title: In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity.
abstract: While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.
authors.affiliation: Laboratory of Cellular Biophysics, Rockefeller University, New York 10021, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: S M
authors.initials: SM
authors.lastname: Simon
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis* / Q000235:genetics*; D000970:Antineoplastic Agents / Q000493:pharmacokinetics; D001693:Biological Transport, Active / Q000235:genetics; D002460:Cell Line; D004396:Coloring Agents / Q000493:pharmacokinetics; D003630:Daunorubicin / Q000493:pharmacokinetics; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004952:Esters / Q000493:pharmacokinetics; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007202:Indicators and Reagents / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D004396:Coloring Agents; D004952:Esters; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003630:Daunorubicin