ABSTRACT
The multidrug resistance (MDR) phenotype is associated with the overexpression of members of the ATP-binding cassette family of proteins. These MDR transporters are expressed at the plasma membrane, where they are thought to reduce the cellular accumulation of toxins over time. Our data demonstrate that members of this family are also expressed in subcellular compartments where they actively sequester drugs away from their cellular targets. The multidrug resistance protein 1 (MRP1), P-glycoprotein, and the breast cancer resistance protein are each present in a perinuclear region positive for lysosomal markers. Fluorescence-activated cell sorting analysis suggests that these three drug transporters do little to reduce the cellular accumulation of the anthracycline doxorubicin. However, whereas doxorubicin enters cells expressing MDR transporters, this drug is sequestered away from the nucleus, its subcellular target, in vesicles expressing each of the three drug resistance proteins. Using a cell-impermeable inhibitor of MRP1 activity, we demonstrate that MRP1 activity on intracellular vesicles is sufficient to confer a drug resistance phenotype, whereas disruption of lysosomal pH is not. Intracellular localization and activity for MRP1 and other members of the MDR transporter family may suggest different strategies for chemotherapeutic regimens in a clinical setting.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cloning, Molecular , Doxorubicin/pharmacokinetics , Doxorubicin/pharmacology , HeLa Cells , Humans , Lysosomes/metabolismABSTRACT
The multidrug resistance protein 1 (MRP1) contributes cellular resistance to a wide array of physiological toxins and chemotherapeutic agents. Its in vivo activity has been studied primarily in cells that have been continuously drug selected, culture conditions that might confound the effects of MRP1 expression with the effects of a cell's detoxification machinery. Transient transfection with a MRP1-green fluorescent protein (EGFP) fusion protein allowed us to measure the activity of MRP1 in cells that had insufficient time to induce other chemoprotective proteins. Furthermore, separate transfections with MRP1-yellow fluorescent protein and a fluorescently tagged P-glycoprotein (MDR1-cyan fluorescent protein) permitted the drug-resistant properties of MRP1-expressing cells to be compared with those of MDR1-expressing cells. Our data showed that the expression of MRP1-EGFP results in significantly decreased cellular accumulation of tetramethylrhodamine ethyl ester (TMRE) and daunorubicin, mildly decreased cellular accumulation of mitoxantrone, and decreased nuclear accumulation of doxorubicin. Additionally, MRP1-EGFP expression protected cells from the microtubule depolymerization caused by vincristine and colchicine, but not by vinblastine.
