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Dynamic and intracellular trafficking of P‐glycoprotein‐EGFP fusion protein: Implications in multidrug resistance in cancer
Author(s) -
Fu Dong,
Bebawy Mary,
Kable Eleanor P.W.,
Roufogalis Basil D.
Publication year - 2004
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.11659
Subject(s) - intracellular , daunorubicin , brefeldin a , green fluorescent protein , p glycoprotein , endoplasmic reticulum , golgi apparatus , transfection , biology , microbiology and biotechnology , fusion protein , multiple drug resistance , cell culture , biochemistry , immunology , recombinant dna , genetics , leukemia , gene , antibiotics
Abstract In our present study, a P‐glycoprotein‐EGFP (P‐gp‐EGFP) fusion plasmid was constructed and functionally expressed in HeLa cells to investigate the intracellular localization and trafficking of P‐glycoprotein (P‐gp). Using immunocytochemistry and fluorescent confocal microscopy techniques, colocalization studies showed that after transfection, P‐gp‐EGFP was progressively transported from the endoplasmic reticulum (ER) to the Golgi and finally to the plasma membrane within 12–48 hr. The degree of intracellular accumulation of daunorubicin was related to the particular localization of P‐gp‐EGFP. Significant daunorubicin accumulation occurred in transfected cells when P‐gp‐EGFP was localized predominantly within the ER, and accumulation remained high when P‐gp‐EGFP was mainly localized in the Golgi. However, there was little or no intracellular accumulation of daunorubicin when P‐gp‐EGFP was localized predominantly on the plasma membrane. Blocking the intracellular trafficking of P‐gp‐EGFP with brefeldin A (BFA) and monensin resulted in inhibition of traffic of P‐gp‐EGFP and retention of P‐gp‐EGFP intracellularly. Intracellular accumulation of daunorubicin also increased in the presence of BFA or monensin. Our study shows that P‐gp‐EGFP can be used to define the dynamics of P‐gp traffic in a transient expression system, and demonstrates that localization of P‐gp on the plasma membrane is associated with the highest level of resistance to daunorubicin accumulation in cells. Modulation of intracellular localization of P‐gp with agents designed to selectively modify its traffic may provide a new strategy for overcoming multidrug resistance in cancer cells. © 2004 Wiley‐Liss, Inc.