テロメラーゼの細胞内局在と活性制御

• Material Type: 文書・図像類
• Title: テロメラーゼの細胞内局在と活性制御
• Author/Editor: 村上, 清史; Murakami, Seishi
• Author Heading: 村上, 清史; Murakami, Seishi
• Publication, Distribution, etc.: 金沢大学がん研究所
• Publication Date: 2008-05
• Publication Date (W3CDTF): 2008-05
• Alternative Title: Subcellular localization and control of activity of telomerase
• Periodical title: 平成19(2007)年度 科学研究費補助金 基盤研究(B) 研究成果報告書 = 2007 Fiscal Year Final Research Report
• No. or year of volume/issue: 2005-2007
• Volume: 2005-2007
• Pages: 12p.-
• Text Language Code: jpn
• Subject Heading: 癌; ウイルス; 蛋白質; 発生・分化; シグナル伝達; テロメラーゼ; ヌクレオリン; 細胞内局在; Cancer; Virus; Protein; Development and Differentiation; Sienal transduction; Telomerase; Nucleolin; Subcellular localization
• Target Audience: 一般
• Note (General): テロメラーゼは抗がん剤研究の有力な標的である。本年度、FLAG標識hTERT発現HeLa細胞のヒトテロメラーゼ複合体とhTERTと相互作用するヌクレオリンの生物的機能の解析(村上)、hTERT発現による不死化ヒト初代細胞系の解析(本多)、更に細胞の増殖・分化などに重要な役割を担う受容体型チロシンキナーゼの解析(善岡)を行った。得られた主な結果は、1)HeLa細胞にFLAG-hTERTを導入して得られた活性型組み換え型テロメラーゼは、昆虫細胞系2成分発現ヒトテロメラーゼ複合体と類似して、分子篩法で2種類の複合体(IとII)に分画された。Hsp90を含まず、Hsp90阻害剤に耐性の複合体Iと、Hsp90を含む後者の複合体IIは、Hsp90阻害剤に感受性を示し内在性及びFLAG標識hTERTは共に不安定であった。また内因性hTERTとFLAG-hTERTの大半は複合体Iに分布した。ヌクレオリンが複合体Iに含まれ、複合体IIには殆ど含まれない。これらの結果は、細胞内で異なる2種類の活性型テロメラーゼ複合体が存在し、テロメラーゼの異なる局在或いは活性型テロメラーゼの異なる機能を示唆した。2)hTERT発現レトロウイルスベクターをヒト正常線維芽(BJ)細胞に感染させ、hTERT発現細胞が不死化した細胞株を樹立した。ジーンチップ解析では、hTERT導入不死化BJ細胞ではBJ細胞に比較し、細胞増殖、細胞周期に関与する因子の発現亢進が認められた。3)hTERTと特異的に相互作用するヌクレオリンは、C型肝炎ウイルス(HCV)複製に必須であることが、ヌクレオリン結合性を消失したHCV NS5B変異を持つHCVサブレプリコン系ではHCV複製が全く起きないことと、RNAi法でヌクレオリン発現が低下した細胞では、HCV複製が大きく低下する結果により示された。; Since telomerase activity is barely detectable in primary cells but clearly detected in cancerous cells, telomerase has been considered to be a strong candidate of the targets to cancer treatment In the fiscal year, we concentrated in several experiments including purification and characterization of recombinant human telomerase complex, and biological function of the specific interaction of nucleolin and hTERT recovered from a HeLa cell line stably expressing FLAG-hTERT (Murakami), analyses of human primary cells immortalized by hTERT (Honda), and the tyrosine kinases of receptor type which play critical odes in cell proliferation and differentiation (Yoshioka). The followings are the main conclusions of the experiments.1) The partially purified active telomerase complexes comprise two different complexities, complex I (around 680 kDa), and complex II (around 400 kDa), that are quite alike to those that can be recovered firm the insect cells co-expressing FLAG-hTERT and hTERC. The com plex I does not contain Hsp90 and is resistant to Hsp90 inhibitors, whereas complex II contains Hsp90 and is sensitive to Hsp90 inhibitors. Human TEAT in the complex II is rather unstable during incubation in vim and in ring. Such unstable property was not observed with the complex I. Since MG132, a specific proteasome inhibitor, but not MG133, a negative control, stabilized hTERT in the complex Z strongly suggesting that hTERT in the complex If is under degradation pathway under the control of proteasome. The two different complexities of human telomerase seem to imply two different entities of active telomerase in different subcellular localization or its different functions. 2) We have established a cell line of the human primary fibroblasts(BJ cells) stably expressing hTERT, and we compared expression profiles of RI cells and the KJ cells immortalized by MERE Some genes involving in cell cycle progression and cell proliferation were evidently expressed higher in the immortalized 131-hTERT than these in in the primary cells. 3) Nucleolin, one of the specific interacting partners of hTERT can bind to Hepatitis C Virus (HCV) NS5B, RNA-dependent RNA replication of HCV. We evaluated the role of the specific interaction of nucleolin and NS5B in HCV replication. The HCV subreplicon system has been applied for the address We found that the HCV subreplicons harboring alanine substitution mutations or mutation at the residue(s) either one of two critical sequences within NS5B both of which are necessary for the nucleolin-binding could not replicate at all in a transient HCV replication system in HepG2 ml/s., although the wild replicon could support efficient HCV. By introducing transiently RNAi of nucleolin, that down regulated expression of nucleolin by half to one third, reduced HCV evidently reduced HCV replication using the HCV subreplicon system. Taken together, the specific interaction of nucleolin and NS5B is critical for HCV replication.; 研究課題/領域番号:17390091, 研究期間(年度):2005-2007; 出典：「テロメラーゼの細胞内局在と活性制御」研究成果報告書 課題番号17390091 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）) 本文データは著者版報告書より作成
• DOI: 10.24517/00050053
• Source: https://kanazawa-u.repo.nii.ac.jp/?action=repository_action_common_download&item_id=43711&item_no=1&attribute_id=26&file_no=1
• Access Restrictions: 限定公開
• Rights (production): CC BY-NC-ND
• Related Material: https://kaken.nii.ac.jp/search/?qm=90019878; https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390091/; https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-17390091/173900912007kenkyu_seika_hokoku_gaiyo/
• Data Provider (Database): 国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）; https://irdb.nii.ac.jp
• Original Data Provider (Database): 金沢大学 : 金沢大学学術情報リポジトリKURA