Fluo-3を用いた、低レベルの抗癌剤多剤耐性細胞の迅速解析と耐性克服剤の検討

Material type: 規格・テクニカルリポート類

Author: 小泉, 晶一ほか

Publisher: 金沢大学医薬保健研究域医学系

Publication date: 1994-03

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Note (General)：: 出版タイプ： AM[目的]P-糖蛋白のefflux pumpとしての機能性を応用して、多剤耐性(MDR)細胞の、新しい蛍光プローベFluo-3を用いた機能的解析の可能性を検討し、Rhodamine-123と比較した。[方法]薬剤感受性K562親株、多剤耐性K562/VCR、K562/ADR細胞、その...

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Material Type: 規格・テクニカルリポート類
Title: Fluo-3を用いた、低レベルの抗癌剤多剤耐性細胞の迅速解析と耐性克服剤の検討
Author/Editor: 小泉, 晶一
Koizumi, Shoichi
Author Heading: 小泉, 晶一
Koizumi, Shoichi
Publication, Distribution, etc.: 金沢大学医薬保健研究域医学系
Publication Date: 1994-03
Publication Date (W3CDTF): 1994
Alternative Title: Flow cytometric analysis of low-grade multidrug resistance using fluo-e : a comparison with rhodamine-123
Periodical title: 平成5(1993)年度科学研究費補助金一般研究(C) 研究成果報告書 = 1993 Fiscal Year Final Research Report
No. or year of volume/issue: 1992-1993
Volume: 1992-1993
Pages: 6p.-
Subject Heading: Multidrug resistance
MDR1 gene
P-glycoprotein
Fluo-3
Rhodamine-123
K562 cells
Leukemia
Chemotherapy
Target Audience: 一般
Note (General): 出版タイプ： AM
[目的]P-糖蛋白のefflux pumpとしての機能性を応用して、多剤耐性(MDR)細胞の、新しい蛍光プローベFluo-3を用いた機能的解析の可能性を検討し、Rhodamine-123と比較した。[方法]薬剤感受性K562親株、多剤耐性K562/VCR、K562/ADR細胞、その感受性復元細胞、MTXのみ耐性で非多剤耐性K562/MTX細胞、多剤耐性であるがP-糖蛋白陰性K562/THPADR細胞を使用した。細胞(10^6/ml 5%FCS加RPMI1640)を37℃保温後、Fluo-3A/M(4μM)またはRh-123(1μM)を添加した。20分後に530nmで平均蛍光強度(mcf)をflow cytometer(Cytoron Absolute)で測定した。さらにP-糖蛋白阻害剤のverapamil(Ver,20μM)やcepharanthine(CP,10μM)を添加してmcfの変化を観察した。Effluxパターンは細胞を洗浄後30分、60分で検討した。P-糖蛋白発現はMRK16抗体を用いた免疫蛍光染色法によった。MDRmRNAはRT-PCR法によった。[結果]K562/VCRとK562/ADRではFluo-3,Rh-123ともに細胞内蓄積低下とVer/CPによる排泄遅延がみられた。P-糖蛋白発現のみられないK562/THPADR,K562/MTXではVer/CPによるFluo-3の排泄遅延が認められたがRh-123ではみられなかった。MDR1mRNA発現はP-糖蛋白陰性の細胞も含めてすべての細胞でみられた。[結論]以上より、Rh-123はP-糖蛋白発現をより明確に表現したが、Fluo-3はMDR1mRNAを発現する低レベルの多剤耐性の検出に有用かもしれない。
Six cell lines including drug-sensitive K562/parent cells, multidrug-resistant(MDR) K562/VCR, K562/ADR and K562/tetrahydropyranyl(THP)-ADR cells, methotrexate-resistant K562/MTX and revertant K562/ADR-R cell lines were used in this study : Although P-glycoprotein(P-gp)was overexpressed only in K562/VCR and K562/ADR cells, the RT-PCR method revealed distinct expression of MDR1mRNA even in the K562/parent cells. Intracellular accumulation of Fluo-3 and rhodamine-123 (Rh-123) was measured by incubation of cells with 4 muM Fluo-3 or 1muM Rh-123 for 20 min. by using a flow cytometer. Verapamil(Ver, 20 muM) or cepharanthine (biscoclaurine alkaloid, CP, 10muM) was added just before the addition of the fluorescent agents, Efflux patterns were also studied 30 and 60 min. after incubation with or without Ver ad CP.Increased intracellular accumulation and delayd efflux pattern of Fluo-3 by Ver and CP were shown in MDR K562/VCR and K562/ADR cells, and most interestingly, similar increase of uptake and delayd efflux pattern of Fluo-3 by Ver and CP butin a lower grade, were also demonstrated in P-gp-non-expressed K562/parent, K562/MTX and K562/THP cells. Accumulation of Rh-123 waas not influenced by Ver and CP.Thirteen of 31 clinical leukemic cell samples expressing MDR1mRNA, but not P-gp demonstrated enhancement of intracellular accumulation of Fluo-3 by Ver and Cp, indicating that flow cytometric functional analysis using Fluo-3 with Ver and CP may be usefl for more sensitive functional assay of low-grade multidrug resistance.
研究課題/領域番号04670582, 研究期間(年度):1992-1993
出典：「Fluo-3を用いた、低レベルの抗癌剤多剤耐性細胞の迅速解析と耐性克服剤の検討」研究成果報告書　課題番号04670582(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: 10.24517/00051051
https://doi.org/10.24517/00051051
Related Material: https://kaken.nii.ac.jp/search/?qm=50019973
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04670582/
https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-04670582/046705821993kenkyu_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