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

Jordi      Petriz; Michael   M.   Gottesman; Josep   M.   Aran

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

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