Y. Chen et Sm. Simon, In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity, J CELL BIOL, 148(5), 2000, pp. 863-870
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.