Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.
Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.
复制标题
DOI：
10.1371/journal.pone.0022429
复制
发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Patton WF
中科院分区：
文献类型：
--
作者：
Lebedeva IV;Pande P;Patton WF
多药耐药(MDR)的一个潜在机制是跨膜三磷酸腺苷结合盒(ABC)转运蛋白上调。ABC转运蛋白还决定了药物在体内的一般命运和作用。ABC转运蛋白的三种主要类型是mdr1(P-gp、P-糖蛋白、ABCB1)、MRP1/2(ABCC1/2)和BCRP/MXR(ABCG2)蛋白。流式细胞术(FCM)可以确定ABC转运蛋白在活细胞中的功能表达水平，但大多数用作指示剂的染料(罗丹明123、DiOC2(3)、Calcein-AM)的适用性有限，因为它们不能检测所有三种主要类型的ABC转运蛋白。覆盖范围广的染料(如阿霉素、柔红霉素和米托蒽醌)由于总体较暗而缺乏敏感度，因此可能产生相当大比例的假阴性结果。我们描述了两个新的荧光探针，它们是所有三种常见类型的ABC转运蛋白的底物，并且可以在使用活细胞的流式细胞术分析中作为MDR的指示物。根据多药耐药活性因子(MAF)值和Kolmogorov-Smirnov统计分析，该探针表现出快速的内化、良好的摄取/外排动力学和高灵敏度的MDR检测。这两种染料与ABC转运蛋白的一般或特定抑制剂结合使用，很容易识别功能外排，并能够检测少量外排以及确定多药耐药的类型。该方法可用于ABC转运蛋白调控因子的筛选，便于对ABC转运蛋白活性的快速、重复性、特异性和相对简单的功能检测，并可在广泛使用的仪器上实现。
An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.
登录
查看更多内容
影响因子：
20.3
作者：
Broxterman, HJ;Sonneveld, P;Schuurhuis, GJ
通讯作者：
Schuurhuis, GJ
DOI：
10.1152/ajpgi.2000.278.4.g522
发表时间：
2000-04-01
影响因子：
4.5
作者：
Cantz, T;Nies, AT;Keppler, D
通讯作者：
Keppler, D
影响因子：
11.2
作者：
McCollum AK;TenEyck CJ;Stensgard B;Morlan BW;Ballman KV;Jenkins RB;Toft DO;Erlichman C
通讯作者：
Erlichman C
影响因子：
5.8
作者：
COLLINGTON, GK;HUNTER, J;HIRST, BH
通讯作者：
HIRST, BH
影响因子：
8.8
作者：
Hunter, J;Hirst, B H;Simmons, N L
通讯作者：
Simmons, N L