FLOW CYTOMETRIC DETERMINATION OF ACTIVITY OF P-GLYCOPROTEIN IN TUMOR SAMPLES

Classical multidrug resistance (MDR) is associated with the overexpres sion of a membrane glycoprotein termed P-glycoprotein (P-gp) that tran sports a variety of apparently unrelated anti-cancer drugs out of cell s. Based on the fluorescent properties of the dye rhodamine 123 (Rh 12 3) we aimed to develop a functional flow cytometric assay for the dete ction of MDR-expressing cells. Using drug sensitive cell lines (KB-3-1 ) and MDR mutants (KB-8-5, KB-C 1) experimental conditions were establ ished enabling demonstration of significant differences in Rh 123 accu mulation/efflux. Using two-colour flow cytometry we subsequently analy sed 42 consecutive patients suffering from B-cell chronic lymphocytic leukemia (B-CLL). 34/42 (81 %) cases showed a marked Rh 123 efflux whi ch was completely abolished in the presence of the MDR inhibitor verap amil. The percentage of Rh 123 effluxing cells ranged from 10 to 70% w ith a median of 32%. In 26 cases extracted RNA was analysed by quantit ative polymerase chain reaction to evaluate the expression of MDR 1 mR NA. In 25 of 26 (96%) cases MDR 1 mRNA was detectable. The levels of M DR 1 mRNA expression correlated well with the results of the Rh 123 ef flux assay (r = 0.72; p < 0.0001). In addition, 37 cases of acute myel oid leukemia were analysed and demonstrated Rh 123 efflux in 18/37 (49 %) cases. In this entity the percentage of Rh 123 effluxing cells rang ed from 12 to 80% (median 45%). Additionally, we evaluated the Rh 123 efflux/retention in peripheral blood cells of healthy volunteers. We f ound Rh 123 efflux in the following subsets: CD16+ natural killer cell s > CD8+ T cells > CD45RA T cells > CD19+ B cells > CD4+ T cells. No a ctive efflux was detectable in granulocytes and monocytes. Our results indicate that flow cytometric measurement of Rh 123 efflux is well su ited for the detection of MDR expressing cells in heterogeneous clinic al samples such as blood or bone marrow.