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).
Pétriz
Jordi
J
Servei d'Hemateràpia i Hemostàsia, Institut d'Investigacions Biomèdiques August Pi i Sunyer (DIBAPS), Hospital Clinic, Universitat de Barcelona Villarroel, Barcelona, Spain.
Gottesman
Michael M
MM
Aran
Josep M
JM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D003630:Daunorubicin / Q000378:metabolism; D004285:Dogs; D004317:Doxorubicin / Q000378:metabolism; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D005453:Fluorescence; D005455:Fluorescent Antibody Technique; D050939:Gene Fusion; D019450:Genes, MDR / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D020112:Rhodamine 123 / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism*; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000903:Antibiotics, Antineoplastic; D017931:DNA Primers; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123; D004317:Doxorubicin; D003630:Daunorubicin