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[Flow cytometric analysis of P-glycoprotein function by rhodamine 123 dye-efflux assay in human leukemia cells]
M Kawabata1, H Kobayashi, S Mori
1Department of Laboratory Medicine, National Defense Medical College, Tokorozawa.
Abstract:
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.
Insights
The rhodamine 123 efflux assay effectively measures P-glycoprotein function in multidrug-resistant leukemia cells. This method correlates with P-gp expression and is suitable for clinical laboratory analysis.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) in cancer cells is often mediated by P-glycoprotein (P-gp), an efflux pump.
- P-gp expression leads to reduced intracellular drug accumulation and treatment failure.
- Assessing P-gp function is crucial for understanding MDR and guiding therapy.
Purpose of the Study:
- To evaluate P-gp function in human leukemia sublines using the rhodamine 123 (Rh123) efflux assay.
- To correlate Rh123 efflux with P-gp expression levels (protein and mRNA) and drug resistance.
- To assess the utility of the Rh123 efflux assay for clinical leukemia samples.
Main Methods:
- Utilized the fluorescent dye Rh123 as a substrate to measure P-gp efflux in MOLT-3 leukemia sublines.
- Quantified P-gp expression via indirect flow cytometry (MRK16 antibody) and MDR1 mRNA levels (Northern blot, RT-PCR).
- Applied the Rh123 efflux assay to 28 clinical leukemia samples, employing CD34 staining for selective analysis.
Main Results:
- MDR leukemia cells demonstrated efficient Rh123 efflux, directly proportional to vincristine resistance.
- Rh123 efflux correlated well with P-gp cell surface expression and MDR1 mRNA levels.
- Nine of 28 clinical samples showed positive Rh123 efflux, correlating with MDR1 expression via RT-PCR.
Conclusions:
- The Rh123 dye-efflux assay is a simple, sensitive method for determining P-gp function and expression.
- The assay is well-suited for clinical laboratory settings for evaluating MDR in leukemia.
- Selective gating using markers like CD34 is essential for accurate analysis of clinical samples.