FLOW-CYTOMETRY EVALUATION OF THE MULTIDRUG-RESISTANT PHENOTYPE WITH FUNCTIONAL TESTS INVOLVING UPTAKE OF DAUNORUBICIN, HOECHST-33342, OR RHODAMINE-123 - A COMPARATIVE-STUDY

Multidrug-resistant (MDR) cells are characterized by a defect in drug accumulation caused by overexpression of a transmembrane glycoprotein, the P-glycoprotein (P-gp). The MDR phenotype can be characterized eit her by use of monoclonal antibodies raised against P-gp or with functi onal tests based on the intracellular accumulation of fluorescent mole cules. The aim of the present study was to compare the effectiveness o f functional tests performed by flow cytometry including uptake of dau norubicin (DNR) (2 mu g/ml), Hoechst 33342 (5 mu g/ml), or rhodamine 1 23 (RH 123) (0.1 mu g/ml); and to evaluate the effect of cell death in duced by heating at 60 degrees C for 2 h on incorporation of DNR and R H 123. Sensitive and resistant human hematopoietic K 562 cells express ing P-gp were identified by monoclonal antibodies C 219 and MRK-16. Fl uorescence of the dyes was always higher in sensitive than in resistan t cells. However, DNR and Hoechst 33342 produced a slight incorporatio n in resistant cells, while RH 123 showed lack of incorporation in res istant cells. Thus, RH 123 allows sensitive and resistant cells to be clearly distinguished. In case of cell death, accumulation of RH 123 a nd DNR were different. With RH 123, fluorescence intensity strongly de creased in sensitive cells. With DNR, fluorescence intensity was enhan ced in resistant cells. Thus, when the MDR phenotype is defined by upt ake of DNR or RH 123, artifactual results due to cell death may be avo ided by using a dye such as propidium iodide to eliminate dead cells.