Comparative study of the importance of multidrug resistance-associated protein 1 and P-glycoprotein to drug sensitivity in immortalized mouse embryonic fibroblasts.
Comparative study of the importance of multidrug resistance-associated protein 1 and P-glycoprotein to drug sensitivity in immortalized mouse embryonic fibroblasts.
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发表时间：
2002-10
影响因子：
5.7
通讯作者：
Z. Lin;Dennis R. Johnson;R. Finch;M. Belinsky;G. Kruh;A. Sartorelli
中科院分区：
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作者：
Z. Lin;Dennis R. Johnson;R. Finch;M. Belinsky;G. Kruh;A. Sartorelli
多药耐药相关蛋白1和P-糖蛋白是主要的三磷酸腺苷结合盒转运体，其功能是作为外排泵，并对多种结构无关的抗癌药物产生耐药性。为了评估这些转运蛋白在抗癌药物方面的相对重要性，我们建立并鉴定了SV40永生化的[mrp1(-/-)](KO)、[mdr1a/1b(-/-)](DKO)以及来自基因敲除小鼠的原代胚胎成纤维细胞的[mrp1(-/-)、mdr1a/1b(-/-)](TKO)缺陷的成纤维细胞系。Western印迹分析表明，KO和DKO成纤维细胞的P-糖蛋白和mrp1的表达水平分别与野生型(WT)成纤维细胞相似。此外，对其他多药耐药相关蛋白(MRP)家族成员的半定量RT-PCR检测表明，TKO成纤维细胞显示出与WT成纤维细胞相似的MRP2-7的表达谱。这些结果表明，mrp1、P-糖蛋白或这两种转运蛋白的丢失不会导致其他已确定的转运蛋白表达的明显代偿性变化。细胞存活率和钙黄绿素积累量测定显示，与WT成纤维细胞相比，KO和DKO成纤维细胞对长春新碱和依托泊苷的敏感性和钙黄绿素积累量有轻度到中度的增加，而TKO成纤维细胞对这些药物的敏感性显著增强，钙黄绿素积累量进一步增加。此外，mrp1和P-糖蛋白的抑制剂维拉帕米可显著增加成纤维细胞对长春新碱和依托泊苷的敏感性，但不影响TKO细胞对这些药物的敏感性。总之，这些发现表明mrp1和P-糖蛋白是永生化小鼠胚胎成纤维细胞药物敏感性的主要决定因素。他们还建议存在一种补偿机制，通过这种机制，在普通药物底物的运输中，一个转运体的损失可以被另一个转运体所抵消。
Multidrug resistance-associated protein 1 and P-glycoprotein are major ATP-binding cassette transporters that function as efflux pumps and confer resistance to a variety of structurally unrelated anticancer agents. To evaluate the comparative importance of these transporters with respect to anticancer agents, we established and characterized SV40-immortalized [mrp1(-/-)] (KO), [mdr1a/1b(-/-)] (DKO), and combined [mrp1 (-/-), mdr1a/1b(-/-)] (TKO) deficient fibroblast lines derived from primary embryonic fibroblasts of knockout mice. Western blot analyses demonstrated that KO and DKO fibroblasts exhibited similar levels of P-glycoprotein and mrp1, respectively, to that of wild-type (WT) fibroblasts. In addition, semiquantitative reverse transcription-PCR measurements of other multidrug resistance-associated protein (mrp) family members demonstrated that TKO fibroblasts displayed expression profiles of mrps 2-7 comparable to that of WT fibroblasts. These results indicate that loss of mrp1, P-glycoprotein, or both transporters does not cause overt compensatory changes in the expression of the other determined transporters. Using cell viability and calcein accumulation assays, we demonstrated that KO and DKO fibroblasts exhibited a low to moderate increase in sensitivity to vincristine and etoposide and in calcein accumulation compared to WT fibroblasts, whereas TKO fibroblasts displayed a markedly enhanced sensitivity to these agents and further elevated calcein accumulation. Furthermore, verapamil, an inhibitor of both mrp1 and P-glycoprotein, significantly sensitized WT fibroblasts to both vincristine and etoposide while having no effect on the sensitivity of TKO cells to these agents. Collectively, these findings indicate that mrp1 and P-glycoprotein are major determinants of drug sensitivity in immortalized mouse embryonic fibroblasts. They also suggest the existence of a compensatory mechanism by which the loss of one transporter can be functionally offset by the other in the transport of common drug substrates.