Validation of a single point flow cytometric assay for determining P-glycoprotein activity in multidrug resistant cell lines
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P-glycoprotein, a transmembrane protein which acts as an energy dependent efflux pump, has been implicated as one mechanism of multidrug resistance (MDR) in human tumours. Commonly employed assays measure P-glycoprotein immunohistochemically or mdr1 messenger RNA. In this study we compared a single point flow cytometric assay for determining activity of P-glycoprotein with cellular expression of P-glycoprotein determined by Western blot. Five cell lines, with varying levels of multiple drug resistance, were incubated with daunorubicin (DNR) in the presence (treated) and absence (control) of cyclosporine or verapamil, agents known to inhibit the activity of P-glycoprotein. The treated cell lines, along with non-treated controls were examined for intracellular concentrations of DNR measured by fluorescence intensity using a flow cytometer. 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 cytometry correlates highly with cellular content of P-glycoprotein measured 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 clinical samples for P-glycoprotein activity.
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