がん細胞におけるアポトーシスの分子解析

• Project/Area Number: 12215156
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University (2001-2004); Osaka Bioscience Institute (2000)
• Principal Investigator: 垣塚 彰 京都大学, 大学院・生命科学研究科, 教授 (80204329)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥46,600,000 (Direct Cost: ¥46,600,000); Fiscal Year 2004: ¥7,800,000 (Direct Cost: ¥7,800,000); Fiscal Year 2003: ¥7,800,000 (Direct Cost: ¥7,800,000); Fiscal Year 2002: ¥10,000,000 (Direct Cost: ¥10,000,000); Fiscal Year 2001: ¥11,000,000 (Direct Cost: ¥11,000,000); Fiscal Year 2000: ¥10,000,000 (Direct Cost: ¥10,000,000)
• Keywords: ポリグルタミン / 神経細胞死 / AAA ATPase / アミノ酸修飾 / VCP / 出芽酵母 / 腺癌 / 扁平上皮癌 / 酸化修飾 / ATPase活性 / 温度感受性株 / 細胞死 / Hsp70 / RNAi / 細胞周期 / 亜砒酸 / 前立腺ガン / p97 / APL / ASK1 / 酸化ストレス / SEK1 / スプライニングバリアント

Research Abstract

細胞死・細胞増殖に関わる新規遺伝子・蛋白質として同定したAAA familyのATPaseであるVCP蛋白質について解析を行い、以下の諸点を明らかにした。
(1)抗VCP抗体を用いて、正常組織及び各種癌組織でVCPの発現を解析した。マウス正常組織では、管腔構造をとる上皮系の細胞や免疫系細胞、乳腺上皮、胃酸合成細胞などの分泌系の細胞に強い発現を認めた。一方、ヒト癌由来の組織では、特に腺癌と扁平上皮癌に強い発現を認めた。
(2)RNAi法を用いて、HeLa細胞で内在性のVCPを98%以上ノックダウンすることに成功した。このHeLa細胞では、紡錘体極の数が不安定になり、娘細胞に正しく染色体を分配することができなくなっていた。その結果、M期の終了後、細胞周期の停止もしくは細胞死に陥っていた。
(3)VCP蛋白質内であるリン酸化を受けたアミノ酸を特異的に認識する抗体を作成し、培養細胞を免疫染色すると紡錘体極やセントロゾームが染色された。
(4)外来性のVCPを誘導発現させることができる出芽酵母を作り、その酵母をEMSで処理してDNA変異を導入後、VCPの存在の有無で発育に差のでる温度感受性株を得た。その温度感受性株をゲノムライブラリーでトランスフォームし、温度感受性をレスキューする8つのゲノム断片を得た。
(5)VCPをアセチル化する酵素の精製を進め、アセチル化活性のある分画に特異的と思われる蛋白質のバンドを数個にまで絞り込むことができた。

Research Products

• [Journal Article] alpha, beta -unsaturated ketone is a core moiety of natural ligands for covalent binding to peroxisome proliferator-activated receptor gamma. (2005)
  • Author(s): Shiraki, T., et al.
  • Journal Title: J.Biol.Chem. 279(In press)
• [Journal Article] Effective treatment of advanced solid tumors by the combination of Arsenic Trioxide and L-Buthionine-Sulfoximine. (2004)
  • Author(s): Maeda, H., et al.
  • Journal Title: Cell Death Differ. 7 Pages: 737-746
• [Journal Article] Alteration in thee cellular response to retinoic acid of a human acute promyelocytic leukemia cell line, UF-1, carrying a patient-deriveed mutant PML-RARα chimeric gene. (2004)
  • Author(s): Sato, A., et al.
  • Journal Title: Leukemia Res. 9 Pages: 959-9667
• [Journal Article] The role of pre-existing aggregates in Hsp104-dependent polyglutamine aggregate formation and epigenetic change of yeast prions. (2004)
  • Author(s): Kimura, Y., et al.
  • Journal Title: Genes Cells 8 Pages: 685-696
• [Journal Article] A mutant ataxin-3 putative-cleavage fragment in brain of Machado-Joseph disease patients and transgenic mice is cytotoxic above a critical concentration. (2004)
  • Author(s): Goti, D., et al.
  • Journal Title: J.Neuroscience 24 Pages: 10266-10279
• [Journal Article] Physical and functional interaction between dorfin and valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders. (2004)
  • Author(s): Ishigaki, S., et al.
  • Journal Title: J.Biol.Chem. 279 Pages: 51376-51385
• [Publications] Maeda, H., et al.: "Effective treatment of advanced solid tumors by the combination of Arsenic Trioxide and L-Buthionine-Sulfoximine."Cell Death Differ.. (In press). (2004)
• [Publications] Matsumoto, M., et al.: "Molecular clearance of ataxin-3 is regulated by a mammalian E4."EMBO J.. 23. 659-669 (2004)
• [Publications] Kobayashi, T., Kakizuka, A.: "Molecular analyses of Machado-Joseph disease."Cytogenet.Genome Res.. 100. 261-275 (2003)
• [Publications] Kimura, Y., Kakizuka, A.: "Polyglutamine diseases and molecular chaperones."IUBMB Life. 55. 337-345 (2003)
• [Publications] Kamei, Y., et al.: "PGC-1β/ERRL1 is an ERR protein ligand, whose expression induces a high-energy expenditure and"Proc.Natl.Acad.Sci.U.S.A.. 100. 12378-12383 (2003)
• [Publications] Mizuno, Y., et al.: "Vacuole-creating protein in neuro-degenerative diseases."Neurosci.Lett.. 343. 77-80 (2003)
• [Publications] Higashiyama, H., et al.: "Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegenerations in Drosophila"Cell Death Differ.. 9. 264-273 (2002)
• [Publications] Nakamoto, M., et al.: "Unequal crossing-over in unique PABP2 Mutations : a possible cause of oculopharyngeal muscular dystrophy"Archives Neurology. 59. 474-477 (2002)
• [Publications] Nishitoh, H., et al.: "ASK1 is essential for endoplasmic reticulum stress-induced Neuronal cell death triggered by expanded polyglutamine repeats"Genes & Dev.. 16. 1345-1355 (2002)
• [Publications] Kimura, Y., et al.: "Circumvention of chaperone requirement for aggregate formation of a short polyglutamine tract by the co-expression of a long polyglutamine tract"J.Biol.Chem.. 277. 37536-37541 (2002)
• [Publications] Kobayashi, T., et al.: "Functional ATPase activity of p97/VCP is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells"J.Biol.Chem.. 277. 47358-47365 (2002)
• [Publications] Mizuno, Y., et al.: "Vacuole-creating protein in neurodegenerative diseases"Neurosci.Lett.. (in press). (2003)
• [Publications] Yamamoto, Y., Hasegawa, H., Tanaka, K., Kakizuka, A.: "Isolation of neuronal cells with high processing activity for the Machado-Joseph disease protein"Cell Death Differ.. 8. 871-873 (2001)
• [Publications] Hirabayashi, M., Inoue, K., Tanaka, K., et al.: "VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes relevant to neurodegeneration"Cell Death Differ.. 8. 977-984 (2001)
• [Publications] Maeda, H., Hori, S., Nishitoh, H., et al.: "Tumor growth inhibition by arsenic trioxide (As2O3) in the orthotopic metastasis model of androgen-independent prostate cancer"Cancer Res.. 61. 5432-5440 (2001)
• [Publications] Kimura, Y., Koitabashi, S., Kakizuka, A., Fujita, T.: "Initial process of polyglutamine aggregate formation in vivo"Genes Cells. 6. 887-897 (2001)
• [Publications] Higashiyama, H., Hirose, F., Yamaguchi, M., et al.: "Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegenerations in Drosophila"Cell Death Differ.. 9. 264-273 (2002)
• [Publications] Nakamoto, M., Nakano, S., Kawashima, S., et al.: "Unequal crossing-over in unique PABP2 Mutations : a possible cause of oculopharyngeal muscular dystrophy"Archives Neurology. 59. 474-477 (2002)
• [Publications] Maeda,H.,Segawa,T.,Kamoto,T.,Yoshida,H.,Kakizuka,A.,Ogawa,O.,& Kakehi,Y.: "Rapid detection of candidate metastatic foci in the orthotopic inoculation model of androgen-sensitive prostate cancer cells introduced with green fluorescent protein."The Prostate. 45. 335-340 (2000)
• [Publications] Kakizuka,A.: "Molecular mechanisms underlying neuronal cell death in polyglutamine diseases."Neurochemical Research. 25. 990 (2000)
• [Publications] Yasuda,S,Hori,S.,Maeda,H.,Maeda,R.,Gotoh,Y.,Nishitoh,H.,Ichijo,H.,& Kakizuka,A.: "As2O3 treatment recruits Daxx and ASK1 to re-organized PML bodies and activates the SEK1-JNK cell death kinase cascade in APL cells."Cell Death & differentiation. (in revision).
• [Publications] Yamamoto,Y.,Hasegawa,H.,Tanaka,K.,& Kakizuka,A.: "Genetical isolation of neuronal cells with high processing activity for the Machado-Joseph disease protein."Cell Death & differentiation. (in revision).