VALIDATION OF A SINGLE-POINT FLOW CYTOMETRIC ASSAY FOR DETERMINING P-GLYCOPROTEIN ACTIVITY IN MULTIDRUG-RESISTANT CELL-LINES

P-glycoprotein, a transmembrane protein which acts as an energy depend ent efflux pump, has been implicated as one mechanism of multidrug res istance (MDR) in human tumours. Commonly employed assays measure P-gly coprotein immunohistochemically or mdr1 messenger RNA. In this study w e compared a single point flow cytometric assay for determining activi ty of P-glycoprotein with cellular expression of P-glycoprotein determ ined by Western blot. Five cell lines, with varying levels of multiple drug resistance, were incubated with daunorubicin (DNR) in the presen ce (treated) and absence (control) of cyclosporine or verapamil, agent s known to inhibit the activity of P-glycoprotein. The treated cell li nes, along with non-treated controls were examined for intracellular c oncentrations of DNR measured by fluorescence intensity using a flow c ytometer. The ratio of fluorescence intensity expressed in the treated /control was used as an index of functional activity of P-glycoprotein . Functional activity of the P-glycoprotein as determined by flow cyto metry correlates highly with cellular content of P-glycoprotein measur ed by western blot (correlation coefficients of r = 0.90-0.98 for the various cell line combinations). This method represents a rapid single point flow cytometric assay which may be suitable for screening clini cal samples for P-glycoprotein activity.