[Functional detection of P-glycoprotein expressing cells with flow cytometry].
Classical multidrug resistance (MDR) is associated with the overexpression of a membrane glycoprotein termed P-glycoprotein (P-gp) that transports a variety of apparently unrelated anti-cancer drugs out of cells. Based on the fluorescent properties of the dye rhodamine 123 (Rh 123) we aimed to develop a functional flow cytometric assay for the detection of MDR-expressing cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established enabling demonstration of significant differences in Rh 123 accumulation/efflux. Using two-colour flow cytometry we subsequently analysed 42 consecutive patients suffering from B-cell chronic lymphocytic leukemia (B-CLL). 34/42 (81%) cases showed a marked Rh 123 efflux which was completely abolished in the presence of the MDR inhibitor verapamil. The percentage of Rh 123 effluxing cells ranged from 10 to 70% with a median of 32%. In 26 cases extracted RNA was analysed by quantitative polymerase chain reaction to evaluate the expression of MDR 1 mRNA. In 25 of 26 (96%) cases MDR 1 mRNA was detectable. The levels of MDR 1 mRNA expression correlated well with the results of the Rh 123 efflux assay (r = 0.72; p < 0.0001). In addition, 37 cases of acute myeloid leukemia were analysed and demonstrated Rh 123 efflux in 18/37 (49%) cases. In this entity the percentage of Rh 123 effluxing cells ranged from 12 to 80% (median 45%). Additionally, we evaluated the Rh 123 efflux/retention in peripheral blood cells of healthy volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)
Ludescher
C
C
Universitätklinik für Innere Medizin, Universität Innsbruck.
Hilbe
W
W
Eisterer
W
W
Preuss
E
E
Hofmann
J
J
Thaler
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics*; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020112:Rhodamine 123; D012235:Rhodamines / Q000493:pharmacokinetics
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015703:Antigens, CD; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D012235:Rhodamines; D020112:Rhodamine 123
Funktionelle Erfassung P-Glykoprotein-exprimierender Zellen mittels Durchflusszytometrie.