Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport
Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport
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DOI：
10.1111/j.1476-5381.2010.00801.x
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发表时间：
2010-07-01
影响因子：
7.3
通讯作者：
Loescher, Wolfgang
中科院分区：
文献类型：
--
作者：
Kuteykin-Teplyakov, Konstantin;Luna-Tortos, Carlos;Loescher, Wolfgang
背景和目的：P-糖蛋白（Pgp）外排试验被广泛用于鉴定Pgp底物。用人MDR 1（ABCB 1）转染的肾细胞系Madin-Darby犬肾（MDCK）-II和LLC-PK 1用于提供药物转运的重组模型。这些细胞中的内源性转运蛋白可能有助于重组转运蛋白的活性，因此，MDR 1转染细胞中的药物转运通常针对亲本（野生型）细胞中获得的转运进行校正。然而，内源性转运蛋白的表达在转染细胞和野生型细胞之间可能不同，因此这种校正可能导致错误的数据。在这里，我们已经测量了内源性外排转运蛋白在转染和野生型MDCK-II或LLC细胞的表达和Pgp介导的药物transport.Experimental方法的后果：使用定量实时RT-PCR，我们确定了内源性Mdr 1 mRNA和其他外排转运蛋白在野生型和MDR 1转染MDCK-II和LLC细胞的表达。跨细胞运输测定与测试基板vinblastine.Key结果：在MDR 1转染MDCK细胞，内源性（犬）Mdr 1和Mrp 2（Abcc 2）mRNA的表达显着低于野生型细胞，而MDR 1转染LLC细胞表现出可比的Mdr 1，但显着较高的Mrp 2 mRNA水平比野生型细胞。因此，当用野生型细胞中获得的转运校正MDR 1转染MDCK细胞中的转运时，外排实验中人Pgp对长春碱的转运被显著低估。这个问题并没有发生在LLC cells.Conclusions and Implications：在转染和野生型MDCK细胞之间的内源性外排转运蛋白的表达差异提供了一个潜在的偏见在体外研究Pgp介导的药物转运。
Background and purpose:P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport.Experimental approach:Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine.Key results:In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells.Conclusions and implications:Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport.