Almquist K C, Loe D W, Hipfner D R, Mackie J E, Cole S P, Deeley R G
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Cancer Res. 1995 Jan 1;55(1):102-10.
Overexpression of multidrug resistance-associated protein (MRP) has been detected in resistant cell lines derived from a variety of tumor types. The deduced amino acid sequence of MRP suggests that it is a member of the ATP-binding cassette transmembrane transporter superfamily that may be glycosylated and/or phosphorylated [S. P. C. Cole et al., Science Washington, DC), 258: 1650-1654, 1992]. Recently, transfection of HeLa cells with MRP expression vectors has demonstrated that the protein is capable of increasing resistance to natural product drugs such as anthracyclines, Vinca alkaloids, and epipodophyllotoxins (C. E. Grant et al., Cancer Res., 54: 357-361, 1994). Although the resistance phenotype of the transfectants is similar to that of the human small cell lung cancer cell line, H69AR, from which MRP was originally cloned, the transfectants differ in their drug accumulation characteristics, relative resistance to certain drugs, and MRP mRNA:protein ratio. Such differences have also been observed among drug-selected cell lines that overexpress MRP, and the underlying causes of these variable phenotypes are presently not known. We have utilized polyclonal anti-MRP-peptide antibodies to compare MRP post-translational modification, stability, processing, and subcellular distribution in the HeLa transfectants and in the drug-selected H69AR cells. These studies establish that MRP in both the transfected and selected cells is an ATP-binding, integral membrane glycophosphoprotein with an apparent molecular weight of 190,000. No obvious differences were detected in the extent or type of glycosylation or the kinetics of processing and turnover of the protein that might contribute to the different characteristics of the transfected and drug-selected cells. Analyses of the subcellular distribution of MRP by isopyknic density gradient centrifugation revealed that approximately 80% of MRP in the HeLa transfectants was associated with a low density plasma membrane fraction while the comparable fraction in the drug-selected H69AR cells contained only approximately 50% of the protein. The remaining MRP and plasma membrane markers were codistributed in higher density fractions consistent with the presence of MRP in endocytotic vesicles. The relatively high proportion of MRP associated with these fractions in H69AR cells may contribute to the lack of an observable accumulation defect in these cells when compared with the transfectants.
在源自多种肿瘤类型的耐药细胞系中已检测到多药耐药相关蛋白（MRP）的过表达。MRP推导的氨基酸序列表明它是ATP结合盒跨膜转运蛋白超家族的成员，可能被糖基化和/或磷酸化[S. P. C. 科尔等人，《科学》（华盛顿特区），258: 1650 - 1654，1992]。最近，用MRP表达载体转染HeLa细胞已证明该蛋白能够增加对天然产物药物如蒽环类抗生素、长春花生物碱和表鬼臼毒素的耐药性（C. E. 格兰特等人，《癌症研究》，54: 357 - 361，1994）。尽管转染细胞的耐药表型与最初克隆MRP的人小细胞肺癌细胞系H69AR相似，但转染细胞在药物蓄积特征、对某些药物的相对耐药性以及MRP mRNA与蛋白比例方面存在差异。在过表达MRP的药物筛选细胞系中也观察到了此类差异，目前尚不清楚这些可变表型的潜在原因。我们利用多克隆抗MRP肽抗体来比较HeLa转染细胞和药物筛选的H69AR细胞中MRP的翻译后修饰、稳定性、加工过程及亚细胞分布。这些研究证实，转染细胞和筛选细胞中的MRP都是一种ATP结合的整合膜糖磷蛋白，表观分子量为190,000。在糖基化程度或类型、蛋白加工及周转动力学方面未检测到明显差异，而这些差异可能导致转染细胞和药物筛选细胞具有不同特征。通过等密度梯度离心分析MRP的亚细胞分布发现，HeLa转染细胞中约80%的MRP与低密度质膜组分相关，而在药物筛选的H69AR细胞中，相应组分仅含有约50%的该蛋白。其余的MRP和质膜标志物共分布于较高密度组分中，这与MRP存在于内吞小泡中一致。与转染细胞相比，H69AR细胞中与这些组分相关的MRP比例相对较高，这可能是这些细胞中未观察到明显蓄积缺陷的原因。
