CHARACTERIZATION OF FUNCTIONAL ASSAYS OF MULTIDRUG-RESISTANCE P-GLYCOPROTEIN TRANSPORT ACTIVITY

P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy. The P-glycopro tein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of che motherapeutic agents. Pharmaceuticals which inhibit P-glycoprotein tra nsport activity are currently being evaluated in clinical trials. Char acterization of P-glycoprotein functional activity is critical in dete rmining if these multidrug resistance reversal agents improve therapeu tic responses of tumors expressing P-glycoprotein. In this report, we directly compare and characterize assays using rhodamine 123, dimethyl oxadicarbocyanine iodide (DIOC2), [H-3]daunorubicin and hexakis(2-meth oxyisobutyl isonitrile)technetium(I) ([Tc-99m]Sestamibi) as P-glycopro tein transport probes to quantitate functional activity. The accumulat ion of certain substrates is concentration dependent and the parameter s which determine probe accumulation are impacted by the level of P-gl ycoprotein expression. In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expr essing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein tran sport activity is higher than generally recognized. Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentratio ns of potent reversal agents. These results highlight the importance o f carefully characterizing assay systems under uniform conditions to q uantitate P-glycoprotein function.