DETECTION OF ACTIVITY OF P-GLYCOPROTEIN IN HUMAN TUMOR SAMPLES USING RHODAMINE-123
DETECTION OF ACTIVITY OF P-GLYCOPROTEIN IN HUMAN TUMOR SAMPLES USING RHODAMINE-123
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DOI：
10.1111/j.1365-2141.1992.tb04608.x
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发表时间：
1992-09-01
影响因子：
6.5
通讯作者：
HOFMANN, J
中科院分区：
文献类型：
--
作者：
LUDESCHER, C;THALER, J;HOFMANN, J
基于膜外排泵P-糖蛋白(P-gp)转运的染料罗丹明123(Rh123)的荧光特性，我们建立了检测多药耐药(MDR)细胞的功能性流式细胞术。利用药物敏感细胞系(KB-3-1)和多药耐药突变株(KB-8-5、KB-C1)，建立了能够显示Rh123外排和蓄积显著差异的实验条件。随后，我们研究了这种功能检测方法在预测人类外周血和骨髓样本中多药耐药的适用性。应用双色流式细胞术对6例急性髓系白血病(AML)患者的白血病原始细胞进行了分析。在3例中，原始细胞有明显的Rh123快速外流。在MDR抑制剂存在的情况下，这些细胞保留了Rh123。为了确定Rh123的外流是否与P-gp的表达有关，用单抗MRK-16对白血病细胞进行了染色。此外，提取的RNA用聚合酶链式反应分析MDR-1mRNA的表达。在3例Rh123+患者中均检测到mdr1mRNA，而仅1例AML患者表达P-gp。通过比较Rh123和柔红霉素，后者也可以检测MDR细胞，蓄积研究证明Rh123是更敏感的用于流式细胞仪MDR筛查的药物。此外，用Rh123进行双色流式细胞术比用柔红霉素更容易进行。我们的结果表明，流式细胞术检测Rh123的蓄积/外排适用于检测异种临床标本中的多药耐药细胞。
Based on the fluorescent properties of the dye rhodamine 123 (Rh123), which is transported by the membrane efflux pump P-glycoprotein (P-gp), we developed a functional flow cytometric assay for the detection of multidrug-resistant (MDR) cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established that enabled demonstration of significant differences in Rh123 efflux and accumulation. Subsequently we investigated the applicability of this functional assay for the prediction of MDR in human peripheral blood and bone marrow samples. Using two-colour flow cytometry, the leukaemic blast cells of six patients suffering from acute myeloid leukaemia (AML) were analysed. In three cases the blast cells showed a rapid and marked Rh123 efflux. In the presence of MDR inhibitors these cells retained Rh123. To determine whether the efflux of Rh123 was associated with P-gp expression, the leukaemic cells were stained with the monoclonal antibody MRK-16. In addition extracted RNA was analysed by polymerase chain reaction to evaluate the expression of mdr 1 mRNA. In all three Rh123+ cases mdr 1 mRNA was detectable whereas only one AML case expressed P-gp. In comparing Rh123 with daunorubicin, which also allows the detection of MDR cells, accumulation studies proved Rh123 to be the more sensitive drug for flow cytometric MDR screening. Additionally, two-colour flow cytometry was much easier to perform with Rh123 than with daunorubicin.Our results indicate that flow cytometric measurement of Rh123 accumulation/efflux proves applicable to detect MDR cells in heterogenous clinical samples.