An MDR-EGFP gene fusion allows for direct cellular localization, function and stability assessment of P-glycoprotein.

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 ATP-dependent 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 (t(1/2 total Pgp-EGFP) = 2.2 days), but revealed that surface-Pgp acquires extra stability as an active pump (t(1/2 surface Pgp-EGFP) = 3.7 days).