A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells.
A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells.
复制标题
肿瘤细胞中P-糖蛋白相关的多药耐药性定量评估的一种新方法。
DOI：
10.1038/bjc.1996.151
复制
发表时间：
1996-04
影响因子：
8.8
通讯作者：
Sarkadi, B
中科院分区：
文献类型：
--
作者：
Homolya, L;Hollo, M;Muller, M;Mechetner, E B;Sarkadi, B
在人类肿瘤的临床治疗中，需要一种快速、功能性和定量的诊断方法来估计P-糖蛋白（P-gp）依赖的多药耐药，因为化疗方案和耐药逆转剂可以相应地应用。在目前的工作中，通过使用钙黄绿素积累的方法结合免疫识别和耐药性的研究，描述了一种新的方法定量估计的多药物转运蛋白的表达和功能。MDR 1转染和药物选择的肿瘤细胞系与不同水平的耐药性进行了检查。通过定量免疫印迹和免疫荧光细胞术评估P-gp的表达及其细胞表面外观。P-gp的转运功能通过用荧光法和流式细胞术测量钙黄绿素乙酰氧基甲酯（AM）的挤出来评估，而在平行实验中，在细胞存活测定中直接检查耐药性。MDR 1活性因子（MAF），计算从钙黄绿素AM挤出试验，被证明提供了一个可靠的定量测量MDR 1的比活性，反映细胞的耐药性。这种相对简单、快速的新型功能性P-gp检测方法在灵敏度和重现性方面都超过了以前使用的技术。
A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug-selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility.