Flow cytometric analysis of low-grade multidrug resistance using fluo-e : a comparison with rhodamine-123

使用 Fluo-E 进行低度多重耐药性的流式细胞术分析：与 Rhodamine-123 的比较

• 批准号: 04670582
• 负责人: KOIZUMI Shoichci
• 金额: $ 1.41万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670582/
• 关键词: Multidrug resistance; MDR1 gene; P-glycoprotein; Fluo-3; Rhodamine-123; K562 cells; Leukemia; Chemotherapy; K562cells; 白血病; 化学療法; 薬剤耐性; P-糖蛋白; 多剤耐性; ベラパミル; セファランチン

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.

本研究采用药物敏感的K562/亲本细胞、多药耐药（MDR） K562/VCR、K562/ADR、K562/四氢吡喃基(THP)-ADR细胞、甲氨蝶呤耐药的K562/MTX和逆转录的K562/ADR- r细胞系6种细胞系进行研究。虽然p -糖蛋白（P-gp）仅在K562/VCR和K562/ADR细胞中过表达，但RT-PCR方法显示，即使在K562/亲本细胞中也有明显的MDR1mRNA表达。用流式细胞仪测定Fluo-3和罗丹明-123 （Rh-123）在细胞内的蓄积，方法是用4 muM Fluo-3或1muM Rh-123孵卵20分钟。在加入荧光剂之前，分别加入维拉帕米（Ver, 20 μ m）或头孢万汀（双氯螯生物碱，CP, 10 μ m），在有或没有Ver和CP的情况下，分别观察30和60分钟的外排模式。在MDR K562/VCR和K562/ADR细胞中，Ver和CP增加了Fluo-3的细胞内积累和延迟外排模式，最有趣的是，Ver和CP增加了Fluo-3的摄取和延迟外排模式，但程度较低。在不表达p- gp的K562/亲本细胞、K562/MTX细胞和K562/THP细胞中也有表达。Rh-123的积累不受Ver和Cp的影响。31个临床白血病细胞样本中有13个表达MDR1mRNA，但P-gp没有表现出Ver和Cp对Fluo-3细胞内积累的增强，这表明使用Ver和Cp对Fluo-3进行流式细胞术功能分析可能有助于更灵敏的低级别多药耐药功能分析。

项目成果

Koizumi, S.: "Good correlation between expression of P-glycoprotein and accumulation of fluorescent Fluo-3 probe." Pro. Jpn. cancer Assoc.52. 638 (1993)

Koizumi, S.：“P-糖蛋白的表达与荧光 Fluo-3 探针的积累之间具有良好的相关性。”

Koizumi,S.: "Rapid and sensitive detection of low-grade multidrug resistance(MDR)in leukemic cells using the fluorescent indicator,Fluo-3." Proc.Am.Assoc.Cancer Res.33. 481-481 (1992)

Koizumi,S.：“使用荧光指示剂 Fluo-3 快速、灵敏地检测白血病细胞中的低度多药耐药性 (MDR)。”

小泉晶一: "蛍光プローベFluo-3を用いた、低レベル多剤耐性細胞の迅速解析" Proc.Jpn.Cancer Assoc.(日本癌学会総会記事). 51. 418-418 (1992)

Shoichi Koizumi：“使用荧光探针 Fluo-3 快速分析低水平多重耐药细胞”Proc.Jpn.Cancer Assoc.（日本癌症协会大会文章）。

Koizumi,S.: "Improvement in treatment of childhood acute lymphoblastic leukemia:a 10-year study by the Children's Cancer and Leukemia Study Group." Intern.J.Hematol.59(in press). (1994)

Koizumi,S.：“儿童急性淋巴细胞白血病治疗的改进：儿童癌症和白血病研究小组的一项为期 10 年的研究。”

Koizumi, S.: "Rapid and sensitive detection of low-grade multidrug resistance (MDR) in leukemic cells using the fluorescent indicator, Fluo-3" Proc. Am. Assoc. cancer Res.33. 481 (1992)

Koizumi, S.：“使用荧光指示剂 Fluo-3 快速、灵敏地检测白血病细胞中的低度多药耐药性 (MDR)”Proc。