Expression of c-kit and Sca-1 and their relationship with multidrug resistance protein 1 in mouse bone marrow mononuclear cells

Abstract

P-glycoprotein (Pgp) and multidrug resistance protein 1 (MRP1) are members of the ATP-binding cassette (ABC) family of transporter proteins. Both molecules are membrane-associated, energy-dependent efflux pumps with different substrate selectivity and they may play a role in the activation, differentiation and function of haematopoietic cells. Mouse haematopoietic cells are characterized by the expression of the cell surface molecules c-kit and Sca-1. Herein, the presence and activities of Pgp and MRP1 in mouse bone marrow mononuclear cells (BMMC) and their relationship with the proteins c-kit and Sca-1 were evaluated. Pgp and MRP activities were measured based on the extrusion of rhodamine 123 (for Pgp) and Fluo-3 (for MRP). Cell populations were assessed by cytometry using anti-c-kit and anti-Sca1 antibodies. Pgp activity was present in 5% of BMMC while 50% of BMMC cells showed MRP activity. These findings agreed with the proportion of cells expressing the MRP1 surface molecule (51.3 +/- 4.17%). About 14% of BMMC were positive for c-kit and/or Sca-1 (9.3% c-kit- Sca-1+, 4.2% c-kit+ Sca-1- and 0.9% c-kit+ Sca-1+). Among these subpopulations only c-kit- Sca-1+ cells presented Pgp activity (21.36%). On the other hand, MRP activity was present in all three subpopulations. Most cells (82.5%) of the c-kit+ Sca-1- subpopulation presented MRP1 activity compared to only 54.1% of c-kit+ Sca-1+ and 38.8% of c-kit- Sca-1+. This study demonstrates the expression and activity of MRP1 in BMMC. While only a small proportion of precursor cells had Pgp activity, MRP1 activity was present among different subpopulations of precursor cells. Further studies are necessary to establish the role of these transporters in haematopoietic cells.

Figure 1

MRP1 expression and activity in murine bone marrow mononuclear cells. (a) Cells were permeabilized, and labelled with rat anti-MRP1. Cells were then washed and stained with goat anti-rat IgG FITC. Cells were washed and acquired by flow cytometry. Left panel: control, cells were labelled only with the secondary antibody. Right panel: gray histogram represents cells labelled with both antibodies. M1 indicates the autofluorescence region and M2 indicates the labelled region. The value in the figure indicates the percentual of bone marrow mononuclear cells expressing MRP1. This figure is representative of three different experiments. (b) Histograms showing the accumulation of Fluo-3 by the cells, in the presence or absence of MRP inhibitors: INDO – indomethacin (75 μm), PRB – probenecid (1000 μm) and MK – MK571 (25 μm). (c) Dose-response curve of the effect of inhibitors: INDO, PRB or MK in MRP1 activity. Results are expressed as mean ± SD of at least 3 independent experiments.

Figure 2

Pgp-related transport activity in murine bone marrow mononuclear cells. (a) Representative dotplots showing the amount of Rho 123 negative cells (left hand side region). (b) Dose-response curve of Pgp inhibitors: VP (verapamil) and CSA (cyclosporin A). Results are expressed as mean ± SD of 3 independent experiments. *Significantly different from control P < 0·05.

Figure 3

(a) Distribution of progenitor cells subpopulations among bone marrow mononuclear cells. Cells were incubated with anti-c-kit TC and anti-Sca-1 PE antibodies. Cells were then washed and acquired by flow cytometry. Representative dotplot. (b) MRP1 transport activity. Bars represent the percentage of cells extruding Fluo-3 (MRP activity) among c-kit⁺Sca-1⁻ (black bar), c-kit⁻Sca-1⁺ (white bar) and c-kit⁺Sca-1⁺ (gray bar). (c) Pgp transport activity. Bars represent the percentage of cells extruding Rho 123 (Pgp activity) among c-kit⁺Sca-1⁻ (black bar), c-kit⁻Sca-1⁺ (white bar) and c-kit⁺Sca-1⁺ (gray bar). Results are expressed as mean ± SD of 3 independent experiments. *Significantly different from the other two groups P < 0·05.