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Multidrug resistance protein (MRP) activity in normal mature leukocytes and CD34-positive hematopoietic cells from
1Laboratoire Universitaire d'Hématologie et de la Biologie des Cellules Sanguines, Faculté de Médecine, Rennes, France.
Abstract:
Multidrug resistance proteins (MRPs) such as MRP1, MRP2 and MRP3 are membrane efflux pumps involved in multidrug resistance and handling organic anions. In the present study, MRP activity was investigated in normal mature leucocytes and CD34-positive hematopoietic cells from peripheral blood using the flow cytometric carboxy-2',7'-dichlorofluorescein (CF) efflux assay. Basal and similar cellular exports of CF, an anionic fluorescent dye substrate for MRP1 and MRP2 transporters, were evidenced in lymphocytes whatever their subsets (CD3, CD4, CD8, CD20 and CD56 cells), in CD14 monocytes and in CD15 granulocytes whereas higher CF efflux was found in CD34 cells. Such outwardly-directed transports of CF were inhibited by known blockers of MRP function such as probenecid whereas the P-glycoprotein modulator verapamil did not alter the retention of the dye in the blood leukocytes. Peripheral mature blood leukocytes were moreover found to express MRP1 mRNAs and MRP1 protein as assessed by Northern-blot and Western-blot analyses, whereas MRP2 and MRP3 transcripts were not present or only at very low levels. Mature leukocytes therefore display basal constitutive MRP-related transport activity regardless of cell lineage and likely related to MRP1 expression whereas higher MRP-related efflux can be detected in peripheral CD34 hematopoietic cells.
Insights
Mature blood leukocytes exhibit basal multidrug resistance protein (MRP) activity, primarily via MRP1. Higher MRP-related efflux is observed in hematopoietic stem cells, indicating their role in drug transport.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- Multidrug resistance proteins (MRPs) are crucial efflux pumps involved in xenobiotic transport and cellular defense.
- MRP1, MRP2, and MRP3 are key members of the MRP family, influencing drug disposition and organic anion handling.
- Understanding MRP activity in leukocytes is vital for predicting drug responses and managing hematological conditions.
Purpose of the Study:
- To investigate the activity and expression of MRPs in mature peripheral blood leukocytes and CD34-positive hematopoietic stem cells.
- To determine the specific MRP transporters involved in the efflux of anionic substrates in these cell types.
- To elucidate the role of MRP1, MRP2, and MRP3 in the drug transport capabilities of blood cells.
Main Methods:
- Flow cytometry using the carboxy-2',7'-dichlorofluorescein (CF) efflux assay to measure MRP activity.
- Utilizing specific MRP inhibitors like probenecid to confirm transporter function.
- Northern blot and Western blot analyses to assess the mRNA and protein expression of MRP1, MRP2, and MRP3.
Main Results:
- Basal efflux of the MRP substrate CF was detected in various mature leukocyte subsets (lymphocytes, monocytes, granulocytes).
- CD34-positive hematopoietic cells exhibited significantly higher CF efflux compared to mature leukocytes.
- MRP1 mRNA and protein were expressed in mature leukocytes, while MRP2 and MRP3 were detected at very low or undetectable levels.
- Probenecid inhibited CF efflux, confirming MRP involvement, whereas verapamil had no effect, ruling out P-glycoprotein mediation.
Conclusions:
- Mature peripheral blood leukocytes possess constitutive MRP-related transport activity, predominantly mediated by MRP1.
- CD34-positive hematopoietic cells demonstrate enhanced MRP-related efflux activity.
- These findings highlight the functional significance of MRPs in both mature and progenitor blood cells for xenobiotic handling.
