Luker G D, Nilsson K R, Covey D F, Piwnica-Worms D
Laboratory of Molecular Radiopharmacology, Mallinckrodt Institute of Radiology, St. Louis, Missouri 63110, USA.
J Biol Chem. 1999 Mar 12;274(11):6979-91. doi: 10.1074/jbc.274.11.6979.
Class I P-glycoproteins (Pgp) confer multidrug resistance in tumors, but the physiologic function of Pgp in normal tissues remains uncertain. In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. We investigated the esterification of plasma membrane cholesterol under basal conditions and in response to sphingomyelinase treatment in transfected and drug-selected cell lines expressing differing amounts of functional class I Pgp. Compared with parental NIH 3T3 fibroblasts, cells transfected with human multidrug resistance (MDR1) Pgp esterified more cholesterol both without and with sphingomyelinase. Esterification also was greater in drug-selected Dox 6 myeloma cells than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively. However, no differences in total plasma membrane cholesterol were detected. Transfection of fibroblasts with the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not generally affected by ATP-binding cassette transporters. Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity mediated by Pgp. In Pgp-expressing cells treated with nonselective and selective inhibitors, both the kinetics and efficacy of inhibition of cholesterol esterification differed from the antagonism of drug transport mediated by Pgp. Thus, although the data show that greater expression of class I Pgp within a given cell type is associated with enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp.
I类P-糖蛋白（Pgp）赋予肿瘤多药耐药性，但Pgp在正常组织中的生理功能仍不确定。在通常表达Pgp的组织来源的细胞中，最近的数据表明Pgp在胆固醇从质膜转运到内质网的过程中可能发挥作用。我们研究了基础条件下以及在转染和药物筛选的表达不同量功能性I类Pgp的细胞系中，鞘磷脂酶处理后质膜胆固醇的酯化情况。与亲本NIH 3T3成纤维细胞相比，转染人多药耐药（MDR1）Pgp的细胞在有无鞘磷脂酶的情况下都能酯化更多的胆固醇。药物筛选的多柔比星6骨髓瘤细胞中的酯化作用也比亲本8226细胞更强，后者分别表达低水平和无法通过免疫检测到的Pgp。然而，未检测到总质膜胆固醇的差异。用多药耐药相关蛋白（MRP）转染成纤维细胞并未改变酯化作用，表明胆固醇转运一般不受ATP结合盒转运体的影响。评估了甾体（孕酮、脱氢表雄酮）和非甾体拮抗剂（维拉帕米、PSC 833、LY335979和GF120918）对胆固醇转运以及99mTc-司他米比净含量的影响，99mTc-司他米比是由Pgp介导的药物转运活性的报告物。在用非选择性和选择性抑制剂处理的表达Pgp的细胞中，胆固醇酯化抑制的动力学和效力与Pgp介导的药物转运拮抗作用不同。因此，尽管数据表明在给定细胞类型中I类Pgp的更高表达与质膜胆固醇酯化增强相关，支持Pgp在促进胆固醇转运中的生理功能，但分子机制与Pgp的传统药物转运活性无关。
