Ｇｅｍｉｎｉｎ発現量を標的とした白血病幹細胞制御法の開発への基盤研究

• Project Area: Development of Novel Treatment Strategies Targeting Cancer Stem Cells
• Project/Area Number: 25130708
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Hiroshima University
• Principal Investigator: 安永 晋一郎 広島大学, 原爆放射線医科学研究所, 准教授 (50336111)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000); Fiscal Year 2014: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2013: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
• Keywords: 白血病幹細胞 / 造血幹細胞 / 自己複製 / 分化誘導 / 細胞周期 / DＮＡ複製制御 / クロマチンリモデリング / Geminin / ＤＮＡ複製クロマチンリモデリング

Outline of Annual Research Achievements

これまでの本研究にける助成金にて、Gemininの発現を可視化することのできるGeminin-EYFPノックインマウスの骨髄細胞を用いた様々な血液学的解析方法を確立し、それを発展させた。特に、造血幹細胞分画であるCD34-KSL （c-Kit+Sca1+Lin-）細胞の中にGeminin-high細胞群を見つけ、その細胞の中には致死的放射線照射を施したマウスに移植すると長期骨髄再構築能を示す細胞が存在することを明らかにし、このノックインマウスを使うことで自己複製中の造血幹細胞を同定することが可能であることを示した。さらにCD34-KSL （c-Kit+Sca1+Lin-）細胞からCD150-high細胞を分画し、より自己複製中の造血幹細胞をエンリッチした集団を分画することに成功した。この実験系は、自己複製中の白血病幹細胞の同定にも使用できるものと考えられる。また、MLL-AF9、Hoxa9 (+Meis1a)、BCR-ABLなどマウスに実験的白血病を引き起こすことのできる遺伝子群を、各種赤色蛍光（RFP; DsRed Express2、tandem Tomato (tdTomato)、humanized Kusabira Orange (hKO)など）をマーカーに持ったレトロウイルスベクターにつなぎ、従来使用していたGFPマーカーと異なりノックインマウスのEYFPと容易に区別が可能なRFPをマーカーとして持った白血病細胞及び白血病幹細胞をGeminin-EYFPノックインマウス由来の骨髄細胞から作製することが可能になり、白血病幹細胞におけるGemininの発現動態を詳細に解析できるようになった。また、ヒト白血病幹細胞の活性解析を行うために免疫不全マウス（BRGS）を九州大学より入手し、その繁殖を行い、それを用いたヒト白血病幹細胞の解析を行うための基礎実験を完了した。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Transcription of the Geminin gene is regulated by a negative-feedback loop. (2014)
  • Author(s): Ohno, Y., Saeki, K., Yasunaga, S., Kurogi. T., Suzuki-Takedachi, K., Shirai, M., Mihara K., Yoshida, K., Voncken J. W, Ohtsubo, M. & Takihara Y.
  • Journal Title: Mol. Biol. Cel Volume: 25(8) Issue: 8 Pages: 1374-1383
  • DOI: 10.1091/mbc.e13-09-0534
  • Peer Reviewed / Open Access
• [Journal Article] Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis. (2014)
  • Author(s): Mizoguchi Y, Tsumura M, Okada S, Hirata O, Minegishi S, Imai K, Hyakuna N, Muramatsu H, Kojima S, Ozaki Y, Imai T, Takeda S, Okazaki T, Ito T, Yasunaga S, Takihara Y, Bryant VL, Kong XF, Cypowyj S, Boisson-Dupuis S, Puel A, Casanova JL, Morio T, Kobayashi M.
  • Journal Title: J. Leukoc. Biol. Volume: 95 Issue: 4 Pages: 667-76
  • DOI: 10.1189/jlb.0513250
  • Peer Reviewed / Open Access
• [Journal Article] Scmh1 has E3 ubiquitin ligase activity for Geminin and histone H2A and regulates Geminin stability directly or indirectly via transcription repression of Hoxa9 and Hoxb4 (2013)
  • Author(s): Yasunaga, S., Ohtsubo, M., Ohno, Y., Saeki, K., Kurogi. T., Tanaka-Okamoto, M., Ishizaki, H., Shirai, M., Mihara, K., Brock, H. W., Miyoshi, J. & Takihara Y
  • Journal Title: Mol. Cell. Biol. Volume: 33(4) Issue: 4 Pages: 644-660
  • DOI: 10.1128/mcb.00974-12
  • Peer Reviewed
• [Journal Article] Hoxa9 transduction induces mouse hematopoietic stem and progenitor cell activity through direct down-regulation of Geminin. (2013)
  • Author(s): Ohno, Y., Yasunaga, S., Janmohamed, S., Ohtubo, M., Mihara, K., Iscove, N., Takihara, Y.
  • Journal Title: PLoS ONE Volume: 8(1) Issue: 1 Pages: e53161-e53161
  • DOI: 10.1371/journal.pone.0053161
  • Peer Reviewed
• [Presentation] Effects of low-dose irradiation on the hematopoietic system. (2015)
  • Author(s): Yasunaga S., Ohno Y., Suzuki-Takedachi K., Kurogi T., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 5th International Symposium of RIRBM, Hiroshima University
  • Place of Presentation: Hiroshima, Japan
  • Year and Date: 2015-03-02 – 2015-03-03
• [Presentation] Cell-penetrating Geminin is exerted to provide a new strategy for manipulating the activity and number of HSCs. (2015)
  • Author(s): Kurogi T., Ohno Y., Yasunaga S., Suzuki-Takedachi K., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 5th International Symposium of RIRBM, Hiroshima University
  • Place of Presentation: Hiroshima, Japan
  • Year and Date: 2015-03-02 – 2015-03-03
• [Presentation] Analysis of molecular rele for Geminin in self-renewal and differentiation of HSCs. (2015)
  • Author(s): Suzuki-Takedachi K., Yasunaga S., Ohno Y., Kurogi T., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 5th International Symposium of RIRBM, Hiroshima University
  • Place of Presentation: Hiroshima, Japan
  • Year and Date: 2015-03-02 – 2015-03-03
• [Presentation] Simple diagnosis of STAT1 gain-of-function alleles in patient with chronic mucocutaneous candidiasis presenting various clinical signs. (2015)
  • Author(s): Mizoguchi Y., Okada S., Tsumura M., Yasunaga S., Takihara Y., Casanova J.-L., Morio T., Kobayashi M.
  • Organizer: The 5th International Symposium of RIRBM, Hiroshima University
  • Place of Presentation: Hiroshima, Japan
  • Year and Date: 2015-03-02 – 2015-03-03
• [Presentation] Geminin-EYFP knock-in mice provide a powerful tool for dissecting a molecular mechanism regulating self-renewal or differentiation of hematopoietic stem cells. (2014)
  • Author(s): Suzuki-Takedachi K., Yasunaga S., Ohno Y., Kurogi T., Ohtsubo M., Takihara Y.
  • Organizer: The 37th Annual Meeting of the Molecular Biology of Japan
  • Place of Presentation: Yokohama, Japan
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] Cell-penetrating recombinant Geminin protein may provide a new strategy for regulating self-renewal or cellular differentiation in hematopoietic stem cells. (2014)
  • Author(s): Kurogi T., Ohno Y., Yasunaga S., Suzuki-Takedachi K., Santo M., Masuhiro Y., Hanazawa S., Ohtsubo M., Takihara Y.
  • Organizer: The 37th Annual Meeting of the Molecular Biology of Japan
  • Place of Presentation: Yokohama, Japan
  • Year and Date: 2014-11-25 – 2014-11-27
• [Presentation] Geminin visualization aids detailed molecular analysis of self-renewing hematopoietic stem cells. (2014)
  • Author(s): Yasunaga S., Ohno Y., Suzuki-Takedachi K., Kurogi T., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 76th Annual Meeting of the Japanese Society of Hematology
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2014-10-31 – 2014-11-02
• [Presentation] Direct transmission of a cell-penetrating form of Geminin, a regulator for hematopoietic stem cells. (2014)
  • Author(s): Ohno Y., Yasunaga S., Kurogi T., Suzuki-Takedachi K., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 76th Annual Meeting of the Japanese Society of Hematology
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2014-10-31 – 2014-11-02
• [Presentation] Direct transduction of a cell-penetrating form of Geminin, a molecule regulating DNA replication and chromatin remodeling. (2014)
  • Author(s): Takihara Y., Yasunaga S., Ohno Y., Kurogi T., Suzuki-Takedachi K., , Santo M., Ohtsubo M.
  • Organizer: ISEH (International Society for Experimental Hematology) 43rd Annual Scientific Meeting
  • Place of Presentation: Montreal , Canada
  • Year and Date: 2014-08-21 – 2014-08-24
• [Presentation] Identification and functional analysis of self-renewing hematopoietic stem cells by using Geminin-EYFP knock-in mice. (2014)
  • Author(s): Yasunaga S., Ohno Y., Suzuki-Takedachi K., Kurogi T., , Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 12th Stem Cell Research Symposium
  • Place of Presentation: Fukuoka, Japan
  • Year and Date: 2014-05-30 – 2014-05-31
• [Presentation] Geminin regulates transcription via chromatin remodeling to govern cellular proliferation and differentiation. (2014)
  • Author(s): Ohno Y., Yasunaga S., Kurogi T., Suzuki-Takedachi K., Santo M., Ohtsubo M., Takihara Y.
  • Organizer: The 12th Stem Cell Research Symposium
  • Place of Presentation: Fukuoka, Japan
  • Year and Date: 2014-05-30 – 2014-05-31
• [Presentation] Visualization and functional analysis of Geminin, a candidate molecule governing self-renewal and cellular differentiation of hematopoietic stem cells (2013)
  • Author(s): Yasunaga S., Ohno Y., Kurogi T., Saeki K., Nakashima Y., Ohtsubo M., Takihara Y.
  • Organizer: The 11th Stem Cell Research Symposium
  • Place of Presentation: Tokyo, Japan
• [Presentation] E2F-mediated transcriptional activation of the Geminin gene is under the regulation of a negative-feedback loop (2013)
  • Author(s): Ohno Y., Saeki K., Yasunaga S., Shirai M., , Kurogi T., Nakashima Y., Ohtsubo M., Takihara Y.
  • Organizer: The 11th Stem Cell Research Symposium
  • Place of Presentation: Tokyo, Japan
• [Presentation] Expression and molecular function of Geminin, which governs hematopoietic stem cell activity (2013)
  • Author(s): Takihara Y., Yasunaga S., Ohno Y., Kurogi T., Suzuki-Takedachi K., Nakashima Y., Ohtsubo M.
  • Organizer: 第８６回日本生化学会大会
  • Place of Presentation: Yokohama, Japan
  • Invited
• [Presentation] Molecular role for Geminin in switching self-renewal and differentiation of hematopoietic cells (2013)
  • Author(s): Yasunaga S., Ohno Y., Kurogi T., Suzuki-Takedachi K., Nakashima Y., Ohtsubo M., Takihara Y.
  • Organizer: The 75th Annual Meeting of the Japanese Society of Hematology
  • Place of Presentation: Sapporo, Japan
• [Presentation] Negative feed-back loop regulates transcription of the Geminin gene, which governs HSC activity (2013)
  • Author(s): Ohno Y., Yasunaga S., Kurogi T., Suzuki-Takedachi K., Nakashima Y., Ohtsubo M., Takihara Y.
  • Organizer: The 75th Annual Meeting of the Japanese Society of Hematology
  • Place of Presentation: Sapporo, Japan
• [Presentation] Negative feed-back loop regulates E2F-mediated transcriptional activation of the Geminin gene (2013)
  • Author(s): Kurogi T., Ohno Y., Yasunaga S., Suzuki-Takedachi K., Shirai M., Ohtsubo M., Takihara Y.
  • Organizer: The 36th Annual Meeting of the Molecular Biology of Japan
  • Place of Presentation: Kobe, Japan
• [Presentation] Molecular analysis of a role for Geminin in regulating proliferation and differentiation of hematopoietic stem/progenitor cells by visualizing Geminin expression (2013)
  • Author(s): Suzuki-Takedachi K., Yasunaga S., Ohno Y., Kurogi T., Ohtsubo M., Takihara Y.
  • Organizer: he 36th Annual Meeting of the Molecular Biology of Japan
  • Place of Presentation: Kobe, Japan
• [Remarks] 広島大学 原爆放射線医科学研究所 幹細胞機能学研究分野
  • URL: http://home.hiroshima-u.ac.jp/dscb/index.html
• [Remarks] 福岡大学 医学部 生化学講座
  • URL: http://www.med.fukuoka-u.ac.jp/biochem1/index-j.htm
• [Remarks] 幹細胞機能学ホームページ
  • URL: http://home.hiroshima-u.ac.jp/dscb