[Flow cytometric analysis of P-glycoprotein function by rhodamine 123 dye-efflux assay in human leukemia cells].
Cells with multidrug resistance(MDR) phenotype express P-glycoprotein(P-gp) on cell membrane, which works as a drug-efflux pump with low selectivity. P-gp function can be determined microfluorometrically using the fluorescent dye rhodamine 123(Rh123), which is an artificial substrate for P-gp. In this study, we assessed P-gp function in human leukemia sublines of MOLT-3 with various magnitude of MDR phenotype using the Rh123-efflux assay. The MDR cells efficiently pumped out Rh123 outside cells in parallel with the magnitude of resistance to vincristine, while the parent MOLT-3 cells scarcely showed dye efflux. The P-gp function determined by the dye efflux assay was correlated with the degree of cell surface expression of P-gp measured by indirect flow cytometric analysis using MRK16 anti-P-gp antibody and with the amount of MDR1 mRNA (encoding P-gp) quantified by Northern blot analysis and by competitive reverse transcription-polymerase chain reaction (RT-PCR) assay. In the evaluation of 28 clinical samples obtained from patients with leukemias, 9 cases exhibited positive results Rh123-efflux. A good correlation of Rh123-efflux with MDR1 expression measured by competitive RT-PCR was observed in these samples. Since subpopulations of normal lymphocytes show low degree of P-gp function, the strict gating of leukemia cells was mandatory in the dye-efflux assay in clinical samples. Although leukemia cells could not be distinguished from normal lymphocytes in the conventional scattergram in some cases, additional staining of the former cells with specific monoclonal antibody such as CD34(labelled with PE-Cy5, a dye without interference with Rh123 fluorescence emission) enabled a selective analysis of a particular subpopulation. The Rh123 dye-efflux assay is a simple and sensitive method for the determination of P-gp expression and its function, and is particularly suitable for the analyses in the clinical laboratory.
Kawabata
M
M
Department of Laboratory Medicine, National Defense Medical College, Tokorozawa.
Kobayashi
H
H
Mori
S
S
Sekiguchi
S
S
Takemura
Y
Y
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000502:physiology*; D018432:Drug Resistance, Multiple; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D020112:Rhodamine 123; D012235:Rhodamines; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D012235:Rhodamines; D020112:Rhodamine 123