activator inhibitor I (PAI-1) promoter activity approximately by 14-fold using an enhancer element. This enhanced PAI-1 promoter has a strong basal activity, comparable to CAG promoter activity, and has a response similar to the PAI-1 promoter with respect to TGF beta1 and TNF alpha stimulation, The characteristics of the enhanced PAI-1 promoter are thought to be suited to timely and tissue-specific expression of anticoagulant molecules in the vascular cells, Thus, we developed recombinant adeno-associated virus (rAAV) vectors using the enhanced PAI-1 promoter and were successful in transducing vascular endothelial cells to express the thrombomodulin transgene under the regulation of the enhanced PAI-1 promoter in vitro. Thromobomodulin transgene expression driven by the enhanced PAI-1 promoter in rAAV vector-transduced cultured endothelial cells was between 600- and 1000-fold higher than constitutive thrombomodulin gene expression in cultured human umbilical vein endothelial cells and was up-regulated by TGF beta1 and TNF alpha stimulation which may down-regulate endogenous thrombomodulin gene expression in endothelial cells. The brain vascular endothelial cells of Mongolian gerbils could also be transduced by the same rAAV vector in vivo. Transduction of endothelial cells by rAAV vectors to express enhanced PAI-1 promoter-driven transgenes may be a useful gene therapy approach for vascular diseases.
  • Y Furukawa, J Kikuchi, M Nakamura, S Iwase, H Yamada, M Matsuda
    BRITISH JOURNAL OF HAEMATOLOGY 110 3 663 - 673 2000年09月 [査読有り][通常論文]
     
    To maintain the fidelity and integrity of blood formation, the cell cycle is under strict regulation during haematopoietic cell differentiation. To elucidate the molecular mechanisms of cell cycle regulation during haematopoiesis, we examined cell cycle control gene expression during lineage-specific differentiation from CD34(+) progenitor cells. Expression of cyclin-dependent kinases (cdks) and cyclins, except cdk4, was generally suppressed in CD34(+) cells freshly isolated from the bone marrow of healthy volunteers. Among four major cdk inhibitors, p16 was expressed more highly in CD34(+) cells than in CD34-negative bone marrow mononuclear cells, whereas the amounts of p21 and p27 transcripts increased in the CD34(-) population. The behaviour of cell cycle control genes during haematopoietic differentiation was classified into four patterns: (i) universal upregulation (cdc2, cdk2, cyclin A, cyclin B and p21): (ii) upregulation in specific lineages (cyclin D1, cyclin D3 and p15): (iii) no induction or stable expression (cdk4, cyclin D2, cyclin E and p27): and (iv) universal downregulation (p16). Lineage-specific changes included the sustained elevation of cdc2 and cyclin A during erythroid differentiation, cyclin D1 and p15 induction in myeloid lineage and selective upregulation of cyclin D3 in megakaryocytes. Blocking induction of cyclin D3 resulted in the inhibition of megakaryocytic differentiation. These results suggest that the expression of cell cycle control genes is distinctively regulated in a lineage-dependent manner, reflecting the cell cycle characteristics of each lineage. Some of these genes play an essential role in the process of differentiation itself.
  • Y Furukawa, S Iwase, J Kikuchi, Y Terui, M Nakamura, H Yamada, Y Kano, M Matsuda
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 28 21661 - 21667 2000年07月 [査読有り][通常論文]
     
    Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.
  • J Kikuchi, Y Furukawa, O Suzuki, N Hayashi, M Nakamura, M Morita, M Matsuda
    MOLECULAR BIOTECHNOLOGY 15 3 193 - 200 2000年07月 [査読有り][通常論文]
     
    In this article, we develop a novel subtraction method using carbodiimide-bound microplates. This method utilizes the high affinity of carbodiimides for both single- and double-stranded nucleic acids. Carbodiimide-mediated end-attachment of driver RNA to microplates allows semisolid phase hybridization between driver RNA and target cDNA, and ensures easy removal of RNA/cDNA hybrids composed of the genes commonly expressed in driver and target. As a result, the target-specific genes are left unhybridized and enriched in the hybridization supernatant. We define the optimal conditions for the method as a target/driver RNA ratio of 1:10 and a period of hybridization of 24 h. There are at least three major advantages with the present method: (1) The entire procedure, which consists of two steps, is very simple; (2) hybridization efficiency can be monitored before further processing of the samples; and (3) rare transcripts can be effectively enriched. This method may be a powerful tool to isolate the genes specifically expressed in particular cell or tissue types, and is easily applicable to many studies in molecular biology and genetics. Isolation of polyploid megakaryocyte-specific genes is shown as an example.
  • J Kikuchi, Y Furukawa, N Kubo, A Tokura, N Hayashi, M Nakamura, M Matsuda, Sakurabayashi, I
    ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY 20 1 128 - 134 2000年01月 [査読有り][通常論文]
     
    Recently, we have found that aggregated low density lipoprotein (agLDL) inhibits apoptosis of lipid-bearing macrophages, thereby facilitating foam cell formation and atherosclerosis, To clarify the mechanisms by which agLDL inhibits apoptosis of macrophages, we isolated the genes specifically induced by agLDL by using a subtraction-based cloning strategy. One of the cloned genes, termed low density lipoprotein (LDL)-inducible gene (LIG), encodes a human homologue of bovine ubiquitin-conjugating enzyme E2-25K. Although LIG mRNA was ubiquitously expressed among human tissues, including hematopoietic cells, the abundance of transcripts was markedly increased by agLDL treatment in activated monocytes. LIG mRNA expression was not enhanced by nonatherogenic lipoproteins such as native LDL and high density lipoprotein, suggesting a role in atherosclerosis, Polyubiquitination of intracellular proteins was observed in monocytes cultured with agLDL, which coincided with upregulation of LIG. Furthermore, ubiquitin-dependent degradation of p53, an inducer of apoptosis, was accompanied by LIG induction in agLDL-treated monocytes. The antiapoptotic effect of agLDL was abrogated by a specific proteasome inhibitor, which also increased the half-life of p53 in monocytes. These results suggest that LIG contributes to foam cell formation by the suppression of apoptosis of lipid-bearing macrophages through ubiquitination and subsequent degradation of p53.
  • J Kikuchi, T Asakura, K Hasuda, T Ito, K Ohwaku, H Araki, MP Williamson
    JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS 42 1-2 35 - 47 2000年01月 [査読有り][通常論文]
     
    Because of the complexity arising from the large molecular size and the amino acid sequence homologies of IgG-binding domains of Staphylococcal Protein A (SpA), we have introduced a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependence of the NMR chemical shifts for its structure analysis. In order to enable selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt = 27 kDa) which consists of E, D, A, B and 13 residues of the C-domain was used in this study. Amide proton chemical shifts, measured using uniformly N-15-labeled LPA and LPA labeled selectively with N-15-alanine, show that the turn between helices 1 and 2, and its tertiary interactions with helix 3, are very similar in all domains. This contradicts previous results obtained using independent structure calculations on isolated domains. The close similarity in NH and N-15 chemical shifts of alanine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical environment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:203-208], even though it is not observed in the crystal structure [Deisenhofer J. Biochemistry 1981;20:2361-2370]. (C) 2000 Elsevier Science B.V. All rights reserved.
  • Y Furukawa, N Kubo, J Kikuchi, A Tokura, N Fujita, Sakurabayashi, I
    ELECTROPHORESIS 21 2 338 - 346 2000年01月 [査読有り][通常論文]
     
    The effect of aggregated low-density lipoprotein (agLDL) on cell viability and macrophage-specific gene expression using human peripheral blood monocytes in culture was investigated. AgLDL suppressed activation-induced cell death of phorbol ester-treated macrophages. The inhibition of apoptosis was accompanied by downregulation of apoptosis-promoting proteases, including interleukin-l beta-converting enzyme (ICE) and CPP32 and upregulation of anti-apoptotic cytokine (interleukin-1 beta (IL-1 beta)). In contrast, macrophage-colony stimulating factor (M-CSF) enhanced cell death of lipid-bearing macrophages, suggesting that the anti-atherogenic action of M-CSF is at least in part mediated through apoptotic elimination of macrophages. Then, we attempted to isolate the genes specifically induced by agLDL in macrophages using a subtraction-based cloning strategy. One of the genes isolated, termed LIG (LDL-inducible gene), encodes a human homolog of E2 ubiquitin-conjugating enzyme. Ubiquitination of multiple intracellular proteins was observed in agLDL-treated macrophages, which coincided with upregulation of LIG. These results suggest that LIG acts as a direct mediator of foam cell formation through polyubiquitination and subsequent degradation of cellular proteins with apoptosis-inducing properties. The regulation of apoptosis by macrophage-specific gene expression may contribute to foam cell formation and atherosclerosis.
  • M Nakamura, T Ishida, J Kikuchi, Y Furukawa, M Matsuda
    FEBS LETTERS 463 1-2 125 - 128 1999年12月 [査読有り][通常論文]
     
    We have investigated the regulation mechanism of the surface sialyl-Le(x) (sLe(x)) expression level in tonsillar B cells during activation, sLe(x) antigen became strongly positive after activation, while resting B cells were weakly positive. sLe(x) structures were mainly located on O-linked oligosaccharide chains of glycoprotein. Transcripts of FucT-VII and core 2 GlcNAc transferase (C2GnT) were up-regulated after activation, while those of ST3GallV and beta 1-->4GalT-I were expressed constitutively, However, the up-regulation of C2GnT was more dramatic than that of FucT-VII, These results suggest that sLe(x) expression level is regulated by C2GnT during tonsillar B cell activation. (C) 1999 Federation of European Biochemical Societies.
  • M Mori, Y Terui, M Ikeda, H Tomizuka, M Uwai, T Kasahara, N Kubota, T Itoh, Y Mishima, M Douzono-Tanaka, M Yamada, S Shimamura, J Kikuchi, Y Furukawa, Y Ishizaka, K Ikeda, H Mano, K Ozawa, K Hatake
    BLOOD 94 8 2744 - 2753 1999年10月 [査読有り][通常論文]
     
    Major histocompatibility complex (MHC) molecules play an important role in antigen presentation for induction of tumor as well as cellular and humoral immunities. Recent studies using anti-MHC antibodies demonstrated that antibodies specific for HLA class I molecules induced cellular activation and a type of apoptosis that may be distinct from Fas-dependent or TNFR (tumor necrosis factor-alpha receptor)-dependent processes. We purified a previously untested apoptosis-inducing factor from HL-60 human leukemic cell-conditioned media to homogeneity and sequenced it. It was identified as beta(2)-microglobulin (beta(2)m), which has been previously known as thymotaxin and is a part of the HLA class I antigen complex. beta(2)m acts on both T-leukemic cells and myeloid leukemic cells to induce apoptosis, which then activates caspase 1 and 3. Cross-linking studies showed that biotinilated beta(2)m recognized an epitope distinct from those recognized by the anti-HLA class I antibody, as reported previously. We demonstrated that beta(2)m plays a previously unrecognized and important role in regulating the elimination of tumor cells, which occurs as a result of the action of beta(2)m as an apoptosis-inducing factor. (C) 1999 by The American Society of Hematology.
  • S Iwase, Y Furukawa, J Kikuchi, S Saito, M Nakamura, R Nakayama, J Horiguchi-Yamada, H Yamada
    FEBS LETTERS 450 3 263 - 267 1999年05月 [査読有り][通常論文]
     
    To investigate mechanisms of interferon (IFN) resistance, we have established an IFN-resistant Daudi subline (Daudi(res)), which is 1x10(4) times more resistant to IFN-alpha than parental cells, Among the IFN-inducible genes examined, only ICE mRNA expression was deficient in Daudi(res) cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN-induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudi(res) cells, and forced expression of IRF-1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance, (C) 1999 Federation of European Biochemical Societies.
  • Y Furukawa, S Iwase, J Kikuchi, M Nakamura, H Yamada, M Matsuda
    ONCOGENE 18 11 2003 - 2014 1999年03月 [査読有り][通常論文]
     
    E2F is a heterodimeric transcription factor composed of one of five E2F subunits (E2F-1 to E2F-5) and a DP subunit, E2F regulates the expression of several growth-promoting genes, and thus, can be a target of antiproliferative action of interferons (IFNs), In this study, me investigated the mechanisms whereby IFN-alpha suppresses transcription of the E2F-1 gene. Transfection studies revealed that E2F-1 promoter was functionally divided into two parts: upstream activation sequences (UAS) and a downstream negative-regulatory element (E2F-binding sites). When cells were proliferating, transcription of the E2F-1 gene was primarily driven by the UAS, while E2F sites were not involved in activation. IFN-alpha markedly reduced E2F-1 promoter activity, but introduction of non-binding mutation at the E2F sites completely abrogated the inhibition. Free E2F-1 was found to be the predominant species bound to the E2F sites in proliferating cells. IFN-alpha induced upregulation of E2F-4 along with dephosphorylation of PRE and p130, which resulted in the formation of E2F-4/pRB and E2F-4/p130 complexes on the E2F-1 promoter, These complexes function as transcriptional repressors to inhibit E2F-1 mRNA expression. Our findings indicate that E2F-4 is a critical regulator of E2F-1, which offer an excellent paradigm for understanding functional diversity within the E2F family.
  • M Nakamura, Y Furukawa, R Sasaki, J Masuyama, J Kikuchi, S Iwase, T Kudo, H Narimatsu, S Asakura, S Fujiwara, J Inokuchi
    GLYCOBIOLOGY 9 1 1 - 12 1999年01月 [査読有り][通常論文]
     
    Expression mechanism of CD15s (sialyl-Le(X), sLe(X)) antigen has been investigated using human B lymphoid cell lines. sLeX structures were not expressed in mature B lymphoids but highly expressed in pre-B leukemia and pre-B lymphoma cell lines, The expression site was mainly on the O-linked oligosaccharide chains and E-selectin mediated-cell adhesion capability of sLe(X)-positive cells were significantly suppressed by benzyl-alpha-GalNAc treatment. Subsequently, the bases of the sLeX expression control mechanism were examined at the levels of enzymatic activities and transcripts of glycosyltransferases, (1) The activities of alpha 1-->3fucosyltransferase, alpha 2-->3sialyltransferase, beta 1-->4Gal-transferase, and elongation beta 1-->3GlcNAc-transferase, did not correlate with sLeX expression levels. (2) The transcripts of Fuc-TVII were not parallel with sLeX expression, and those of ST3Gal IV and beta 1-->4Gal-transferase were constitutively detected in all cell lines tested, (3) There was no detectable enzyme activity for core 3 and 4 backbone structure synthesis in human B cell lines. (4) By contrast, the enzyme activities and transcripts of UDP-GlcNAc: Gal beta 1-->3GalNAc (GlcNAc to GalNAc) beta 1-->6N-acetylglucosaminyltransferase (Core2GnT) had significant correlation with the cell surface expression of sLe(X) antigen. (5) Moreover, Western blot analysis revealed the presence of a major similar to 150 kDa glycoprotein that carries O-linked oligosaccharides recognized by anti-sLe(X) monoclonal antibody in sLe(X)-positive pre-B leukemia cell lines. This correlation of Core2GnT with CD15s expression suggests that Core2GnT is a regulator of the cell surface expression of sLe(X) in human pre-B lymphoid cells.
  • M Nakamura, T Kudo, H Narimatsu, Y Furukawa, J Kikuchi, S Asakura, W Yang, S Iwase, K Hatake, Y Miura
    JOURNAL OF BIOLOGICAL CHEMISTRY 273 41 26779 - 26789 1998年10月 [査読有り][通常論文]
     
    Sialyl-Le(x) (sLe(x)) antigen expression recognized by KM93 monoclonal antibody was significantly down-regulated during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human pre-B lymphocytic leukemia cell line KM3. The sLe(x) determinants were almost exclusively expressed on O-linked oligosaccharide chains of an O-glycosylated 150-kDa glycoprotein (gp150). A low shear force cell adhesion assay showed that TPA treatment significantly inhibited E-selectin-mediated cell adhesion. Transcript and/or enzyme activity levels of alpha 1-->3-fucosyltransferase, alpha 2-->3-sialyltransferase, beta 1-->4-galactosyltransferase, and elongation beta 1-->3-N-acetylglucosaminyltransferase did not correlate with sLe(x) expression levels. However, transcript and enzyme activity levels of core 2 GlcNAc-transferase (C2GnT) were significantly down-regulated during TPA treatment. Following transfection and constitutive expression of full-length exogenous C2GnT transcript, C2GnT enzyme activities were maintained at high levels even after TPA treatment and down-regulation of cell surface sLe(x) antigen expression by TPA was completely abolished. Furthermore, in the transfected cells, the KM93 reactivity of gp150 was not reduced by TPA treatment, and the inhibition of cell adhesion by TPA was also blocked. These results suggest that sLe(x) expression is critically regulated by a single glycosyltransferase, C2GnT, during differentiation of KM3 cells.
  • S Iwase, Y Furukawa, J Horiguchi-Yamada, T Nemoto, S Takahara, T Kawano, T Sekikawa, K Ito, Y Yamazaki, J Kikuchi, K Morishita, H Yamada
    INTERNATIONAL JOURNAL OF HEMATOLOGY 67 4 361 - 368 1998年06月 [査読有り][通常論文]
     
    Chromosomal translocation often results in aberrant activation of the genes with oncogenic potential and, thus, plays an important role in leukemogenesis. We report a unique case of acute myelomonocytic leukemia carrying a rare reciprocal translocation, t(3;12)(q26;p13). This patient displayed typical clinical features of 3q21q26 syndrome such as abnormal thrombopoiesis and rapid disease progression. Elastic cells from the patient strongly expressed the EVI1 gene, which is located on 3q26 and is normally suppressed in bone marrow cells. Expression of the TEL gene, located on 12p13, was also observed, but fusion transcript between two genes was not found. No structural alterations of the EVI1 and TEL genes were detected by Southern blot and PCR analyses. We reviewed previous literature and found 10 other cases with t(3;12)(q26;p13). These patients comprise a unique disease group with features including dyshematopoiesis and poor prognosis. However, characteristics related to 3q21q26 syndrome were observed only in the present case. Further investigation is required to elucidate the molecular basis of this particular entity. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.
  • Y Terui, Y Furukawa, J Kikuchi, S Iwase, K Hatake, Y Miura
    EXPERIMENTAL HEMATOLOGY 26 3 236 - 244 1998年03月 [査読有り][通常論文]
     
    Differentiation- and lineage-related differences in the expression of two anti-apoptotic molecules, bcl-x and bcl-2, were examined using various human hematopoietic cell lines. Bcl-x was strongly expressed in cell lines with erythroid and megakaryocytic properties (K562, HEL, CMK, and Mo7E), and was moderately expressed in immature myeloid cell lines (KG-1 and KCL-22). Bcl-2 expression was relatively weak in these cells. On the other hand, bcl-x was not expressed in more mature myeloid cell lines (HL-60 and PL-21), but bcl-2 was strongly expressed in these cells and in monocytoid cell lines (U937, THP-1, and JOSK-I). We investigated the biological significance of high levels of bcl-x expression in erythroid and megakaryocytic lineage cells. When K562 cells were specifically differentiated into megakaryocytic lineage by phorbol ester, the amounts of bcl-x increased by 10-fold. In contrast, bcl-x was gradually downregulated during erythroid differentiation induced by cytosine arabinoside. Apoptosis was observed following erythroid differentiation of K562 cells, but it was not associated with megakaryocytic differentiation in consistent with the increase in bcl-x. Moreover, phorbol ester-induced megakaryocytic differentiation was facilitated by the overexpression of bcl-x in K562 cells. Finally, in situ hybridization revealed that bcl-x mRNA expression was strongest in megakaryocytes among normal bone marrow cells. These results suggest that bcl-x is a regulatory factor in the apoptosis and differentiation of megakaryocytes.
  • M Nakamura, A Tsunoda, K Yanagisawa, Y Furukawa, J Kikuchi, S Iwase, T Sakai, G Larson, M Saito
    JOURNAL OF LIPID RESEARCH 38 9 1795 - 1806 1997年09月 [査読有り][通常論文]
     
    Using mammalian gene-overexpression system, in vitro catalytic activities of CMP-NeuAc:Gal beta 1-->4GlcNAc alpha 2-->6sialyltransferase on glycosphingolipid accepters were analyzed. We transfected the mammalian expression vector containing the cDNA that was cloned from Daudi cells into COS-I cells, and selected monoclonal transfectants in the presence of G418. Although the transfected alpha 2-->6sialyltransferase can catalyze NeuAc transfer onto glycoprotein accepters more than glycolipids based on kinetic analyses, the substantial synthesis of IV(6)NeuAc-nLcOse(4)Cer was observed and the activities were 7- to 9-times higher in the transfected cells than in the mock transfectants. In addition, the transfected COS-I cells with alpha 2-->6sialyltransferase cDNA were revealed to contain a higher amount of ganglioside that has the terminal NeuAc alpha 2-->6Gal sequence in the in situ situation than the mock transfectants. These results using transfectants, together with those using the purified enzyme protein, suggest that the alpha 2-->6sialyltransferase enzyme from Daudi cells can also catalyze NeuAc transfer in alpha 2-->6 linkage onto glycosphingolipid acceptors.
  • J Kikuchi, Y Furukawa, S Iwase, Y Terui, M Nakamura, S Kitagawa, M Kitagawa, N Komatsu, Y Miura
    BLOOD 89 11 3980 - 3990 1997年06月 [査読有り][通常論文]
     
    The mechanism of megakaryocytic differentiation was investigated using human megakaryocytic leukemia cell line UT-7. Polyploidization of UT-7 cells was induced by the microtubule-depolymerizing agent, nocodazole, and 12-O-tetradecanoylphorbol-13-acetate (TPA), but the effect was much more striking with nocodazole. By contrast, induction of cytoplasmic maturation, as judged by beta-thromboglobulin production and platelet factor 4 expression, was more prominent in TPA-treated cells than in nocodazole-treated cells, Nocodazole and TPA could act synergistically to increase ploidy and to enhance the expression of mature phenotypes. Human thrombopoietin induced functional maturation but not polyploidization in UT-7 cells and also acts synergistically with nocodazole. Cyclin-dependent kinase inhibitor p21 was upregulated at the early stage of megakaryocytic differentiation, and overexpression of p21 resulted in an increase in ploidy of UT-7 cells. This suggests that p21 is implicated in polyploidization via suppression of CDC2 activity at mitosis, UT-7 but not HL-60 cells could incorporate [H-3]thymidine in the presence of TPA, indicating the presence of megakaryocyte-specific licensing factor to allow DNA replication during differentiation, Taking these data together, we propose that megakaryocytic differentiation consists of two distinct processes, polyploidization and functional maturation, and that these two processes are independently regulated. (C) 1997 by The American Society of Hematology.
  • N Kubo, J Kikuchi, Y Furukawa, T Sakai, H Ohta, S Iwase, H Yamada, Sakurabayashi, I
    FEBS LETTERS 409 2 177 - 182 1997年06月 [査読有り][通常論文]
     
    In order to investigate the mechanisms holy modified lipoproteins enhance foam cell formation, we cultured peripheral blood monocytes with various stimulants and examined the effects of aggregated low-density lipoprotein (agLDL) on cell viability and lipid metabolism, AgLDL could completely inhibit phorbol ester-induced apoptosis, which was accompanied by intracellular cholesterol accumulation, Suppression of apoptosis-promoting proteases, ICE and CPP32, was observed in agLDL-treated cells, This indicates that agLDL accelerates foam cell formation through inhibition of apoptosis and enhancement of lipid accumulation in activated monocytes, By contrast, apoptosis was enhanced when monocytes were cultured with agLDL and M-CSF. Intracellular cholesterol accumulation was not significant in M-CSF treated cells, This suggests that M-CSF may act anti-atherogenic through apoptotic elimination of lipid-baring macrophages and enhanced lipid turnover, Our observation supports the novel hypothesis that regulation of apoptosis may play an important role in the development of atherosclerosis. (C) 1997 Federation of European Biochemical Societies.
  • S Iwase, Y Furukawa, J Kikuchi, M Nagai, Y Terui, M Nakamura, H Yamada
    JOURNAL OF BIOLOGICAL CHEMISTRY 272 19 12406 - 12414 1997年05月 [査読有り][通常論文]
     
    E2F is a heterodimeric transcription factor that controls transcription of several growth-regulatory genes including cdc2, To investigate the mechanism of interferon-alpha (IFN-alpha)-mediated growth suppression of hematopoietic cells, we examined the effect of IFN-alpha on the expression and function of E2F using IFN-sensitive Daudi cells, Down-regulation of E2F-1, a subunit of E2F, was observed after 8 h of culture with IFN-alpha; expression of E2F-4, another subunit of E2F, and DP-1, a heterodimeric partner of E2F, was unaffected, Gel shift assays revealed that the DNA binding activity of free E2F, which is composed of E2F-1 and E2F-4, was inhibited by IFN-alpha. In contrast, IFN-alpha did not affect the DNA binding ability of E2F-1 and E2F-4 in a complex with retinoblastoma (RE) susceptibility gene family proteins including pRB, p107, and p130, IFN-alpha could induce dephosphorylation of pRB, thereby turning active E2F-pRB complexes into transcriptional repressors, Transient chloramphenicol acetyltransferase assays revealed that the activity of the E2F-dependent cdc2 promoter was suppressed by IFN-alpha. These results suggest that the antiproliferative action of IFN-alpha is mediated through the modulation of E2F activity in two different ways: down-regulation of transcriptionally active free E2F and conversion of E2F-pRB complexes into transcriptional repressors.
  • N Komatsu, K Kirito, Y Kashii, Y Furukawa, J Kikuchi, N Suwabe, M Yamamoto, Y Miura
    BLOOD 89 4 1182 - 1188 1997年02月 [査読有り][通常論文]
     
    To understand the regulatory mechanism of erythropoietin (EPO) receptor (EPOR) gene expression, the effect of EPO on the steady-state level of EPOR mRNA was examined using the human EPO-dependent cell line UT-7 as a model system. We found that the treatment of UT-7 cells with EPO resulted in a transient decrease of the EPOR mRNA level. This transient downregulation was also induced by stimulation with granulocyte-macrophage colony-stimulating factor (GMCSF), another stimulator of UT-7 cell growth. These results raised the possibility that EPOR gene expression is in part related to cell growth. Moreover, it was found that EPO-induced downregulation of EPOR mRNA level was preceded by a transient downregulation of GATA-1 mRNA. To examine the relationship between the expression of EPOR, GATA-1, and GATA-2 mRNA levels and the cell cycle, logarithmically growing UT-7 cells were centrifugically fractionated according to the cell-cycle phase. Both EPOR and GATA-1 mRNA levels, but not the GATA-2 mRNA level, concomitantly decreased at the G(0)/G(1) phase and increased at the S and G(2)/M phases. An electrophoretic mobility shift assay (EMSA) showed that in EPO-stimulated UT-7 cells, the dynamic changes in EPOR gene expression paralleled the GATA-1 DNA-binding activity to the oligonucleotide probe containing a GATA-binding site located at the promoter region of the EPOR gene. These findings suggest that the regulation of EPOR mRNA level is mainly associated with GATA-1 gene expression in UT-7 cells undergoing proliferation, and that these serial events are under the control of, or related to, the cell cycle. (C) 1997 by The American Society of Hematology.
  • Y Furukawa, S Iwase, Y Terui, J Kikuchi, T Sakai, M Nakamura, S Kitagawa, M Kitagawa
    JOURNAL OF BIOLOGICAL CHEMISTRY 271 45 28469 - 28477 1996年11月 [査読有り][通常論文]
     
    Apoptosis has recently been hypothesized to be the result of aberrant cell cycle control. In this study, we have investigated the role of cell cycle regulatory elements in Fas-induced apoptosis of hematopoietic cells. When HL-60 cells were treated with anti-Fas antibody, rapid activation of growth-associated histone H1 kinase was observed without any change in cell cycle distribution, This was accompanied by the increase in cdc2 mRNA expression and Cdc2 kinase activity. Up-regulation of cdcs mRNA was similarly induced in BCL-2-over-expressing HL-60 subline by anti-Fas treatment independently of the appearance of apoptotic phenotypes. Fas-induced apoptosis was completely inhibited by butyrolactone I, a specific inhibitor of Cdc2 kinase. Moreover, the same phenomenon was observed during Fas-induced but not spontaneous apoptosis of postmitotic granulocytes. Finally, we have found that ''Fas-responsive element'' was located between nucleotides -730 and -552 of the cdc2 promoter and was responsive for transcriptional activation of the cdc2 gene during Fas-induced apoptosis. These results indicate that aberrant activation of Cdc2 is associated with Fas-induced apoptosis of hematopoietic cells, and that the mechanism of cdc2 transcription during Fas-induced apoptosis is different from that in normal cell cycle control.
  • Y Terui, Y Furukawa, T Sakai, J Kikuchi, H Sugahara, Y Kanakura, S Kitagawa, Y Miura
    JOURNAL OF IMMUNOLOGY 156 5 1981 - 1988 1996年03月 [査読有り][通常論文]
     
    Interaction between fibronectin (FN) and very late activation Ag-5 (VLA-5) integrin was recently reported to be involved in apoptosis of hematopoietic cells, In an effort to clarify the physiologic role of FN in the regulation of biologic behavior of terminally differentiated hematopoietic cells, we have examined the change of VLA-5 expression during myeloid cell differentiation and its effects on monocytes and granulocytes. VLA-5 alpha mRNA was up-regulated during monocytic differentiation, but not during granulocytic differentiation of HL-60 cells, Flow cytometric and immunocytochemical analysis revealed that surface expression of VLA-5 was selectively increased upon monocytic differentiation and that it was strongly positive on peripheral blood monocytes, Susceptibility to FN-induced apoptosis was greatly increased upon monocytic differentiation, and it was almost completely abrogated by anti-VLA-5 Ab or RGD peptide, Similarly, FN could significantly enhance apoptosis of normal monocytes but not of granulocytes. Finally, we have shown that anti-FN Ab could suppress spontaneous apoptosis of normal monocytes in culture and prolong their survival, These results suggest that FN might play an important role in negative regulation of the survival of monocytes through its interaction with VLA-5, which is selectively up-regulated during monocytic differentiation.
  • Y TERUI, Y FURUKAWA, J KIKUCHI, M SAITO
    JOURNAL OF CELLULAR PHYSIOLOGY 164 1 74 - 84 1995年07月 [査読有り][通常論文]
     
    The cell cycle-associated differences in the susceptibility to apoptosis were examined in HL-60 cells before and after differentiation with phorbol 12, 13-dibutyrate (PDBu). HL-60 cells in various phases of the cell cycle were separated by the counterflow centrifugal elutriation and the susceptibility to apoptosis was measured by the morphological examination and by DNA fragmentation assay. Undifferentiated HL-60 cells in S phase showed a significantly higher susceptibility to apoptosis than those in G0/G1 or G2/M phase either in the absence or presence of apoptosis-inducing reagents such as A23187, actinomycin D (Act D), and cycloheximide (CHX). In contrast, PDBu-treated HL-60 cells preferentially underwent apoptosis in G0/G1 phase. When untreated HL-60 cells enriched for G0/G1 phase were recultured in a complete medium, the percentage of apoptotic cells increased after 6-12 h in correlation with the increase in S-phase cells. When the same experiment was performed with PBDu-treated cells, spontaneous increase of apoptotic cells was observed while almost all cells remained in G0/G1 phase. Northern blot analysis revealed that undifferentiated cells expressed the same amounts of bcl-2 mRNA in each cell cycle phase, whereas G0/G1-predominant reduction of bcl-2 mRNA was noted in PDBu-treated cells. There was no difference in the amounts of CD11b mRNA between G0/G1 fraction and S + G2/M fraction of differentiated HL-60 cells. BCL-2 overexpression could almost completely abrogate the G0/G1-predominant induction of apoptosis in differentiated HL-60 cells. These results suggest that G0/G1 cell cycle arrest and down-regulation of bcl-2 mRNA in G0/G1 phase might be associated with apoptosis in differentiated HL-60 cells whereas the weakness of chromatin structure in S phase might be related to apoptosis in undifferentiated HL-60 cells. (C) 1995 Wiley-Liss, Inc.
  • Y FURUKAWA, J KIKUCHI, Y TERUI, S KITAGAWA, M OHTA, Y MIURA, M SAITO
    JAPANESE JOURNAL OF CANCER RESEARCH 86 2 208 - 216 1995年02月 [査読有り][通常論文]
     
    Normal human monocytes were isolated in a nascent state by centrifugal elutriation and used for the study of interleukin-1 (IL-1) and interleukin-1 receptor antagonist (IL-1ra) expression. Neither IL-1 beta nor IL-1ra mRNA was present in monocytes just after the isolation, but they were induced simultaneously in response to various stimulants. In contrast, only IL-1 beta mRNA was expressed in monocytic leukemia cell line JOSK-1, while little or no IL-1ra mRNA was detected even after stimulation. Dominant expression of IL-1 beta over IL-1ra was also observed in fresh leukemia cells including monocytic leukemias, i.e., IL-1 beta mRNA was constitutively expressed in 26 out of 36 cases (72.2%), whereas IL-1ra mRNA was present only in 8 cases (22.2%). The signal intensity of IL-1 beta mRNA was stronger than that of IL-1ra even in IL-1ra-positive cases. Apoptotic cell death of monocytes was significantly inhibited by IL-1 beta, and it was enhanced by IL-1ra. In fresh leukemia cells, H-3-thymidine uptake was generally higher in IL-1-producing cases than in IL-1ra-producing cases, and was increased by the addition of IL-1 beta in all cases tested. Cell proliferation was inhibited by either IL-1ra or anti-IL-1 beta antibody in IL-1-producing cases, while it was enhanced by anti-IL-1ra antibody in IL-1ra-producing cases. These results suggest that the balance between IL-1 and IL-1ra is necessary for homeostasis of the mononuclear phagocytosis system. The imbalance between these two counter-acting cytokines might contribute to the altered growth and accumulation of leukemic cells.

講演・口頭発表等

  • 作用機序から考察する移植後維持療法におけるニンラーロの有用性  [招待講演]
    骨髄腫セミナー(武田薬品工業招待講演) 2020年11月 公開講演,セミナー,チュートリアル,講習,講義等
  • 抗体薬併用の作用機序から考える投与のタイミング  [招待講演]
    第25回東海骨髄腫フォーラム(ヤンセンファーマ招待講演) 2020年01月 公開講演,セミナー,チュートリアル,講習,講義等
  • Epigenetic mechanisms of cell adhesion mediated drug resistance in multiple myeloma.  [招待講演]
    第81回日本血液学会学術集会シンポジウム講演 2019年10月 シンポジウム・ワークショップパネル(指名)
  • 微小環境内の骨髄腫細胞に対するプロテアソーム阻害剤の効果  [招待講演]
    甲信骨髄腫セミナー in 松本(武田薬品工業招待講演) 2019年10月 公開講演,セミナー,チュートリアル,講習,講義等
  • 抗体薬併用の作用機序から考える投与のタイミング  [招待講演]
    DARAZALEX BASIC SEMINAR in 秋田 (ヤンセンファーマ招待講演) 2019年07月 公開講演,セミナー,チュートリアル,講習,講義等
  • 抗体薬併用の作用機序から考える投与のタイミング  [招待講演]
    Multiple Myeloma Frontier Seminar in Sapporo (ヤンセンファーマ招待講演) 2019年03月 公開講演,セミナー,チュートリアル,講習,講義等
  • 免疫調節薬と抗体薬併用の新たな作用機序  [招待講演]
    東海大学 Elotuzumab Conference (ブリストルマイヤーズスクイブ招待講演) 2019年03月 公開講演,セミナー,チュートリアル,講習,講義等
  • 免疫調節薬と抗体薬併用の新たな作用機序  [招待講演]
    エムプリシティ2周年記念講演会 in 神戸 (ブリストルマイヤーズスクイブ招待講演) 2018年11月 公開講演,セミナー,チュートリアル,講習,講義等
  • IMiDsの病態の進展予防効果  [招待講演]
    関東IMiDsフォーラム (セルジーン招待講演) 2018年06月 公開講演,セミナー,チュートリアル,講習,講義等
  • 転写因子Ikarosの新たな標的分子とその機能  [招待講演]
    第43回日本骨髄腫学会学術集会モーニングセミナー(ブリストルマイヤーズスクイブ招待講演) 2018年05月 公開講演,セミナー,チュートリアル,講習,講義等
  • 転写因子Ikarosの新たな標的分子とその機能  [招待講演]
    第2回Breakthrough Myeloma Summit (ブリストルマイヤーズスクイブ招待講演) 2017年11月 公開講演,セミナー,チュートリアル,講習,講義等
  • Modulation of innate immunity as a mechanism of Lenalidomide maintenance for multiple myeloma.  [招待講演]
    第79回日本血液学会学術集会シンポジウム講演 2017年10月 シンポジウム・ワークショップパネル(指名)
  • Toll様受容体CD180を介した多発性骨髄腫の感染による病態の進展機序  [通常講演]
    第42回日本骨髄腫学会学術集会シンポジウム講演 2017年05月 シンポジウム・ワークショップパネル(公募)
  • 3つのサブユニット(β1、β2、β5)を阻害する化合物の基礎研究について  [招待講演]
    Kyprolis Meet The Expert (小野薬品工業招待講演) 2016年09月 公開講演,セミナー,チュートリアル,講習,講義等
  • リン酸化によるヒストンメチル化酵素EZH2の不活性化が多発性骨髄腫の薬剤耐性を誘導する  [招待講演]
    第12回麒麟塾 (協和発酵キリン招待講演) 2016年07月 公開講演,セミナー,チュートリアル,講習,講義等
  • 多発性骨髄腫の薬剤耐性機序とその克服  [招待講演]
    第20回ILYH研究会 (ヤンセンファーマ招待講演) 2015年12月 公開講演,セミナー,チュートリアル,講習,講義等
  • Epigenetic mecha- nisms of cell adhesion mediated drug resistance in multiple myeloma  [招待講演]
    第77回日本血液学会学術集会シンポジウム講演 2015年10月 シンポジウム・ワークショップパネル(指名)
  • プロテアソーム阻害剤の作用機序解明と新規阻害剤の開発  [招待講演]
    血液疾患フォーラム (ヤンセンファーマ招待講演) 2013年11月 公開講演,セミナー,チュートリアル,講習,講義等
  • プロテアソーム阻害剤の作用機序解明と新規阻害剤の開発  [招待講演]
    血液疾患講演会 (中外製薬招待講演) 2012年11月 公開講演,セミナー,チュートリアル,講習,講義等
  • プロテアソーム阻害剤の作用機序解明と新規阻害剤の開発  [招待講演]
    第10回先端血液セミナー (協和発酵キリン招待講演) 2012年07月 公開講演,セミナー,チュートリアル,講習,講義等
  • ボルテゾミブの抗骨髄腫作用はヒストン脱アセチル化酵素発現の抑制を介する  [招待講演]
    第7回麒麟塾 (協和発酵キリン招待講演) 2011年06月 公開講演,セミナー,チュートリアル,講習,講義等
  • 転写因子Sp1・Sp3・Sp4によるコア2GlcNac転移酵素-1の転写制御  [通常講演]
    第24回日本糖質学会年会シンポジウム講演 2003年07月 シンポジウム・ワークショップパネル(公募)

MISC

  • 【多発性骨髄腫をめぐる病態解析研究と治療の進展】多発性骨髄腫の分子病態に応じた治療選択
    古川 雄祐, 菊池 次郎 血液内科 81 (3) 304 -312 2020年09月
  • 菊池 次郎, 古川 雄祐 臨床血液 61 (7) 832 -841 2020年07月 
    多発性骨髄腫の薬剤耐性獲得における骨髄微小環境の役割と機序の解明は重要な課題である。筆者らはストローマ細胞との接着時,転写抑制に働くヒストン修飾であるヒストンH3の27番目のリジンのトリメチル化抑制が耐性獲得の鍵となることを明らかにした。メチル化抑制はメチル化酵素EZH2のリン酸化を介した活性抑制によるものであった。下流ではアポトーシス抑制分子に加えて,細菌由来のリポ多糖(LPS)を認識するToll様受容体CD180の発現亢進が見られた。LPSはストローマ細胞との接着かつ低酸素下におけるCD180発現亢進に応じて増殖促進に働いた。また,CD180遺伝子が転写因子Ikarosにより転写活性化されること,lenalidomideがCD180発現抑制を介してLPSによる増殖を抑制することを明らかにした。LenalidomideにはCD180発現抑制により感染をきっかけとした病態悪化の予防を介した生存期間延長効果が示唆される。(著者抄録)
  • 【多発性骨髄腫-クロストークの腫瘍】骨髄微小環境とのクロストークから考察する多発性骨髄腫の発症・進展・薬剤抵抗性のメカニズム
    古川 雄祐, 菊池 次郎 カレントテラピー 38 (6) 514 -521 2020年06月 
    多発性骨髄腫はB細胞系列の最終分化段階にあるpost-germinal center B-cellを発生母地とし、14q転座ないし染色体高二倍体化をドライバー変異として発症する。これらの変異によって発生した骨髄腫幹細胞が骨髄微小環境とのクロストークによって多様なサブクローンを産生し、初期段階から複雑な階層構造を取る。骨髄腫細胞、特に幹細胞画分はニッチとの相互作用によってdormancyや抗がん剤抵抗性を獲得している。VLA-4を介する細胞接着によって活性化されたPI3K-AKTがヒストンメチル化酵素EZH2を不活化し、Bcl-2・IGF-1・HIF-1αなどを脱抑制して薬剤耐性を賦与する。またEZH2の下流にはIKZF1があり、未分化骨髄腫細胞のIMiDs高感受性を良く説明する。さらにIKZF1はCD38やSLAMF7の発現も制御しており、治療戦略を立てる際の重要なファクターとなる。(著者抄録)
  • 菊池 次郎, 長田 直希, 小山 大輔, 黒田 芳明, 安井 寛, 古川 祐雄 International Journal of Myeloma 9 (1) 89 -89 2019年05月 [査読無し][通常論文]
  • 多発性骨髄腫の病状が感染をきっかけに悪化する新たなメカニズム 多発性骨髄腫細胞にToll様受容体関連分子CD180が発現
    菊池 次郎 Medical Science Digest 44 (13) 707 -710 2018年11月 
    多発性骨髄腫細胞は現在も難治性の造血器腫瘍である。骨髄腫細胞は骨髄微小環境、特に骨髄間質細胞との細胞間接着を介した相互作用により増殖能や薬剤耐性能を獲得する。したがって、その機序の解明は重要な研究課題である。筆者らは、骨髄間質細胞との接着シグナルが、1)転写抑制に働くヒストンH3の27番目のリジン(H3-K27)のメチル化とくにトリメチル化を抑制し接着耐性の腱になること、2)下流で抗アポトーシスに働くIGF-1やBcl-2の発現が亢進すること、3)Toll様受容体(TLR)CD180の発現を亢進させ細菌感染をきっかけとした病態の悪化に働くこと、4)骨髄腫治療のキードラッグの一つ免疫調節薬がCD180発現抑制を介してその機序の阻害に働くこと、を明らかにした。ここから、細菌感染の予防と免疫調節薬による維持療法が多発性骨髄腫の予後改善に有効となる可能性を明らかにした。(著者抄録)
  • 脳移行性を有するLSD1阻害剤によるT-リンパ芽球性白血病中枢神経病変の治療(Eradication of central nervous system leukemia of T-cell origin by a brain-permeable LSD1 inhibitor)
    菊池 次郎, 斎藤 詩緒里, 小山 大輔, 佐藤 心, 小山 裕雄, 長田 直希, 黒田 芳明, 赤羽 弘資, 犬飼 岳史, 梅原 崇史, 古川 雄祐 臨床血液 59 (9) 1534 -1534 2018年09月
  • 造血器悪性腫瘍におけるSLAMF7の発現様式とその機序、臨床応用への意義(SLAMF7 expression profiles in hematologic malignancies: an interpretation for clinical significance)
    伊波 英克, 池辺 詠美, ファジャド・リンゼイ, 鈴木 敦, 菊池 次郎, 古川 雄祐, 堀 光雄 臨床血液 59 (9) 1696 -1696 2018年09月
  • 古川 雄祐, 黒田 芳明, 菊池 次郎 臨床血液 59 (8) 1048 -1057 2018年08月 
    多発性骨髄腫(MM)においては骨髄間質細胞との接着と低酸素(骨髄微小環境)によってdormancyと抗がん剤耐性が誘導されるが、そのメカニズムはまだ完全には解明されていない。そこで骨髄微小環境にあるdormant MM細胞が再増殖するメカニズムと治療的介入の可能性について研究を行った。MM細胞には非定型的Toll-like receptor(TLR)であるCD180/MD-1複合体が発現しており、間質細胞との接着および低酸素によって発現が亢進した。CD180/MD-1複合体は細菌由来のリポ多糖体(LPS)を認識し、MAPキナーゼ(ERK・JNK)を介してMM細胞の増殖を促進した。CD180遺伝子の転写調節領域にはIKZF結合配列が存在し、MM細胞においてはIKZF1(Ikaros)が結合してCD180の発現を促進する。間質細胞との接着および低酸素環境においてはIkarosの発現が上昇するため、CD180の転写が活性化される。LenalidomideはIkarosの分解を介して骨髄腫細胞のCD180発現とLPSによる増殖を抑制した。CD180の発現抑制はlenalidomideによる継続治療・維持療法の有効性の根拠の1つと考えられる。(著者抄録)
  • 古川 雄祐, 黒田 芳明, 菊池 次郎 International Journal of Myeloma 8 (2) 53 -53 2018年05月
  • 黒田 芳明, 菊池 次郎, 小山 大輔, 一戸 辰夫, 古川 雄祐 International Journal of Myeloma 8 (2) 58 -58 2018年05月
  • 菊池 次郎, 黒田 芳明, 小山 大輔, 長田 直希, 和泉 透, 安井 寛, 川瀬 孝和, 一戸 辰夫, 古川 雄祐 International Journal of Myeloma 8 (2) 91 -91 2018年05月
  • 菊池 次郎, 黒田 芳明, 古川 雄祐 International Journal of Myeloma 8 (2) 179 -179 2018年05月
  • 古川 雄祐, 菊池 次郎 血液フロンティア 28 (5) 747 -754 2018年04月 
    <文献概要>免疫調節薬(IMiDs)とくにlenalidomideは,継続的な使用により骨髄腫患者の予後を改善する。これはIMiDsが治療後に残存する骨髄腫幹細胞をターゲットとするためである。そのメカニズムの1つとして,骨髄間質細胞との接着によるEZH2不活化によりIKZF1の転写が亢進することが考えられる。一方,プロテアソーム阻害剤はMタンパク産生の旺盛な成熟クローンに有効であるが,継続して用いることによって成熟クローンの供給源である骨髄腫幹細胞を枯渇させる。プロテアソーム阻害剤による継続治療は,17p欠失を有する例には必須である。分子病態を理解した上で適切な薬剤を継続的に使用することは,多発性骨髄腫の治療成績向上に大きく貢献する。
  • 古川 雄祐, 菊池 次郎 血液フロンティア 28 (4) 553 -563 2018年03月 
    <文献概要>多発性骨髄腫は,14q転座ないし染色体高二倍体化をinitiating mutationとして発症する。これらの変異によって生じた骨髄腫幹細胞が初期から多様なサブクローンを産生し,複雑な階層構造をとることが明らかになっている。高二倍体化によって発症した骨髄腫はダーウィン型の枝分かれ進化をし,骨髄微小環境や免疫応答が進展に影響するため免疫調節薬(IMiDs)が有効である。一方,約20%の症例は中立進化をするため,IMiDsの効果が弱く,プロテアソーム阻害剤を必要とする。また,成熟したクローンはプロテアソーム阻害剤に感受性が高く,骨髄腫幹細胞を含む未分化なクローンはIMiDsによって排除される。多様なクローンの存在と薬剤感受性の違いは,治療戦略を立てる際に重要なファクターとなる。
  • 多発性骨髄腫におけるToll様受容体CD180の機能解明と臨床応用
    菊池 次郎 先進医薬研究振興財団研究成果報告集 2017年度 132 -133 2018年03月
  • 菊池 次郎, 黒田 芳明, 古川 雄祐 血液内科 75 (4) 456 -464 2017年10月
  • 【多発性骨髄腫の最新治療】骨髄腫細胞のエピゲノム異常とHDAC阻害剤の臨床効果
    古川 雄祐, 黒田 芳明, 菊池 次郎 BIO Clinica 32 (9) 875 -880 2017年08月 
    ヒストン脱アセチル化酵素(HDAC)はコア・ヒストンN末端のリジン残基からアセチル基を除去することでクロマチン構造をコンパクトにし、転写を抑制する一群の酵素である。また組織特異的転写因子やチュブリンを脱アセチル化するサブタイプも存在し、細胞分化やオートファジーの制御に関与する。多発性骨髄腫においては、種々のHDACが強発現しており、予後との関連も示されている。機能的にはHDAC1とHDAC3が骨髄腫細胞の増殖に必須で、これらを標的とするHDAC阻害剤は骨髄腫に有効と考えられる。またHDAC6はプロテアソームでは分解できない凝集タンパク質をオートファジーで処理する際に必須の役割を果たしている。したがってHDAC6阻害剤はプロテアソーム阻害剤と相乗的に抗骨髄腫効果を発揮する。この3つのHDACを抑制する汎HDAC阻害剤panobinostatが難治性骨髄腫の治療に用いられている。(著者抄録)
  • 古川 雄祐, 黒田 芳明, 菊池 次郎 血液フロンティア 27 (9) 1239 -1246 2017年08月 
    多発性骨髄腫はclass I HLA,soluble MICA,PD-L1を発現して,NK細胞やキラーTリンパ球などのエフェクター細胞による免疫排除を免れている。免疫調節薬(IMiDs)はPD-L1の発現を抑制するとともにエフェクター細胞を活性化し,免疫環境を改善することで抗骨髄腫効果を発揮する。したがってIMiDsと治療用抗体の併用はきわめて合理的かつ有効な治療法である。また,プロテアソーム阻害剤もclass I HLAの発現やMICAのsheddingを抑制して抗骨髄腫免疫を増強するため,IMiDs耐性例などにおける抗体療法への組み込みが期待される。すでに抗SLAMF7抗体エロツズマブが臨床的に成果を上げているが,今後,抗CD38抗体daratumumabなども加わり,抗体医薬は治癒を目指す骨髄腫治療戦略に不可欠な地位を占めると考えられる。(著者抄録)
  • 菊池 次郎, 黒田 芳明, 小山 大輔, 長田 直希, 和泉 透, 安井 寛, 川瀬 孝和, 一戸 辰夫, 古川 雄祐 International Journal of Myeloma 7 (1) 34 -34 2017年04月 [査読無し][通常論文]
  • 【形質細胞性疾患の病態と治療】多発性骨髄腫におけるエピゲノム異常と薬剤抵抗性
    菊池 次郎, 古川 雄祐 血液内科 74 (2) 131 -137 2017年02月
  • 菊池 次郎, 古川 雄祐 血液内科 73 (4) 534 -538 2016年10月
  • 古川 雄祐, 和田 妙子, 菊池 次郎 血液内科 72 (6) 857 -862 2016年06月
  • 開俊樹, 菊池次郎, 喜多俊介, 前仲勝実, 古川雄祐, 柴山修哉 日本農芸化学会大会講演要旨集(Web) 2016 2C004 (WEB ONLY) 2016年03月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 Pharma Medica 33 (12) 9 -13 2015年12月
  • 古川 雄祐, 菊池 次郎 血液内科 71 (6) 742 -747 2015年12月
  • 古川 雄祐, 菊池 次郎 腫瘍内科 16 (6) 558 -565 2015年12月
  • ヒストン脱メチル化酵素LSD1による前白血病幹細胞形成
    和田 妙子, 小山 大輔, 菊池 次郎, 本田 浩章, 古川 雄祐 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3T18 -04(3P1036)] 2015年12月
  • 高橋 和也, 犬飼 岳史, 黄 媚賢, 黒田 格, 廣瀬 衣子, 渡邊 敦, 杣津 晋平, 合井 久美子, 友安 千紘, 矢野 未央, 今村 俊彦, 細井 創, 菊池 次郎, 古川 雄祐, 加賀美 恵子, 阿部 正子, 杉田 完爾 日本小児血液・がん学会雑誌 52 (4) 261 -261 2015年10月 [査読無し][通常論文]
  • Taeko Wada, Daisuke Koyama, Jiro Kikuchi, Hiroaki Honda, Yusuke Furukawa EXPERIMENTAL HEMATOLOGY 43 (9) S63 -S63 2015年09月 [査読無し][通常論文]
  • Jiro Kikuchi, Daisuke Koyama, Taeko Wada, Tohru Izumi, Peter O. Hofgaard, Bjarne Bogen, Yusuke Furukawa EXPERIMENTAL HEMATOLOGY 43 (9) S64 -S64 2015年09月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 臨床検査 58 (13) 1560 -1568 2014年12月 
    non-IgM MGUSの発症にはcyclin Dの強発現が関与している.non-IgM MGUSから多発性骨髄腫への進展には,Rasの突然変異・c-Mycの強発現・ゲノムの低メチル化が重要である.次世代シークエンシングによって多発性骨髄腫の進展にかかわる突然変異の出現機構が明らかにされつつある.IgM MGUSからマクログロブリン血症への進展にはMYD88の変異によるNF-κBの恒常的活性化が関与している.(著者抄録)
  • 菊池 次郎, 古川 雄祐 血液内科 69 (4) 562 -567 2014年10月
  • 古川 雄祐, 菊池 次郎 血液フロンティア 24 (6) 869 -879 2014年05月 
    多発性骨髄腫は形質細胞がドライバー変異(14q転座・高二倍体化)によってがん幹細胞化し、そこにstepwiseに付加的遺伝子異常が加わることで、monoclonal gammopathy of undetermined significance(MGUS)→無症候性骨髄腫(smoldering myeloma)→症候性骨髄腫→形質細胞性白血病と、linearにevolutionしていくと考えられてきた。ところが、最近の次世代シークエンスや高感度アレイによる解析の結果、すでにMGUSの段階で多様なクローンが存在し、枝分かれ状の複雑な階層構造を有することが明らかになってきた。病態進展の本態は、変異の蓄積よりもクローン構造の変化が主体と考えられる。治療成績のさらなる向上のためには、クローン多様性を考慮した治療戦略が必要であろう。(著者抄録)
  • 菊池 次郎, 小山 大輔, 向井 陽美, 古川 雄祐 International Journal of Myeloma 4 (2) 73 -73 2014年05月
  • 菊池 次郎, 古川 雄祐 血液内科 67 (1) 112 -122 2013年07月 [査読無し][通常論文]
  • 菊池 次郎, 古川 雄祐 BIO Clinica 27 (13) 1212 -1217 2012年12月 [査読無し][通常論文]
     
    骨髄腫細胞は骨髄間質系細胞とのサイトカイン刺激や接着シグナルを介した相互作用により薬剤耐性を獲得する。これまでに間質系細胞の産生するIL-6、SDF-1や、骨髄腫細胞に発現するVLA-4、PSGL-1、CXCR4等の接着分子の関与が示唆されている。これら分子に対する抗体や阻害剤は前臨床試験において、抗がん剤の感受性向上に有効性が示されている。しかしながら、骨髄腫細胞は間質系細胞を含む種々の細胞と複雑なネットワークを形成し薬剤耐性を獲得している。さらなる分子機構の解明は今後の重要な研究課題である。(著者抄録)
  • WADA Taeko, KIKUCHI Jiro, HIRAOKA Nobuya, KOYAMA Daisuke, FURUKAWA Yusuke 臨床血液 53 (9) 1042 2012年09月 [査読無し][通常論文]
  • HIRAOKA Nobuya, KIKUCHI Jiro, WADA Taeko, MORI Shigehisa, NAKAMURA Yuichi, KANO Yasuhiko, FURUKAWA Yusuke 臨床血液 53 (9) 1146 2012年09月 [査読無し][通常論文]
  • 菊池 次郎, 古川 雄祐 医学のあゆみ 242 (13) 1209 -1214 2012年09月 [査読無し][通常論文]
     
    ヒストン脱アセチル化酵素(HDAC)はヒストン修飾を介した遺伝子発現の制御因子であり、ヒストン脱アセチル化を介しておもに転写抑制に働いている。多発性骨髄腫を含む悪性腫瘍ではHDACの強発現がみられ、分化やアポトーシス関連分子の発現が抑制されている。HDAC阻害剤はこの発現抑制の是正を介して抗腫瘍効果を誘導する。多発性骨髄腫に対するHDAC阻害剤の効果は前臨床試験では有効性がみられたが、臨床試験ではほとんど効果がみられなかった。ところが、最近になってプロテアソーム阻害剤ボルテゾミブと併用した複数の臨床試験における有効性が報告された。現在、第III相試験による検証が進められているが、ボルテゾミブとHDAC阻害剤の併用は発現抑制の是正を介した機構と変成蛋白の蓄積に伴う小胞体ストレスを介した機構の両者を介して相加的に作用することから、有効な治療法と考えられる。(著者抄録)
  • 菊池 次郎, 古川 雄祐 血液フロンティア 22 (S-1) 625 -633 2012年04月 [査読無し][通常論文]
     
    抗がん剤耐性は多発性骨髄腫の予後不良の主因である。骨髄腫細胞は骨髄微小環境との相互作用によって薬剤耐性(接着耐性)を獲得する。筆者らは、ボルテゾミブが薬剤耐性の鍵分子であるVLA4やHDACを標的にすることから、高い抗骨髄腫作用を示すことを明らかにした。このような作用はボルテゾミブに特異的であり、多発性骨髄腫の治療成績向上にはボルテゾミブを用いた併用療法の有効性が示唆される。本稿では、これまでに得た理論的基盤をもとに、特にHDAC阻害剤併用の有効性について考察を加えたので紹介する。(著者抄録)
  • 古川 雄祐, 菊池 次郎 血液内科 64 (4) 424 -431 2012年04月 [査読無し][通常論文]
  • BCR-ABL Regulates Death Receptor Expression for TNF-Related Apoptosis-Inducing Ligand (TRAIL) in Philadelphia Chromosome-Positive Leukemia
    Kuroda Itaru, Inukai Takeshi, Zhang Xiaochun, Kikuchi Jiro, Furukawa Yusuke, Nemoto Atsushi, Akahane Koshi, Hirose Kinuko, Honna-Oshiro Hiroko, Goi Kumiko, Kagami Keiko, Yagita Hideo, Tauchi Tetsuzo, Maeda Yasuhiro, Sugita Kanji BLOOD 118 (21) 1178 -1179 2011年11月 [査読有り][通常論文]
  • E2A-HLFキメラ転写因子によるLMO2誘導異常と、t(17:19)を持つB前駆細胞性ALLの白血病誘発におけるその意味(Aberrant induction of LMO2 by the E2A-HLF chimeric transcription factor and its implication in leukemogenesis of B-precursorALL with t(17:19))
    廣瀬 衣子, 犬飼 岳史, 菊池 次郎, 古川 雄祐, 伊川 友活, 河本 宏, Oram S. Helen, Goettgens Berthold, 清河 信敬, 宮川 世志幸, 大喜多 肇, 赤羽 弘資, 張 暁春, 黒田 格, 大城 浩子[本名], 加賀美 恵子, 合井 久美子, 黒澤 秀光, Look A. Thomas, 松井 啓隆, 稲葉 俊哉, 杉田 完爾 小児がん 48 (プログラム・総会号) 194 -194 2011年11月
  • HIRAOKA Nobuya, KIKUCHI Jiro, WADA Taeko, MORI Shigehisa, NAKAMURA Yuichi, KANO Yasuhiko, FURUKAWA Yusuke 臨床血液 52 (9) 1018 2011年09月 [査読無し][通常論文]
  • 古川 雄祐, 平岡 信弥, 和田 妙子, 菊池 次郎, 加納 康彦 日本薬理学雑誌 138 (1) 26 -32 2011年07月 [査読無し][通常論文]
     
    ベンダムスチン(トレアキシン)はプリンアナログ様骨格にアルキル基が結合したハイブリッドな抗がん薬である。作用機序はDNAアルキル化が主体と考えられ、代謝拮抗作用については明確な結論は出ていない。がん細胞に作用させた場合に他のアルキル化薬に比べて多彩な作用を示すのが特徴で、(1)架橋形成〜DNA鎖切断によるネクローシス誘導、(2)DNA損傷チェックポイント活性化によるp53依存性アポトーシスおよび活性酸素種(ROS)を介するp53非依存性アポトーシスの誘導、(3)分裂期チェックポイントの阻害による分裂期細胞死(mitotic catastrophe)誘導、(4)DNA修復阻害、(5)遺伝子発現調節、(6)早期のS期停止誘導などが報告されている。このような多様な作用機序を有することが、アルキル化薬を含む他の抗がん薬と交差耐性を示さない、単剤で従来の標準併用化学療法を上回る成績を示すなどの優れた特徴を生んでいると考えられる。ベンダムスチンは1963年に旧東ドイツにおいて開発されたが、90年代に入ってから本格的に臨床試験が行われ、低悪性度B細胞性非ホジキンリンパ腫(瀘胞性リンパ腫、小細胞リンパ腫)、マントル細胞リンパ腫、慢性リンパ性白血病に対する有効性が確立している。現在は再発・難治例が適応となっているが、初発の低悪性度非ホジキンリンパ腫を対象としてリツキシマブ+ベンダムスチンと現在の標準的治療であるリツキシマブ+CHOP(シクロフォスファミド、ドキソルビシン、ビンクリスチン、プレドニゾロン)を比較する第III相試験が行われ、無増悪性生存期間の中央値において前者が有意に優れていた。さらに現在、中悪性度非ホジキンリンパ腫(びまん性大細胞性リンパ腫、末梢性T細胞リンパ腫)、多発性骨髄腫への適応拡大のための臨床試験が行われている。副作用としては血液毒性、リンパ球減少による日和見感染、消化器毒性(食欲不振、悪心、便秘)などがあるが、重篤なものではない。脱毛、末梢神経障害は認めない。ベンダムスチンは今後、さまざまな悪性腫瘍において第一選択の薬剤となる可能性がある。(著者抄録)
  • 古川 雄祐, 清水 瑠美, 和田 妙子, 菊池 次郎 血液内科 62 (4) 499 -506 2011年04月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 最新医学 66 (3) 432 -440 2011年03月 [査読無し][通常論文]
     
    一見均一に見える骨髄腫細胞集団であるが,クローン性の増殖を示す細胞はごく少数である.骨髄腫幹細胞のマーカーはCD138-/CD19+/CD27+/CD38-で,メモリーB細胞の段階で染色体転座やNF-κB活性化により骨髄腫幹細胞が生じ,骨髄微小環境において骨髄腫として発症すると考えられる.VLA-4を介する骨髄間質細胞との接着が,骨髄腫幹細胞の細胞周期停止,薬剤抵抗性に重要である.従来の抗がん剤のみで骨髄腫幹細胞を駆逐することは難しいが,プロテアソーム阻害薬,IMiDs,HDAC阻害薬,抗VLA-4抗体などとの併用は有効と考えられる.(著者抄録)
  • 古川 雄祐, 菊池 次郎 血液内科 62 (2) 178 -184 2011年02月 [査読無し][通常論文]
  • 古川雄祐, 平岡信弥, 和田妙子, 菊池次郎, 加納康彦 日本薬理学雑誌 138 (1) 26-32 (J-STAGE) 2011年 [査読無し][通常論文]
  • 和田 妙子, 菊池 次郎, 古川 雄祐 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 4T12 -2 2010年12月 [査読無し][通常論文]
  • HIRAOKA Nobuya, KIKUCHI Jiro, NOBUYOSHI Masaharu, WADA Taeko, MITSUNAGA Kanae, KANO Yasuhlko, FURUKAWA Yusuke 臨床血液 51 (9) 995 2010年09月 [査読無し][通常論文]
  • 白血病細胞の生存・増殖にRCAN1は重要な役割を果たす(Crucial role of RCAN1 in leukemia progression)
    藤原 慎一郎, 永井 正, 菊池 悟, 上澤 光世, 山本 千裕, 大嶺 謙, 上原 英輔, 菊池 次郎, 古川 雄祐, 小澤 敬也 日本癌学会総会記事 69回 161 -162 2010年08月
  • 菊池 次郎, 古川 雄祐 血液・腫瘍科 61 (2) 206 -211 2010年08月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 Annual Review血液 2010 139 -145 2010年01月 [査読無し][通常論文]
     
    抗がん剤耐性は骨髄腫細胞の予後不良の主因である.骨髄腫の薬剤耐性は自然耐性(de novo resistance)と獲得耐性(acquired resistance)の2つに大別され,前者はinterleukin-6やSDF-1などの液性因子によるもの(soluble factor-mediated drug resistance)と主にVLA4を介する骨髄微小環境との相互作用によって誘導されるもの[接着耐性(cell adhesion-mediated drug resistance)]がある.メカニズムとして,CDK抑制因子の誘導による細胞周期停止やBimの分解,Bcl-xの発現誘導によるアポトーシス抵抗性の賦与が明らかにされている.後者は薬剤の長期曝露によって耐性が獲得される場合で,DNA修復能の亢進や薬物代謝の促進を介する.最近,bortezomib,抗VLA4抗体,低分子CXCR4阻害剤などが骨髄腫細胞の接着耐性を解除し,薬剤感受性を向上させることが報告されており,臨床応用が期待される.(著者抄録)
  • 古川 雄祐, 菊池 次郎 最新医学 64 (12) 2506 -2511 2009年12月 [査読無し][通常論文]
     
    骨髄腫の薬剤耐性は自然耐性と獲得耐性に大別され,前者は液性因子によるものとVLA4を介する骨髄微小環境との接着によるものがある.メカニズムとして,細胞周期停止とアポトーシス抵抗性が重要である.後者は薬剤の長期暴露によって耐性が獲得される場合で,DNA修復能や薬物代謝の亢進による.最近,ボルテゾミブ,抗VLA4抗体,低分子CXCR4阻害薬などが接着耐性を解除し,薬剤感受性を向上させることが報告されている.(著者抄録)
  • Shin-ichiro Fujiwara, Tadashi Nagai, Satoru Kikuchi, Mitsuyo Uesawa, Chihiro Sakurai, Ken Ohmine, Jiro Kikuchi, Yusuke Furukawa, Keiya Ozawa BLOOD 114 (22) 523 -523 2009年11月 [査読無し][通常論文]
  • 造血前駆細胞の分化におけるホスファチジルグルコシドの発現
    岡 智子, 長塚 靖子, 菊池 次郎, 平林 義雄, 横手 耐治, 花房 俊昭, 小澤 敬也, 室井 一男 臨床血液 50 (9) 1210 -1210 2009年09月
  • 菊池 次郎, 和田 妙子, 清水 瑠美, 和泉 透, 光永 佳奈枝, 畑野 かおる, 信吉 正治, 小澤 敬也, 加納 康彦, 古川 雄祐 臨床血液 50 (9) 931 -931 2009年09月 [査読無し][通常論文]
  • オドゲレル・ツォグバドラク, 菊池 次郎, 和田 妙子, 加納 康彦, 古川 雄祐 臨床血液 50 (9) 965 -965 2009年09月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 Biotherapy 23 (5) 379 -385 2009年09月 [査読無し][通常論文]
     
    一見均一にみえる骨髄腫細胞集団であるが、クローン性の増殖を示す細胞は100〜100000個に1個しか存在しないことは1970年代から示されていた。最近の研究なら骨髄腫幹細胞のマーカーはCD138-/CD19+/CD27+/CD38-であることが示されており、これはメモリーBリンパ球に相当する。メモリーBリンパ球の段階で、免疫グロブリン重鎖を含む染色体転座やNF-κB活性化によって骨髄腫幹細胞が生じ、形質細胞に分化した段階で、微小環境との相互作用により骨髄腫として発症する。骨髄微小環境との相互作用により細胞周期停止、抗癌剤抵抗性(接着耐性)が獲得されるが、その責任分子はVLA4と考えられる。骨髄腫幹細胞を標的とする治療により、治療成績の大いなる改善が期待される。(著者抄録)
  • 菊池 次郎, 古川 雄祐 血液・腫瘍科 59 (3) 315 -320 2009年09月 [査読無し][通常論文]
  • ヒトKlotho遺伝子の転写調節機構の解明と薬剤による発現制御の試み
    東 昌広, 古川 雄祐, 菊池 次郎, 中澤 英子, 草野 英二 日本腎臓学会誌 51 (3) 340 -340 2009年04月
  • 菊池 次郎, 古川 雄祐 血液・腫瘍科 58 (2) 219 -224 2009年02月 [査読無し][通常論文]
  • 古川 雄祐, 菊池 次郎 最新医学 63 (12) 2375 -2380 2008年12月 [査読無し][通常論文]
     
    骨髄腫細胞の増殖・生存には,骨髄微小環境との相互作用が重要である.特に骨芽細胞との接着により,細胞周期停止,抗癌剤抵抗性(接着耐性)が獲得される.骨髄腫の細胞表面には種々の接着分子が発現しているが,接着耐性の責任分子はVLA4である.骨髄腫幹細胞のマーカーはCD138-/CD19+/CD27+/CD38-で,メモリーB細胞に相当する.メモリーB細胞の段階で染色体転座やNF-κB活性化によって骨髄腫幹細胞が生じ,形質細胞に分化した段階で微小環境との相互作用により骨髄腫として発症する.(著者抄録)
  • Kaoru Hatano, Jiro Kikuchi, Masaaki Takatoku, Rumi Shimizu, Taeko Wada, Masuzu Ueda, Masaharu Nobuyoshi, Iekuni Oh, Kazuya Sato, Takahiro Suzuki, Katsutoshi Ozaki, Masaki Mori, Tadashi Nagai, Kazuo Muroi, Yasuhiko Kano, Yusuke Furukawa, Keiya Ozawa BLOOD 112 (11) 577 -577 2008年11月 [査読無し][通常論文]
  • 白血病細胞におけるCalcipressin 1(DSCR-1)の異常発現
    菊池 悟, 永井 正, 菊池 次郎, 藤原 慎一郎, 上澤 光世, 吉田 こず恵, 古川 雄祐, 小澤 敬也 臨床血液 49 (9) 992 -992 2008年09月
  • PDGF受容体による受容体型チロシンキナーゼSTK/RONのトランス活性化
    小林 高久, 菊池 次郎, 草野 英二, 古川 雄祐 臨床血液 49 (9) 1197 -1197 2008年09月
  • 多発性骨髄腫と骨髄ストローマ細胞間相互作用が誘導する薬剤耐性における鍵分子の探索(Bortezomib overcomes CAM-DR via down-regulation of VLA-4 expression in multiple myeloma)
    畑野 かおる, 菊池 次郎, 高徳 正昭, 清水 瑠美, 和田 妙子, 上田 真寿, 鈴木 隆浩, 尾崎 勝俊, 永井 正, 室井 一男, 加納 康彦, 古川 雄祐, 小澤 敬也 日本癌学会総会記事 67回 353 -353 2008年09月
  • 畑野 かおる, 菊池 次郎, 高徳 正昭, 清水 瑠美, 和田 妙子, 上田 真寿, 鈴木 隆浩, 翁 家国, 佐藤 一也, 尾崎 勝俊, 森 政樹, 永井 正, 室井 一男, 加納 康彦, 古川 雄祐, 小澤 敬也 臨床血液 49 (9) 1203 -1203 2008年09月 [査読無し][通常論文]
  • 菊池 次郎, 清水 瑠美, 和田 妙子, 安藤 秀信, 中村 充, 小澤 敬也, 古川 雄祐 臨床血液 49 (9) 912 -912 2008年09月 [査読無し][通常論文]
  • 清水 瑠美, 和田 妙子, 菊池 次郎, 加納 康彦, 小澤 敬也, 古川 雄祐 臨床血液 49 (9) 999 -999 2008年09月 [査読無し][通常論文]
  • Odgerel Tsogbadrakh, 菊池 次郎, 和田 妙子, 加納 康彦, 古川 雄祐 臨床血液 49 (9) 990 -990 2008年09月 [査読無し][通常論文]
  • 和田 妙子, 菊池 次郎, 清水 瑠美, 北村 俊雄, 古川 雄祐 臨床血液 49 (9) 1195 -1195 2008年09月 [査読無し][通常論文]
  • メサンギウム増殖性腎炎における受容体型チロシンキナーゼRONの発現
    小林 高久, 古川 雄祐, 菊池 次郎, 伊藤 千春, 武藤 重明, 草野 英二 日本腎臓学会誌 50 (3) 340 -340 2008年04月
  • 菊池 次郎 自治医科大学紀要 30 189 -190 2007年12月
  • 菊池 次郎 血液フロンティア 17 (11) 1647 -1655 2007年10月 [査読無し][通常論文]
     
    E2Fは9つのE2Fサブユニット、2つのDPサブユニットのそれぞれ1つからなるヘテロ二量体の転写因子であり、細胞周期、細胞分化及びアポトーシス制御に関わっている。ヒトCD34陽性の造血幹/前駆細胞にはE2F-1,-2,-4、及び-6が発現しており、主にE2F-4が細胞周期を抑制し、E2F-2はS期への進行に働いている。E2F-1の作用は主にアポトーシス誘導であり、E2F-6はそれを拮抗阻害している。E2F-6は、造血幹/前駆細胞の増殖に伴うアポトーシス抑制により造血システムの維持に重要である。(著者抄録)
  • 17;19転座型ALLにおけるLMO2の過剰発現と細胞死の抑制作用
    廣瀬 衣子, 犬飼 岳史, 菊池 次郎, 黒田 格, 張 暁春, 本名 浩子, 合井 久美子, 加賀美 恵子, 稲葉 俊哉, 黒澤 秀光, 遠藤 幹也, 後藤 裕明, 古川 雄祐, 中澤 眞平, 杉田 完爾 臨床血液 48 (9) 873 -873 2007年09月
  • リンパ系腫瘍に対するBortezomib(ベルケード)と抗腫瘍薬の併用効果について-in vitroにおける検討
    畑野 かおる, 加納 康彦, 阿久津 美百生, 菊池 次郎, 上田 真寿, 高徳 正昭, 佐藤 一也, 阿部 正文, 永井 正, 古川 雄祐, 小澤 敬也 臨床血液 48 (9) 1093 -1093 2007年09月
  • 菊池 次郎, 清水 瑠美, 和田 妙子, 安藤 秀信, 中村 充, 小澤 敬也, 古川 雄祐 臨床血液 48 (9) 914 -914 2007年09月 [査読無し][通常論文]
  • Tsogbadrakh Odgerel, 菊池 次郎, 和田 妙子, 清水 瑠美, 松尾 良信, 加納 康彦, 古川 雄祐 臨床血液 48 (9) 878 -878 2007年09月 [査読無し][通常論文]
  • リンパ系腫瘍に対するBortezomib(ベルケード)と抗腫瘍薬の併用効果について in vitroにおける検討(Effects of bortezomib in combination with conventional anticancer drugs against human lymphoid cell lines)
    畑野 かおる, 加納 康彦, 阿久津 美百生, 菊池 次郎, 上田 真寿, 高徳 正昭, 鈴木 隆浩, 佐藤 一也, 阿部 正文, 永井 正, 古川 雄祐, 小澤 敬也 日本癌学会総会記事 66回 525 -526 2007年08月
  • メサンギウム細胞増殖における受容体型チロシンキナーゼRONの役割
    小林 高久, 古川 雄祐, 菊池 次郎, 伊藤 千春, 宮田 幸雄, 武藤 重明, 草野 英二 日本腎臓学会誌 49 (3) 307 -307 2007年04月
  • ヒトプレB細胞のセレクチンカウンターリガンドの探索と同定
    野々村 智尋, 菊池 次郎, 尾崎 秀徳, 安藤 秀信, 中村 充 日本免疫学会総会・学術集会記録 35 165 -165 2005年11月
  • ヒトプレB細胞セレクチンカウンターリガンドCD43の翻訳後糖鎖修飾とセレクチン反応性制御メカニズム
    菊池 次郎, 尾崎 秀徳, 野々村 智尋, 安藤 秀信, 中村 充 日本免疫学会総会・学術集会記録 35 165 -165 2005年11月
  • J Kikuchi, J Mimuro, K Ogata, T Tabata, Y Ueda, A Ishiwata, K Kimura, K Takano, S Madoiwa, H Mizukami, Y Hanazono, A Kume, M Hasegawa, K Ozawa, Y Sakata JOURNAL OF GENE MEDICINE 7 (6) 836 -836 2005年06月 [査読無し][通常論文]
  • J Mimuro, K Ogata, J Kikuchi, T Tabata, Y Ueda, M Naito, S Madoiwa, M Hasegawa, K Ozawa, Y Sakata BLOOD 102 (11) 742A -742A 2003年11月 [査読無し][通常論文]
  • J Mimuro, M Naito, H Endo, S Madoiwa, K Ogata, J Kikuchi, T Sugo, T Yasu, Y Kariya, Y Hoshino, Y Sakata BLOOD 102 (11) 314A -314A 2003年11月 [査読無し][通常論文]
  • J Mimuro, J Kikuchi, T Tabata, Y Ueda, A Kume, Y Hanazono, H Mizukami, K Ogata, S Madoiwa, T Sugo, M Hasegawa, K Ozawa, Y Sakata BLOOD 100 (11) 441A -441A 2002年11月 [査読無し][通常論文]
  • F Mimuro, K Ogata, H Mizukami, J Kikuchi, S Madoiwa, T Sugo, Y Hanazono, A Kume, A Yoshioka, K Ozawa, Y Sakata BLOOD 100 (11) 90B -90B 2002年11月 [査読無し][通常論文]
  • 河村 喜雄, 吉羽 洋周, 田中 伸司, 佐藤 一雄, 菊池 次郎, 高橋 かほる 育種学雑誌 43 (1) 91 -99 1993年03月 [査読無し][通常論文]
     
    The survivability of protoplasts that dominates biological cell operation efficiency of an automated processing apparatus was investigated. A method of evaluating survivability of protoplasts is newly developed. The method is to observe the decay in the number of living protoplasts loaded under mechanical stress. Several hundreds of protoplasts were individually placed in microchambers which provide weak suction force intermittently to each protoplast. The suction force accelerated the breakage of protoplasts . The quantity of living protoplasts decreased exponentially. A time constant of the decreasing function was defined as a time constant of survivability. Using the time constant of survivability, the survivability of protoplasts could be evaluated quantitatively. The half decay in the nurnber of living protoplasts was in the range of 30 to 60 minutes; the conventional evaluation method of observing the cell wall regeneration capability and the multiple cell division capability has taken more than several weeks.

産業財産権

  • 特願2017-134173:リジン特異的脱メチル化酵素1阻害活性を有する新規化合物、その製造方法及びその用途  2017年07月07日
    梅原崇史, 佐藤 心, 小山裕雄, 山本博文, 近藤 豊, 勝島啓佑, 古川雄祐, 菊池次郎
  • 特願2017-133702:多能性幹細胞からのテラトーマ形成抑制剤及びその用途  2017年07月07日
    古川雄祐, 菊池次郎
  • 特願2017-133951:多能性幹細胞からのテラトーマ形成抑制剤及びその用途  2017年07月07日
    菊池次郎, 古川雄祐, 梅原崇史, 佐藤心
  • 中村 充, 菊池 次郎, 野々村 智尋, 安藤 秀信  独立行政法人産業技術総合研究所, 独立行政法人科学技術振興機構  200903030025079614
  • 岡本 治正, 菊池 次郎  独立行政法人産業技術総合研究所, 日立工機株式会社, 岡本 治正  200903004658544153
  • 菊池 次郎  日立工機株式会社  200903007712758562
  • 河村 喜雄, 吉羽 洋周, 田中 伸司, 佐藤 一雄, 菊池 次郎, 高橋 かほる  株式会社日立製作所, 日立工機株式会社  201103013435715662

受賞

  • 2020年11月 自治医科大学 最優秀論文賞
     Eradication of central nervous system leukemia of T-cell origin with a brain-permeable LSD1 inhibitor.
  • 2019年11月 自治医科大学 優秀論文賞
     Myeloma cells are activated in bone marrow microenvironment by the CD180/MD-1 complex, which senses lipopolysaccharide.
  • 2016年11月 自治医科大学 優秀論文賞
     Overexpression of the shortest isoform of histone demethylase LSD1 primes hematopoietic stem cells for malignant transformation.
  • 2016年11月 自治医科大学 最優秀論文賞
     Phosphorylation- mediated EZH2 inactivation promotes drug resistance in multiple myeloma.
  • 2015年11月 自治医科大学 優秀論文賞
     Proteasome inhibitors exert cytotoxicity and increase chemosensitivity via transcriptional repression of Notch1 in T-cell acute lymphoblastic leukemia.
  • 2012年11月 自治医科大学 優秀論文賞
     Latexin regulates the abundance of multiple cellular proteins in hematopoietic stem cells.
  • 2011年11月 自治医科大学 優秀論文賞
     Histone deacetylases are critical targets of bortezomib-induced cytotoxicity in multiple myeloma.
  • 2010年11月 自治医科大学 優秀論文賞
     Bortezomib overcomes cell adhesion-mediated drug resistance via down-regulation of VLA-4 expression in multiple myeloma.
  • 2010年07月 日本白血病基金 クレディセゾン賞
  • 2009年11月 自治医科大学 優秀論文賞
     The FLT3 inhibitor PKC412 exerts differential cell cycle effects on leukemic cells depending on the presence of FLT3 mutations.
  • 2008年10月 第70回日本血液学会第50回日本臨床血液学会総会 優秀ポスター賞
  • 2007年11月 自治医科大学 優秀論文賞
     E2F-6 suppresses growth-associated apoptosis of human hematopoietic progenitor cells by counteracting proapoptotic activity of E2F-1.
  • 2005年10月 第67回日本血液学会第47回日本臨床血液学会総会 優秀ポスター賞
  • 2004年10月 第66回日本血液学会第46回日本臨床血液学会総会 優秀ポスター賞

共同研究・競争的資金等の研究課題

  • 多発性骨髄腫の髄外増幅形成と薬剤耐性獲得機構の解明
    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 : 菊池 次郎
  • 難治性がんに対する VHH 抗体薬物複合体の開発
    日本医療研究開発機構(AMED):橋渡し研究戦略的推進プログラムシーズ A
    研究期間 : 2021年
  • 日本学術振興会:科学研究費補助金基盤研究C
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 菊池次郎
  • ヒストンメチル化酵素MMSETを標的としたがん治療薬の創成
    テルモ生命科学振興財団:研究開発助成金
    研究期間 : 2020年
  • 多発性骨髄腫細胞同士の相互作用による薬剤耐性獲得機構の解明
    高松宮妃癌研究基金:研究助成金
    研究期間 : 2020年
  • 完全人工合成ライブラリーを用いた治療用VHH抗体の開発
    武田科学振興財団:ビジョナリーリサーチ 研究助成
    研究期間 : 2020年
  • ヒストンメチル化酵素 KMT3/NSD2/MMSET を標的とした難治性がんに対する新規治療薬の開発
    日本医療研究開発機構(AMED):橋渡し研究戦略的推進プログラムシーズ A
    研究期間 : 2018年
  • MMSET阻害剤の開発
    骨髄腫患者の会:骨髄腫患者の会研究助成
    研究期間 : 2018年
  • 中枢神経浸潤を伴う小児白血病に対するエピジェネティク療法の開発
    がんの子どもを守る会:研究助成
    研究期間 : 2017年
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2016年 
    代表者 : 菊池 次郎
  • ヒストンメチル化酵素NSD2/MMSETを分子標的とする新規抗がん剤の開発
    理化学研究所:創薬・医療技術基盤プログラム
    研究期間 : 2016年
  • 多発性骨髄腫におけるToll様受容体関連分子CD180の機能 解明
    ノバルティスファーマ:研究助成
    研究期間 : 2016年
  • 多発性骨髄腫におけるToll様受容体CD180の機能解明と 臨床応用
    先進医薬研究振興財団:血液医学研究助成
    研究期間 : 2016年
  • 母子健康協会:小児医学研究助成
    研究期間 : 2016年
  • 中枢神経浸潤を伴う小児白血病に対するエピジェネティク療法の開発
    日本白血病研究基金:一般研究賞
    研究期間 : 2016年
  • 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 : 2011年04月 -2015年03月 
    代表者 : 黒澤 秀光, 菊池 次郎, 古川 雄祐, 犬飼 岳史, 稲葉 俊哉
     
    t(17;19) B前駆細胞性急性リンパ性白血病(t(17;19)+ALL)は、転座により生じる融合転写因子E2A-HLFが、白血病化とともに骨浸潤、高カルシウム血症、凝固線溶異常を誘導する。E2A-HLFの下流遺伝子PTHrPを同定した。t(17;19)-ALL細胞株にE2A-HLFを導入するとPTHrPが誘導され、E2A-HLFの変異体ではPTHrPは誘導しなかった。t(17;19)+ALL細胞株でPTHrPをノックダウンするとアポトーシスが誘導された。以上よりt(17;19)+ALLにおいてPTHrPはE2A-HLFの下流遺伝子で、高カルシウム血症と白血病化あるいは難治化に関与している。
  • ホモピペラジン化合物による新規プロテアソーム阻害剤経口薬の開発
    三井生命厚生事業団:医学研究助成特別助成
    研究期間 : 2014年
  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2011年 -2013年 
    代表者 : 菊池 次郎
     
    本研究では、造血器悪性腫瘍の抗がん剤耐性の克服を目指し、腫瘍細胞が抗がん剤耐性を獲得する際のヒストン修飾を介した遺伝子発現調節機構の解明を進めた。 まず、造血器悪性腫瘍細胞を抗がん剤にて処理の後、耐性化した細胞におけるヒストンコードの特徴を解析した。今回は、造血器悪性腫瘍の中でも抗がん剤耐性の克服が重要な課題であり、現在も難治性の疾患である多発性骨髄腫をモデルに用いて検討を進めた。抗がん剤としては、多発性骨髄腫の治療に用いられるデキサメタゾン、ビンクリスチン、ドキソルビシンやボルテゾミブを用いた。これらの抗がん剤を骨髄腫細胞株に作用させ、アセチル化やメチル化などの修飾を受けるヒストンH3のlysine-4(以下H3K4と略)やH3K9、H3K18、H3K27、H3K36及びH4K12のヒストン修飾様式を、それぞれの特異抗体を用いてウェスタンブロットにより検出した。 4種類の骨髄腫細胞株に先記した4種類の抗がん剤を作用させた結果、H3K27のメチル化の亢進が特徴的に観察された。また、H3K27のメチル化酵素であるEZH2の発現も検出された。EZH2はH3K27をメチル化して転写抑制的に働くことが報告されている。従って、EZH2が骨髄腫細胞のアポトーシス誘導に働く遺伝子の発現抑制を介して抗がん剤耐性に関与している可能性が示唆された。今後、EZH2遺伝子の過剰発現とノックダウンや、EZH2ヒストンメチル化阻害剤などを用いて抗がん剤耐性における機能解明を進める方針である。
  • 武田科学振興財団:医学系研究奨励継続助成
    研究期間 : 2013年
  • ヒストンメチル化を介した造血器悪性腫瘍の薬剤耐性獲得機構の解明と臨床応用
    安田記念医学財団:癌研究助成
    研究期間 : 2013年
  • プロテアソーム阻害作用を有するホモピペラジン化合物の探索(継続)
    科学技術振興機構:研究成果最適展開支援プログラムA-STEP探索タイプ
    研究期間 : 2013年
  • ホモピペラジン化合物による新規プロテアソーム阻害剤経口薬の開発
    三井生命厚生事業団:医学研究助成
    研究期間 : 2012年
  • ホモピペラジン化合物による新規プロテアソーム阻害剤経口薬の開発
    大阪癌研究会:研究助成
    研究期間 : 2012年
  • プロテアソーム阻害作用を有するホモピペラジン化合物の探索
    科学技術振興機構:研究成果最適展開支援プログラムA-STEP探索タイプ
    研究期間 : 2012年
  • ヒストン修飾を介した造血器悪性腫瘍細胞の耐性化機構の解明
    武田科学振興財団:医学系研究奨励
    研究期間 : 2011年
  • 新規プロテアソーム阻害剤経口薬の開発
    加納基金:研究助成
    研究期間 : 2011年
  • 文部科学省:科学研究費補助金(萌芽研究, 挑戦的萌芽研究)
    研究期間 : 2008年 -2010年 
    代表者 : 菊池 次郎
     
    1. 目的;骨髄移植など造血幹細胞を用いた治療法における問題解決を目指し、末梢血白血球(T-リンパ球)から造血幹細胞を誘導する方法を開発する。2. 方法;T-リンパ球に転写調節因子を始めとする遺伝子群を導入し、その発現様式を造血幹細胞に限りなく近づけることによって、造血幹細胞の誘導を目指す。3. 結果;これまでの検討から、造血幹細胞に高発現する遺伝子数種類を導入したT-リンパ球を数日間培養すると、造血幹細胞特異的マーカーであるCD34やc-kit発現細胞が誘導できることを明らかにした。さらに、ピストン脱アセチル化(HDAC)阻害剤とDNAメチル化阻害剤(アザシチジン)の併用により、CD34陽性細胞の誘導効率を向上させた。今年度はさらなる効率の向上を目指し、HDAC阻害剤とT-リンパ球に特異的に発現する遺伝子のノックダウンの併用について検討した。まず、HDAC阻害剤であるバルプロ酸、ロミデプシン及びトリコスタチンA(TSA)の中では、TSAが細胞毒性が少なく、最も高いHDAC阻害作用を示した。続いて、T-リンパ球に高発現する遺伝子として、転写因子GATA3を同定した。GATA3はリンパ球分化の制御因子であり、分化の方向付けに重要なことが報告されている。そこで、sh-RNAのレンチウイルス導入系を作成し、GATA3の発現ノックダウンの併用を試みた。その結果、CD34陽性細胞の誘導効率の改善は見られなかったが、メチルセルロース半固形培地において造血幹細胞由来のより大型のコロニー形成を誘導することができた。以上の結果から、末梢血白血球(T-リンパ球)にホメオボックス転写因子群及びGATA3に対するsh-RNAをレンチウイルスを用いて導入し、TSA及びアザシチジン添加下で培養することで、CD34陽性細胞とコロニー形成能を有する造血幹細胞様の細胞を誘導することができた。
  • 日本白血病研究基金:クレディセゾン賞
    研究期間 : 2010年
  • 末梢血リンパ球脱分化による造血幹細胞の誘導
    金原一郎記念医学医療 振興財団:研究助成
    研究期間 : 2008年
  • 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 : 2006年 -2007年 
    代表者 : 古川 雄祐, 菊池 次郎, 和田 妙子
     
    1)血液細胞におけるHDAC発現とその制御機構の解明:(1)正常造血幹細胞/前駆細胞(CD34陽性骨髄、臍帯血単核細胞)においてHDACの発現はきわめて低く、特に蛋白レベルでは検出限度以下であった。一方、committed progenitors(CD34陰性骨髄単核細胞)においては発現が認められたが、顆粒球・単球では再度発現が低下した。この発現パターンはin vitroの培養系でも再現された。(1)急性白血病においては細胞株(HL-60、U937、K562)、臨床検体ともにHDACの強発現を認めた。HL-60、U937を顆粒球ないし単球系に分化させると、HDACの発現は著明に低下した。一方、K562を赤芽球・巨核球系に分化させた場合は、HDACの発現は抑制されなかった。(3)HDAC1プロモーターはTATA lessで、GC box、CCAAT box、GATAおよびMZF-1の結合配列を認めた。プロモーター活性はSp-1、GATA-1によって活性化され、GATA-2、MZF-1、C/EBPα、C/EBPβによって抑制された。またGATA-1によるHDAC1転写活性化は、GATA-2+MZF-1によって著明に抑制された。 2)造血幹細胞におけるHDACの機能と白血化への関与の解明:(1)純化した造血幹細胞にHDAC1を強発現させたが、明らかな増殖の促進は認めなかった。(2)C57BL/6(Ly-5.1)マウス由来の造血幹細胞(c-Kit陽性細胞)にレトロウイルスを用いてHDAC1を発現させ、放射線照射したC57BL/6(Ly-5.2)マウスに移植した。現在、約9か月の観察であるが、HDAC1発現群において有意の白血球減少が認められている。一方、赤血球数、血小板数は対照群(Mock)、無処置マウスと差を認めていない。
  • 新規高脂血症原因遺伝子PCSK9の血中LDL濃度調節機構解明と冠動脈疾患早期診断への応用
    黒住医学研究振興財団:研究助成金
    研究期間 : 2007年
  • 幹細胞の骨髄ホーミングを誘導する糖鎖性分子の探索と幹細胞純化への応用
    自治医科大学:医学部 研究奨励金
    研究期間 : 2006年

委員歴

  • 2021年05月   日本骨髄腫学会   代議員
  • 2018年   日本白血病基金   研究助成金予備審査委員
  • 2017年10月   日本血液学会   評議員

メディア報道

  • 多発性骨髄腫の抗体薬エロツズマブ、新たな 作用メカニズムを発見-自治医科大
    報道 : 2019年08月
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