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An MDR-EGFP Gene Fusion Allows for Direct Cellular Localization, Function and Stability Assessment of P-Glycoprotein

[ Vol. 1 , Issue. 1 ]

Author(s):

Jordi Petriz, Michael M. Gottesman and Josep M. Aran   Pages 43 - 56 ( 14 )

Abstract:


In cancer and AIDS, overexpression of the MDR1 gene has important implications in the design of chemotherapy protocols because of the ability of its product, the ATPdependent drug efflux pump P-glycoprotein (Pgp), to confer selective advantage to tumor and HIV-infected cells in the form of multidrug resistance. To study Pgp expression and physiology, we designed a translational fusion between the MDR1 and enhanced green fluorescent protein (EGFP) genes. The chimeric protein, Pgp-EGFP, was concentrated mainly in the plasma membrane and in the Golgi when expressed in drug-sensitive KB- 3-1 cells. Doxorubicin, daunorubicin and rhodamine-123 efflux assays confirmed function of the chimeric pump. Also, at the single-cell level, an inverse relationship between Pgp-EGFP expression and nuclear doxorubicin accumulation was demonstrated. Polarized Pgp expression on the apical cell surface was confirmed by transfection of the MDR-EGFP fusion gene into MDCK cells. However, after colchicine selection, Pgp-EGFP was also detectable in the lateral domain of the transfected MDCK monolayers. These results indicate that drug selection affects not only expression, but cellular localization of Pgp. Furthermore, using a tet-based inducible expression system for Pgp-EGFP, we confirmed the stable nature of Pgp (t1 / 2 total Pgp-EGFP= 2.2 days), but revealed that surface-Pgp acquires extra stability as an active pump (t1 / 2 surface Pgp-EGFP= 3.7 days).

Keywords:

p-glycoprotein, green fluorescent protein, drug selection, turnover, subcellular localization

Affiliation:

Centre de Genetica Medicai Molecular Institut de Recerca Oncologica (IRO) Hospital Duran i ReynalsGran Vía s / n, km 2,7 08907 L'Hospitalet de Llobregat Barcelona, SPAIN.



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