DETECTION OF P-GLYCOPROTEIN WITH A RAPID FLOW CYTOMETRIC FUNCTIONAL ASSAY USING FLUO-3 - EVALUATION OF SENSITIVITY, SPECIFICITY AND FEASIBILITY IN MULTIPARAMETRIC ANALYSIS

The specificity and sensitivity of a flow cytometric assay simultaneou sly measuring expression and transport function of the multidrug resis tance associated P-glycoprotein (Pgp) was evaluated. The monoclonal an tibody (mAb), MRK16 was used to detect phenotypic Pgp expression while Fluo-3-AM was used as a fluorescent substrate in a Pgp functional tra nsport assay. The specificity of the functional assay was examined in two vinblastine selected human leukemic cell lines (K562/VLB2.5 and CC RF-CEM/VLB5O) with acquired Pgp overexpression. Downmodulation of Pgp function in these cell lines could be demonstrated with different subs tances (verapamil, vinblastine, trifluoperazine, cyclosporin A, proges terone and quinidine) and was proven to be consistently higher in the vinblastine selected cells than in their non-selected drug sensitive c ounterparts. Unexpectedly, modulator activity was also observed in dru g sensitive K562 and CCRF-CEM cell lines despite the inability to dete ct Pgp in those cells by MRK16 flow cytometrically. Low level expressi on of the MDR1 gene encoding Pgp in sensitive K562 cells was however d emonstrated with a sensitive RT-PCR procedure. The small effect of Pgp modulators in non-drug selected cells could therefore be attributed t o low level basal expression of Pgp and illustrates the sensitivity of the functional assay. Also, the effect of various Pgp modulators on P gp function was more pronounced in a subpopulation of Pgp expressing l ymphocytes than in lymphocytes which did not express Pgp. Finally, a c orrelation was found between discrete variations in Pgp expression and Pgp function of CD4(+) lymphocytes, underscoring the feasibility of t he functional assay in a triple parametric procedure. The triple param etric assay holds promise to detect Pgp expression and function in cli nical samples containing mixtures of malignant and non-malignant cells .