In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity

While P-glycoprotein (Pgp) is the most studied protein involved in resistan ce to anti-cancer drugs, its mechanism of action is still under debate. Stu dies 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 ce lls transiently transfected with a Pgp-green fluorescent protein (GFP) fusi on protein. This method generated a mixed population of unselected cells wi th a wide range of Pgp-GFP expression levels and allowed simultaneous measu rements of Pgp level and drug accumulation in living cells. The results sho wed that Pgp-GFP expression was inversely related to the accumulation of ch emotherapeutic drugs. The reduction in drug concentration was reversed by a gents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp an tibody. Quantitative analysis revealed an inverse linear relationship betwe en the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzym e, 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.