Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells.
Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells.
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DOI：
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发表时间：
1999-05
影响因子：
3.6
通讯作者：
Yunhai Cui;J. König;U. Buchholz;Herbert Spring;I. Leier;D. Keppler
中科院分区：
文献类型：
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作者：
Yunhai Cui;J. König;U. Buchholz;Herbert Spring;I. Leier;D. Keppler
多药耐药蛋白 MRP1 作为 ATP 依赖性缀合物输出泵发挥作用，并赋予多药耐药性。我们克隆了 MRP2（符号 ABCC2），它是定位于极化细胞顶膜的 MRP 家族成员。组蛋白脱乙酰酶抑制剂增强了转染的人胚肾 (HEK-293) 和 Madin-Darby 犬肾 (MDCK) 细胞中 MRP2 的稳定表达。在极化的 MDCK 细胞中，大鼠和人的 MRP2 均被分选至顶端质膜。针对大鼠 MRP2 氨基末端产生的抗体识别非透化细胞顶端表面的重组蛋白，为 MRP2 氨基末端的细胞外定位提供了直接证据。使用稳定转染细胞的膜囊泡测量重组人和大鼠 MRP2 的 ATP 依赖性转运。人 MRP2 的 Km 值对于白三烯 C4 为 1.0 +/- 0.1 microM，对于 17β-葡萄糖醛酸基雌二醇为 7.2 +/- 0.7 microM；人MRP1的Km值对于白三烯C4为0.1+/-0.02μM，对于17β-葡萄糖醛酸基雌二醇为1.5+/-0.3μM。因此，缀合物转运 ATP 酶 MRP2 和 MRP1 的不同不仅在于它们的域特异性定位，而且还在于它们的动力学特性。使用细胞活力测定在 MDCK 和 HEK-293 细胞中研究了重组 MRP2 赋予的耐药性。人和大鼠MRP2的表达使MDCK细胞对依托泊苷的耐药性分别增强5.0倍和3.8倍，对长春新碱的耐药性分别增强2.3倍和6.0倍。丁硫氨酸亚砜亚胺降低了对这些药物的耐药性。 HEK-293 细胞中过表达的人 MRP2 增强了对依托泊苷（4 倍）、顺铂（10 倍）、阿霉素（7.8 倍）和表阿霉素（5 倍）的耐药性。这些结果表明 MRP2 赋予对细胞毒性药物的抗性。
The multidrug resistance protein MRP1 functions as an ATP-dependent conjugate export pump and confers multidrug resistance. We cloned MRP2 (symbol ABCC2), a MRP family member localized to the apical membrane of polarized cells. Stable expression of MRP2 in transfected human embryonic kidney (HEK-293) and Madin-Darby canine kidney (MDCK) cells was enhanced by inhibitors of histone deacetylase. In polarized MDCK cells, both rat and human MRP2 were sorted to the apical plasma membrane. An antibody raised against the amino terminus of rat MRP2 recognized the recombinant protein on the apical surface of nonpermeabilized cells, providing direct evidence for the extracellular localization of the amino terminus of MRP2. ATP-dependent transport by recombinant human and rat MRP2 was measured with membrane vesicles from stably transfected cells. The Km value of human MRP2 was 1.0 +/- 0.1 microM for leukotriene C4 and 7.2 +/- 0.7 microM for 17beta-glucuronosyl estradiol; the Km values of human MRP1 were 0.1 +/- 0.02 microM for leukotriene C4 and 1.5 +/- 0.3 microM for 17beta-glucoronosyl estradiol. Thus, the conjugate-transporting ATPases MRP2 and MRP1 differ not only by their domain-specific localization but also by their kinetic properties. Drug resistance conferred by recombinant MRP2 was studied in MDCK and HEK-293 cells using cell viability assays. Expression of human and rat MRP2 enhanced the resistance of MDCK cells to etoposide 5.0-fold and 3.8-fold and to vincristine 2.3- and 6.0-fold, respectively. Buthionine sulfoximine reduced resistance to these drugs. Human MRP2 overexpressed in HEK-293 cells enhanced the resistance to etoposide (4-fold), cisplatin (10-fold), doxorubicin (7.8-fold), and epirubicin (5-fold). These results demonstrate that MRP2 confers resistance to cytotoxic drugs.