造血幹細胞におけるテロメラーゼ活性の制御機構の解析と制御分子の単離の試み

• 研究課題/領域番号: 14657245
• 研究種目: 萌芽研究
• 配分区分: 補助金
• 研究分野: 血液内科学
• 研究機関: 大阪大学
• 研究代表者: 金倉 譲 大阪大学, 医学系研究科, 教授 (20177489)
• 研究分担者: 柴山 浩彦 大阪大学, 医学系研究科, 助手 (60346202) ; 水木 満佐央 大阪大学, 医学系研究科, 講師 (80283761) ; 松村 到 大阪大学, 医学系研究科, 助教授 (00294083)
• 研究期間 (年度): 2002 – 2003
• 研究課題ステータス: 完了 (2003年度)
• 配分額: 3,300千円 (直接経費: 3,300千円); 2003年度: 1,300千円 (直接経費: 1,300千円); 2002年度: 2,000千円 (直接経費: 2,000千円)
• キーワード: テロメラーゼ / 造血幹細胞 / c-Myc / 自己複製 / 不死化 / Notch / HOXB4 / サイトカイン / 細胞接着 / TERT

研究概要

テロメアとは染色体末端部分に存在する(TTAGGG)nの繰り返し配列である。DNA合成の際に再生されず、細胞分裂を繰り返すと短縮していき、細胞分裂の回数を規定している。造血幹細胞は自己複製能と多分化能を有し、個体の生存期間、造血幹細胞集団を維持しつつ血液細胞を供給するが、この細胞集団は一般の正常体細胞とは異なり、テロメア配列を補充するテロメラーゼ活性を有している。
本研究においては、造血幹細脚の自己複製・不死化機構とその際のテロメラーゼ活性の制御機構について解析を行い、以下の結果を見出した。
1.マウス骨髄より単離したLin^-Sca-1^+の造血幹細胞に活性型NotchやHOXB4を導入すると、自己複製がもたらされ、この際にはテロメラーゼ活性の上昇が認められた。
2.Notch、HOXB4による自己複製の際の各種細胞周期制御分子の発現変化を調べたところ、G1/S期に関わる細胞周期制御分子の中でcyclin D1やE2F1などと比較してc-mycの発現上昇が最も早期から認められた。また、両分子はルシフェラーゼアッセイにおいてC-mycプロモーターを活性化した。
3.マウス造血幹細胞に4-hydrxytamoxifen(4-HT)によってc-Mycの活性が誘導可能なc-Mycと変異型エストロゲン受容体(ERT)のキメラ分子c-Myc/ERTを導入したところ、c-Myc/ERTは造血幹細胞に自己複製をもたらし、テロメラーゼ活性を上昇させた。
以上の結果から、Notch、HOXB4による造血幹細胞の自己複製、テロメラーゼ活性の制御において、c-Mycが中心的な役割を担うと考えられた(J.Biol.Chem.,in revision)。

研究成果

• [文献書誌] Tanaka H, et al.: "E2F-1 and c-Myc potentiate apoptosis through inhibition NF-κB that facilitates MnSOD-mediated ROS elimination"Molecular Cell. 9. 1017-1029 (2002)
• [文献書誌] Ezoe S, et al.: "GATA-2/estrogen receptor chimera regulates cytokine-dependentgrowth of hematopoiesis through accumulation of p21WAF1 and p27Kip1 proteins"Blood. 100. 3512-3520 (2002)
• [文献書誌] Ueda S, et al.: "Critical roles of c-kit tyrosine residues 567 and 719 in stem cell cellfactor-induced chemotaxis : contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration"Blood. 100. 3342-3349 (2002)
• [文献書誌] Sonoyama J, et al.: "Functional cooperation among Ras, STAT5 and PI3-K is required for fulloncogenic activities of BCR/ABL in K562 cells"J.Biol.Chem.. 277. 8076-8082 (2002)
• [文献書誌] Nishimura Ji, et al.: "Long-term support of hematopoiesis by a single stem cell clone inpatients with paroxysmal nocturnal hemoglobinuria"Blood. 99. 2748-2751 (2002)
• [文献書誌] Nojima J, et al.: "Acquired activated protein C resistance, associated with anti-protein Santibody as a strong risk factor for DVT in non-SLE patients"Thromb.Haemost.. 88. 716-722 (2002)
• [文献書誌] Nojima J, et al.: "Acquired activated protein C resistance is associated with the co-existence of anti-prothrombin antibodies and lupus anticoagulant activity in patients with systemic lupus erythematosus"Br.J.Haematol.. 118. 577-583 (2002)
• [文献書誌] Matsumura I, et al.: "Molecular control of megakaryopoiesis and thrombopoiesis"Int.J.Hematol.. 75. 473-483 (2002)
• [文献書誌] Ueda S, et al.: "Constitutive activation of c-kit by the juxtamembrane but not thecatalytic domain mutations is inhibited selectively by tyrosine kinase inhibitors STI571 and AG1296"Int.J.Hematol.. 76. 427-435 (2002)
• [文献書誌] Kabutomori O, et al.: "Movement of toxic granulation neutrophils and C-reactive protein ininflammatory processes"Am.J.Med.. 112. 595-596 (2002)
• [文献書誌] Kabutomori O, et al.: "Induction of toxic granulation in neutrophils by granulocyte colony-stimulating factor"Eur.J.Haematol.. 69. 187-188 (2002)
• [文献書誌] Yamanishi H, et al.: "Modification of fully automated total iron-binding capacity (TIBC) assayin serum and comparison with dimension TIBC method"Thromb.Haemost.. 88. 716-722 (2002)
• [文献書誌] Yamanishi H, et al.: "Relation between iron content of serum ferritin and clinical status factorsextracted by factor analysis in patients with hypefferritinemia"Clin.Biochem.. 35. 523-529 (2002)
• [文献書誌] Muller-Tidow C, et al.: "Mutations of growth factor receptor Flt3 in acute myeloid leukemia : transformation of myeloid cells by Ras-dependent and Ras-independentmechanisms"Dtsch.Med.Wochenschr.. 127. 2195-2200 (2002)
• [文献書誌] Yang Q, et al.: "Hepatocyte nuclear factor-1a modulates pancreatic β-cell growth byregulating the expression of insulin-like growth factor-1 in INS-1 cells"Diabetes. 51. 1785-1792 (2002)
• [文献書誌] Nanbu-Wakao, R, et al.: "Stimulation of 3T3-L1 adipogenesis by signal transducer and activator of transcription 5"Mol.Endocrinol.. 16. 1565-1576 (2002)
• [文献書誌] Imai Y, et al.: "High prevalence of HCV infedtion in patients with B-cell non-Hodgkin's lymphoma : comparision with birth cohort- and sex-matched blood donors in a Japanese population"Hepatology. 35. 974-976 (2002)
• [文献書誌] Kuratsune H, et al.: "Brain regions involved in fatigue sensation : reduced acetylcarnitine uptake into the brain"Neuroimage. 17. 1256-1265 (2002)
• [文献書誌] Hashimoto K, et al.: "Necessity of tyrosine 719 and phosphatidylinositol 3'-kinase-mediated signal pathway inconstitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the D814V mutation"Blood. 101. 1094-1102 (2003)
• [文献書誌] Kawasaki A, et al.: "Opposing effects of PML and PML/RAR α on STAT3 activity"Blood. 101. 3668-3673 (2003)
• [文献書誌] Mizuki M, et al.: "Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations"Blood. 101. 3164-3173 (2003)
• [文献書誌] Kataoka Y, et al.: "Reciprocal inhibition between MyoD and STAT3 in the regulation of growth and differentiation of myoblasts"J.BIol.Chem.. 278. 44178-44187 (2003)
• [文献書誌] Saeki Y, et al.: "Enhanced production of osteopontin in multiple myeloma : clinical and pathogenicimplications"Br.J.Haematol.. 123. 263-270 (2003)
• [文献書誌] Zhang X, et al.: "Constitutively activated Rho GTPases regulate the growth and morphology of hairy cellleukemia (HCL) cells"Int.J.Hematology. 77. 263-273 (2003)
• [文献書誌] Takakuwa T, et al.: "Establishment and characterization of unique cell lines derived from pyothorax-associated lymphoma which develops in long-standing pyothorax and is stronglyassociated with Epstein-Barr virus infection"Cancer Sci.. 94. 858-863 (2003)
• [文献書誌] Matsumura I, et al.: "Molecular mechanisms of E2F1- and c-Myc-enhanced apoptosis"Rec.Res.Dev.in Mol.& Cell.Biol.. 4. 311-324 (2003)
• [文献書誌] Matsumura I, et al.: "Roles for E2F1 and c-Myc in the regulation of cell growth and survival"Cell Cycle. 2. 333-338 (2003)
• [文献書誌] Mizuki M, et al.: "Constitutive active receptor tyrosine kinase in hematological neoplasia"Rec.Res.Dev.in Mol.& Cell.Biol.. 4. 85-100 (2003)
• [文献書誌] Mizuki M, et al.: "Oncogenic receptor tyrosine kinase in leukemia"Cellular and Molecular Biology. 49. 907-922 (2003)
• [文献書誌] Matsumura I, et al.: "Cell cycle regulation in hematopoietic stem cells"Res.Adv.in Blood. 2. 39-49 (2003)
• [文献書誌] Okawa Y, et al.: "Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei : probable proteolytic products of ubiquitinated H2A"Int.J.Biochem.Cell Biol.. 35. 1588-1600 (2003)
• [文献書誌] Yamanishi H, et al.: "Total iron-binding capacity calculated from serum transferrin concentration or serumiron concentration and unsaturated iron-binding capacity"Clin.Chem.. 49. 175-178 (2003)
• [文献書誌] Yamanishi H, et al.: "Modification of the colorimetric assay for serum unsaturated iron-binding capacity"Clin.Chem.. 49. 1023-1025 (2003)
• [文献書誌] Kimura S, et al.: "Enzymatic assay for determination of bicarbonate ion in plasma using urea amidolyase"Clin.Chim.Acta.. 328. 179-184 (2003)
• [文献書誌] Kimura S, et al.: "New enzymatic assay for serum urea nitrogen using urea amidolyase"J.Clin.Lab.Anal.. 17. 52-56 (2003)
• [文献書誌] Kabutomori O, et al.: "Marked decreases of total and immature reticulocytes in myelodysplastic syndromeamong patients with pancytopenia"Acta Haematol.. 109. 212-213 (2003)
• [文献書誌] Kabutomori O, et al.: "Incorrect measurement of leukocyte counts in post-bone marrow transplantation (P-BMT) patients"Ann.Hematol.. 82. 529-530 (2003)
• [文献書誌] Ueda Y, et al.: "New homogeneous HDL-cholesterol assay without the influence of high TG sample using the selective detergent to lipoproteins"J Clin Lab Anal.. 17. 201-208 (2003)
• [文献書誌] Tanaka S, et al.: "Autoantibodies against muscarinic cholinergic receptor in chronic fatigue syndrome"Int.J.Mol.Med.. 12. 225-230 (2003)
• [文献書誌] Shibayama H, et al.: "Identification of a cytokine-induced antiapoptotic molecule Anamorsin essential for definitive hematopoiesis"J.Exp.Med.. 199. 581-592 (2004)
• [文献書誌] Ezoe S, et al.: "Roles for CDK inhibtors in hematopoietic system"Cell Cycle. (in press). (2004)
• [文献書誌] Tanaka H, et al.: "E2F-1 and c-Myc potentiate apoptosis through inhibition NF-κB that facilitates MnSOD-mediated ROS elimination"Molecular Cell. 9・5. 1017-1029 (2002)
• [文献書誌] Ezoe S, et al.: "GATA-2/estrogen receptor chimera regulates cytokine-dependent growth of hematopoiesis through accumulation of p21^<WAF1> and p27^<Kip1> proteins"Blood. 100・10. 3512-3520 (2002)
• [文献書誌] Ueda S, et al.: "Critical roles of c-kit tyrosine residues 567 and 719 in stem cell cell factor-induced chemotaxis : contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration"Blood. 99・2. 3342-3349 (2002)
• [文献書誌] Sonoyama J, et al.: "Functional cooperation among Ras, STAT5 and PI3-K is required for full oncogenic activities of BCR/ABL in K562 cells"J.Biol.Chem.. 277・10. 8076-8082 (2002)
• [文献書誌] Matsumura I, et al.: "Molecular control of megakaryopoiesis and thrombopoiesis"Int.J.Hematol.. 75・5. 473-483 (2002)
• [文献書誌] Ueda S, et al.: "Constitutive activation of c-kit by the juxtamembrane but not the catalytic domain mutations is inhibited selectively by tyrosine kinase inhibitors ST1571 and AG1296"Int.J.Hematol.. 76・5. 427-435 (2002)
• [文献書誌] Hashimoto K, et al.: "Necessity of tyrosine 719 and phosphatidylinositol 3'-kinase-mediated signal pathway inconstitutive activation and oncogenic potential of c-kit receptor tyrosine kinase with the D814V mutation"Blood. 101・3. 1094-1102 (2003)
• [文献書誌] Mizuki M, et al.: "Suppression of myeloid trascription factors and induction of STAT response genes by AML-specific Flt3 mutations"Blood. (in press).
• [文献書誌] Kawasaki A, et al.: "Opposing effects of PML and PML/RARα on STAT3 activity"Blood. (in press).
• [文献書誌] Matsumura I, et al.: "Molecular mechanisms of E2F1-and-c-Myc-enhanced apoptosis"Rec.Res.Dev.in Mol.& Cell.Biol.. (in press).
• [文献書誌] Matsumura I, et al.: "Roles for E2F1 and c-Myc in the regulation of cell grwoth and apoptosis"Cell Cycle. (In press).
• [文献書誌] Zhang X, et al.: "Constitutively activated Rho GTPases regulate the growth and morphology of hairy cell leukemia(HCL) cells"Int.J.Hematol.. (In press).