研究者総覧

和田 妙子 (ワダ タエコ)

  • 臨床研究支援センター 講師
Last Updated :2021/12/04

研究者情報

通称等の別名

    和田 妙子

学位

  • 医学博士(自治医科大学)

ホームページURL

科研費研究者番号

  • 30382956

ORCID ID

J-Global ID

研究キーワード

  • レギュラトリーサイエンス   エピジェネティクス   癌   

研究分野

  • ライフサイエンス / 分子生物学
  • ライフサイエンス / 腫瘍生物学
  • ライフサイエンス / 血液、腫瘍内科学

所属学協会

  • 日本血液学会   DIA   日本臨床試験学会   日本臨床薬理学会   

研究活動情報

論文

  • Junya Tamaoki, Miki Takeuchi, Ryo Abe, Hiroshi Kaneko, Taeko Wada, Shinjiro Hino, Mitsuyoshi Nakao, Yusuke Furukawa, Makoto Kobayashi
    Scientific reports 10 1 8521 - 8521 2020年05月 [査読有り][通常論文]
     
    LSD1/KDM1A is a widely conserved lysine-specific demethylase that removes methyl groups from methylated proteins, mainly histone H3. We previously isolated the zebrafish LSD1 gene and demonstrated that it is required for primitive hematopoiesis. Recently, a neuron-specific splicing variant of LSD1 was found in mammals and its specific functions and substrate specificities were reported. To our surprise, zebrafish LSD1 cDNA, which we previously analyzed, was corresponded to the neuron-specific variant in mammals. In this study, we investigated the structures and expression of LSD1 splicing variants in zebrafish and found all 4 types of LSD1 isoforms: LSD1, LSD1+2al, LSD1+8al and LSD1+2al8al. Interestingly, LSD1+8al/LSD1+2al8al, which correspond to mammalian neuron-specific variants, expressed ubiquitously in zebrafish. We also performed phenotypic rescue experiments of a zebrafish LSD1 mutant (kdm1ait627) using human and zebrafish LSD1 variants to identify which variant is involved in primitive hematopoiesis. Unexpectedly, the overexpression of all types of human and zebrafish variants was able to rescue the hematopoietic phenotypes in LSD1 mutants. Furthermore, enzymatic-deficient LSD1K661A (human) and K638A (zebrafish) were also able to rescue the mutant phenotypes. These results suggest that the LSD1 functions in zebrafish primitive hematopoiesis are free from any splicing-dependent regulation or demethylation reaction.
  • Taeko Wada, Jiro Kikuchi, Daisuke Koyama, Hiroaki Honda, Yusuke Furukawa
    Leukemia research 82 29 - 32 2019年07月 [査読有り][通常論文]
  • 古川 雄祐, 和田 妙子, 菊池 次郎
    血液内科 72 6 857 - 862 (有)科学評論社 2016年06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)] (公社)日本生化学会 2015年12月
  • Jiro Kikuchi, Daisuke Koyama, Taeko Wada, Tohru Izumi, Peter O Hofgaard, Bjarne Bogen, Yusuke Furukawa
    The Journal of clinical investigation 125 12 4375 - 90 2015年10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/lymphoma 2 (BCL2), and hypoxia inducible factor 1, α subunit (HIF1A). Pharmacological and genetic inhibition of the IGF-1R/PI3K/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 - 46 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 HoxA family 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 γ-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.
  • 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 [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.
  • D. Koyama, J. Kikuchi, N. Hiraoka, T. Wada, H. Kurosawa, S. Chiba, Y. Furukawa
    Leukemia 28 6 1216 - 1226 2014年 [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-κB (NF-κB) (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. © 2014 Macmillan Publishers Limited.
  • N. Hiraoka, J. Kikuchi, D. Koyama, T. Wada, S. Mori, Y. Nakamura, Y. Furukawa
    BLOOD CANCER JOURNAL 3 2013年12月 [査読有り][通常論文]
  • Jiro Kikuchi, Satoshi Yamada, Daisuke Koyama, Taeko Wada, Masaharu Nobuyoshi, Tohru Izumi, Miyuki Akutsu, Yasuhiko Kano, Yusuke Furukawa
    The Journal of biological chemistry 288 35 25593 - 602 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 β5-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.
  • 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 [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 (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 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 ß5-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.
  • Kanae Mitsunaga, Jiro Kikuchi, Taeko Wada, Yusuke Furukawa
    Journal of cellular physiology 227 3 1138 - 47 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.
  • Taeko Wada, Jiro Kikuchi, Yusuke Furukawa
    EMBO reports 13 2 142 - 9 2012年02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.
  • Yusuke Furukawa, Nobuya Hiraoka, Taeko Wada, Jiro Kikuchi, Yasuhiko Kano
    Nihon yakurigaku zasshi. Folia pharmacologica Japonica 138 1 26 - 32 2011年07月 [査読有り][通常論文]
  • R. Shimizu, J. Kikuchi, T. Wada, K. Ozawa, Y. Kano, Y. Furukawa
    LEUKEMIA 24 10 1760 - 1768 2010年10月 [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 mM, 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. Leukemia (2010) 24, 1760-1768; doi: 10.1038/leu.2010.157; published online 5 August 2010
  • 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 - 17 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-kappaB 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.
  • 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]
  • Taeko Wada, Jiro Kikuchi, Noriko Nishimura, Rumi Shimizu, Toshio Kitamura, Yusuke Furukawa
    The Journal of biological chemistry 284 44 30673 - 83 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 erythro-megakaryocytic 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 erythro-megakaryocytic 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.
  • Motohiro Sekiya, Jun-Ichi Osuga, Shuichi Nagashima, Taichi Ohshiro, Masaki Igarashi, Hiroaki Okazaki, Manabu Takahashi, Fumiko Tazoe, Taeko Wada, Keisuke Ohta, Mikio Takanashi, Masayoshi Kumagai, Makiko Nishi, Satoru Takase, Naoya Yahagi, Hiroaki Yagyu, Ken Ohashi, Ryozo Nagai, Takashi Kadowaki, Yusuke Furukawa, Shun Ishibashi
    Cell metabolism 10 3 219 - 28 2009年09月 [査読有り][通常論文]
     
    Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1(Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.
  • 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.
  • 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
  • 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.
  • Jiro Kikuchi, Rumi Shimizu, Taeko Wada, Hidenobu Ando, Mitsuru Nakamura, Keiya Ozawa, Yusuke Furukawa
    Stem cells (Dayton, Ohio) 25 10 2439 - 47 2007年10月 [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. Disclosure of potential conflicts of interest is found at the end of this article.
  • Yutaka Furukawa, Krittaya Sutheesophon, Taeko Wada, Miki Nishimura, Yasushi Saito, Hideshi Ishii, Yusuke Furukawa
    Molecular cancer research : MCR 3 6 325 - 34 2005年06月 [査読有り][通常論文]
     
    Apaf-1 is important for tumor suppression and drug resistance because it plays a central role in DNA damage-induced apoptosis. Inactivation of the Apaf-1 gene is implicated in disease progression and chemoresistance of some malignancies. In this study, we attempted to clarify the role of Apaf-1 in leukemogenesis. Apaf-1 mRNA levels were below the detection limit or very low in 5 of 20 human leukemia cell lines (25%) and 5 of 12 primary acute myeloblastic leukemia cells (42%). There were no gross structural abnormalities in the Apaf-1 gene in these samples. Expression of factors regulating Apaf-1 transcription, such as E2F-1, p53, and Sp-1, did not differ between Apaf-1-positive and Apaf-1-negative cells. Methylation of CpG in the region between +87 and +128 of the Apaf-1 gene was almost exclusively observed in Apaf-1-defective cell lines. Treatment of these cells with 5-aza-2'-deoxycytidine, a specific inhibitor of DNA methylation, restored the expression of Apaf-1. Furthermore, we showed that the region between +87 and +128 could act as a repressor element by recruiting corepressors such as methylated DNA-binding domain 2 and histone deacetylase 1 upon methylation. Overexpression of Dnmt1, a mammalian maintenance DNA methyltransferase, was associated with Apaf-1 gene methylation. DNAs from Dnmt1-overexpressing cells were more resistant to digestion with methylation-sensitive enzyme HpaII than those from cells with low Dnmt1 expression, suggesting that Dnmt1 mediates aberrant methylation of multiple genes. In conclusion, methylation silencing is a mechanism of the inactivation of Apaf-1 in acute leukemia, and Dnmt1 overexpression may underlie hypermethylation of the Apaf-1 gene.
  • Yasuo Yamazaki, Hisashi Koike, Yusuke Sugiyama, Kazuko Motoyoshi, Taeko Wada, Shigeru Hishinuma, Mitsuo Mita, Takashi Morita
    European journal of biochemistry 269 11 2708 - 15 2002年06月 [査読有り][通常論文]
     
    In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.

MISC

共同研究・競争的資金等の研究課題

  • 文部科学省:科学研究費補助金(基盤研究(C))
    研究期間 : 2015年 -2017年 
    代表者 : 和田 妙子
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2013年 -2014年 
    代表者 : 和田 妙子
     
    LSD1はヒストン脱メチル化する酵素で、染色体構造をダイナミックに改変し、別々の染色体に位置するプロモーターを会合させ、転写を複合的に調節する。申請者らは、造血幹細胞において放射線等によってDNA二重鎖切断が惹起されると、切断部位からの転写を抑制するためにLSD1複合体がリクルートされるが、LSD1に量的・質的異常が存在すると染色体転座が誘導されるという仮説を立て研究を開始した。哺乳類のLSD1にはalternative splicingによりエクソン2と8に挿入が入ることで生じる4種のアイソフォームが存在することが報告されている。私たちはエクソン2の挿入(2a)の有無により生じる2つのアイソフォーム、2a-/8a- (-/-) と2a+/8a- (+/-) が存在していることを確認した。さらに、このもっとも短いアイソフォーム (-/-) はヒストン脱メチル化活性の本体であり、その発現は造血幹細胞において極めて低く、白血病細胞で強発現していることを確認した。私たちは造血幹・前駆細胞特異的にLSD1 (-/-) を強発現するトランスジェニック・マウスを作製した。これにより、LSD1 (-/-) を低レベルおよび高レベルに発現する2ラインを得た。LSD1 (-/-) の発現量依存的にLSK細胞の増加がみられ、さらにLSD1の低発現ラインの骨髄細胞は幹細胞性を示し、高発現のラインでは造血幹・前駆細胞の増加を確認した。これはヒト造血幹・前駆細胞におけるLSD1アイソフォームの発現パターンと一致していた。これらトランスジェニック・マウスは一見して正常ではあるが、造血幹細胞の自己複製能が亢進していることから、さらなる遺伝子異常を追加することで白血病の発症や染色体転座の頻度が増加するのではないかと考えた。実際、T細胞性リンパ芽球性リンパ腫・白血病をより早期かつ高頻度に発症した。
  • 文部科学省:科学研究費補助金(若手研究(B))
    研究期間 : 2011年 -2012年 
    代表者 : 和田 妙子
     
    ヒストン脱メチル化酵素 LSD1 は正常細胞にほとんど発現しておらず、白血病細胞では強発現していた。特に染色体転座を有する白血病細胞ではより強発現していた。さらに、血液細胞において LSD1 は2種のアイソフォームが存在し、そのうちの一方が未分化な造血前駆細胞の自己複製能を亢進することがわかった。このアイソフォームの量的異常が白血病化や染色体転座に関与しており治療標的になることが推測され、造血幹細胞特異的に LSD1 を強発現するトランスジェニックマウスと骨髄移植マウスモデルを用いて造血器異常について解析している。


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