Hematopoietic stem cell differentiation affects expression and function of MRP4 (ABCC4), a transport protein for signaling molecules and drugs

Authors

  • Lena Oevermann,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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    • Lena Oevermann, Jan Scheitz, Gabriele Jedlitschky, and Christoph A. Ritter contributed equally to this work.

  • Jan Scheitz,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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    • Lena Oevermann, Jan Scheitz, Gabriele Jedlitschky, and Christoph A. Ritter contributed equally to this work.

  • Katharina Starke,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Kathleen Köck,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Thomas Kiefer,

    1. Department of Hematology and Oncology, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Gottfried Dölken,

    1. Department of Hematology and Oncology, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Juliane Nießen,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Andreas Greinacher,

    1. Department of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Werner Siegmund,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Marek Zygmunt,

    1. Department of Gynecology and Obstetrics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Heyo K. Kroemer,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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  • Gabriele Jedlitschky,

    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
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    • Lena Oevermann, Jan Scheitz, Gabriele Jedlitschky, and Christoph A. Ritter contributed equally to this work.

  • Christoph A. Ritter

    Corresponding author
    1. Department of Pharmacology, Research Center of Pharmacology and Experimental Therapeutics, Ernst-Moritz-Arndt-University, Greifswald, Germany
    2. Institute of Pharmacy, Ernst-Moritz-Arndt-University, Greifswald, Germany
    • Institute of Pharmacology, Ernst-Moritz-Arndt-University of Greifswald, Friedrich-Loeffler-Str. 23D, Greifswald 17487, Germany
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    • Lena Oevermann, Jan Scheitz, Gabriele Jedlitschky, and Christoph A. Ritter contributed equally to this work.

    • Fax: +49–3834–864–802,–5631.


Abstract

Several types of peripheral blood cells express ABC transporters. ABCC4 (MRP4) and ABCC5 (MRP5) localize to different cellular sites and fulfill lineage-specific functions such as mediator storage in platelets' dense granules. All mature blood cells originate from the same precursor and specific functionalities arise during differentiation. To characterize this process, expression, localization and function of MRP4 and MRP5 were assessed in differentiating human CD34+ progenitors and leukemia cell lines using real time polymerase chain reaction (PCR), immunofluorescence microscopy and cell viability assays. Median MRP4 mRNA copy numbers were significantly enhanced by megakaryocytic differentiation from 7.9 × 103 to 5.8 × 104 copies per nanograms of total RNA (p < 0.05) in CD34+ progenitors and in M-07e cells (MRP4 mRNA/18S rRNA ratios: 5.4 ± 3.8 × 10−4 vs. 2.7 ± 0.9 × 10−3 for native and differentiated cells, respectively, p < 0.05), and MRP4 protein was localized to granular structures and to the plasma membrane both in differentiated progenitors and bone marrow megakaryocytes. In contrast, expression of MRP4 decreased during maturation to leukocytes (MRP4 mRNA/18S rRNA ratios: 5.2 × 10−3 for native vs. 3.5 × 10−3 for CD34+ cells in the presence of G-CSF, p < 0.05) and was significantly reduced in mature monocytes and granulocytes compared with progenitors (MRP4 mRNA/18S rRNA ratios: 8.1 ± 5.4 × 10−5 and 2.8 ± 1.6 × 10−4vs. 1.2 ± 0.7 × 10−3, respectively, p < 0.05). Expression of MRP5 was not significantly altered under all differentiation conditions. These results indicate that MRP4 expression is differentially regulated during hematopoiesis. The increase of MRP4 together with its specific localization during differentiation toward megakaryocytes supports the concept of platelet specific functions whereas decreased transporter expression in leukocyte differentiation may have implications for chemotherapy. © 2008 Wiley-Liss, Inc.

ATP binding cassette (ABC) drug transporters are expressed in a range of mature peripheral blood cells and fulfill different functions. In lymphocytes, several studies have indicated substantial variability of P-glycoprotein (P-gp, ABCB1) expression1–3 and a relationship between P-gp and IL-2 release.4, 5 In addition to P-gp and BCRP (ABCG2), members of the C-branch of ABC transporters termed MRPs, which mediate the unidirectional transport of amphiphilic anions are expressed in peripheral blood cells.6 The role of MRP1 has been investigated in detail in various populations of human lymphocytes.7 Inhibition of MRP1 abrogated the secretion of interferon-γ, interleukin (IL)-10, IL-2 and tumor necrosis factor (TNF)-α, and reduced T-cell activation, indicating a functional role of MRP1-associated transport activity for T-cell function.8 MRP1 has also been detected in human erythrocytes,9 playing a role in the maintenance of lipid asymmetry10 and the efflux of reduced and oxidized glutathione or glutathione conjugates,11, 12 as well as in platelets.13

The cyclic nucleotides cGMP and cAMP have been identified as substrates for MRP4 (ABCC4) and MRP5 (ABCC5).14–19 Furthermore, they confer resistance to nucleoside-based cytotoxic drugs.20 Both transporters have been detected in human erythrocytes and are suggested to be the predominant mechanism of eliminating cGMP from these cells.14, 21, 22 We recently identified MRP4 in mature platelets.23 Interestingly, expression of this transporter is restricted to platelet dense-granules and plasma membrane. Based on this localization, MRP4 has been suggested to play an essential role in storage and release of mediators, including cGMP, eicosanoids and especially ADP, the major aggregation-stimulating factor of platelets.23 Dense-granules are formed at the early stage of megakaryocyte maturation and arise from multivesicular bodies that originate either by direct targeting from the Golgi complex or by endocytosis from the plasma membrane.24, 25 Whether the specific localization of MRP4 in platelets changes during differentiation process, is currently unknown.

In addition, MRP proteins have been shown to be directly involved in hematopoietic differentiation. Dendritic cells required MRP1 transporter activity for optimal differentiation26 and intracellular levels of cyclic nucleotides, substrates of MRP4 and MRP5, modulated differentiation of monocytes as well as megakaryoblastic and leukemia cells.27–30

Taken together, current data suggests that localization and function of MRP-type transport proteins in peripheral blood cells contribute to specific cellular functions thereby suggesting regulation during differentiation processes in the hematopoietic system. Based on the fact that all mature blood cells arise from the same pluripotent progenitor cells, we used model systems of hematopoietic differentiation and characterized expression, localization and function of MRP4 and MRP5 in CD34+ cells. Moreover, leukemia cell lines differentiated toward megakaryocytes and leukocytes were assessed using real time RT-PCR, confocal laser scanning immunofluorescence microscopy as well as a cellular sensitivity assay. In this study, we demonstrate that MRP4 expression increases significantly during CD34+ cell differentiation toward megakaryocytes and is present in mature bone marrow megakaryocytes. In contrast, expression of MRP4 decreased during differentiation toward mature leukocytes.

Abbreviations

As2O3, arsenic trioxide; GC, granulocytes; G-CSF, granulocyte colony stimulating factor; IL, interleukin; MC, monocytes; MRP multidrug resistance (associated) protein; PMA, phorbol myristate acetate; SCF stem cell factor; TPO thrombopoietin.

Material and methods

Chemicals

RPMI-1640, fetal calf serum (FCS), penicillin/streptomycin solution, L-glutamin solution were purchased from Pan Biotech (Aidenbach, Germany). Recombinant human growth factors flt-3 ligand, granulocyte colony stimulating factor (G-CSF), IL-3, stem cell factor (SCF) and thrombopoietin (TPO) were all obtained from Promokine, Promocell (Heidelberg, Germany). Ethylene diamine tetraacetic acid (EDTA), 1,25 dihydroxy vitamin D3, arsenic trioxide (As2O3), phorbol myristate acetate (PMA), 6-mercaptopurine, and sodium hydroxide were from Sigma (Deisenhofen, Germany). TOTO-3- iodide was purchased from Molecular Probes (Göttingen, Germany).

Antibodies

The anti-MRP4 (SNG)31, 32 and anti-MRP5 (AMF)14 polyclonal antibodies are directed against the intracellular C-termini of both human proteins and were kindly provided by Prof. Keppler, Deutsches Krebsforschungszentrum, Heidelberg. Phytoerythrin-conjugated mouse monoclonal antibodies against CD34, CD41a, CD61 and CD71 were purchased from BD Pharmingen (San Diego, CA), unconjugated IgG1 mouse monoclonal antibodies detecting CD11b, CD14, CD15, CD16 and CD71 were from Ancell, Immunology Research Products (Bayport), and a FITC-conjugated goat anti-mouse IgG1 antibody was obtained from Bethyl Laboratories Inc. (San Diego, CA).

CD34+ progenitor cells, peripheral blood cells and leukemia cell lines

CD34+ cells were isolated from umbilical cord blood samples collected in heparinized tubes at the Department of Gynecology and Obstetrics in Greifswald, Germany. Mobilized peripheral blood cells were gained from leukapheresis samples in the Department of Hematology and Oncology in Greifswald, Germany. A Lymphoprep® (Sigma, Munich, Germany) density gradient was used following cell separation with MIDI MACS® (magnetic activated cell sorting) LS+ separation columns and a CD34+ progenitor isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Monocytes (MCs) and granulocytes (GCs) were isolated from peripheral whole blood samples collected in EDTA-anticoagulated tubes from healthy volunteers. To gain MCs, a Lymphoprep® density gradient was used following cell separation with MIDI MACS® LS+ separation columns and CD14 MicroBeads (Miltenyi Biotec). To isolate GCs, the sediment consisting of GCs and erythrocytes after centrifugation over a Lymphoprep® density gradient was diluted in equivoluminous amounts of RPMI medium and a 0.4% dextrane sulfate solution in RPMI. Sedimentation for 30 min at 37°C was followed by lysis of erythrocytes with pure deionized water for 30 sec.

M-07e is a cytokine-dependent subline of the myeloid leukemic cell line M-07 derived from a patient with megakaryoblastic leukemia.33, 34 An increase of platelet-specific antigens after incubation with TPO (c-mpl ligand) has been described previously suggesting megakaryocytic differentiation.35 HL60 is a promyeloblastic leukemic cell line of a patient with acute promyeloblastic leukemia without translocation. The cells can be differentiated by vitamin D3 into monocytoid36 and by arsenic trioxide (As2O3) into granulocytoid cells.37 U937 is a myelomonocytic cell line established from a patient with histiocytic lymphoma. It can be differentiated to monocyte/macrophage-like cells by various agents including vitamin D3 and the phorbol ester PMA.38–40

HL60 and U937 cell lines were purchased from DSMZ (Braunschweig, Germany), the M-07e cell line was kindly provided by Dr. Joachim Boos (Dept. of Pediatric Oncology, University of Münster, Germany). All cell lines were cultivated in RPMI-1640 medium supplemented with 10% FCS, 1% penicillin/streptomycin and 1% L-glutamine and passaged every 2 to 3 days. For the M-07e cell line the medium was additionally supplemented with 10 ng/ml IL-3.

CD34+ and leukemia cell differentiation cultures

To differentiate CD34+ progenitors toward the megakaryocytic/thrombocytic lineage cells were isolated from cord blood samples and cultured for 14 days in serum-free X-vivo-15® medium (Cambrex, Nottingham, UK) containing 20% FCS, 1% penicillin/streptomycin and 100 nM TPO. For the myelomonocytic lineage, cells were isolated from leukapheresis samples or cord blood and cultured in 6-well plates for 14 days in X-vivo-15® serum-free medium supplemented with 20% FCS, 1% penicillin/streptomycin, 100 nM G-CSF, 20 ng/ml SCF and 30 ng/ml IL-3. Medium was changed every 3 days. Control cultures were cultivated in X-vivo-15®medium supplemented with 20% FCS, 1% penicillin/streptomycin and 20 ng/ml SCF.

M-07e cells were incubated for 3 days with 50 ng/ml TPO to provoke megakaryocytic development. HL60 cells were incubated for 3 days with 1 μM vitamin D3 for differentiation toward monocytes and for 3 days with 0.5 μM As2O3 for differentiation toward granulocytes. U937 cells were incubated for 3 days with 1 μMvitamin D3 and for 3 days with 50 nM PMA. After differentiation cells were harvested and counted with a Neubauer hemacytometer.

Cell surface labeling by flow cytometry

The state of differentiation was measured by direct or indirect flow cytometry in a FACScalibur® (Becton Dickinson, Heidelberg, Germany). Adherent cells were detached from the surface by incubation with 1 mM EDTA for 10 min at 37°C. Harvested adherent and suspension cells were then either directly labeled using phytoerythrin-conjugated mouse monoclonal antibodies against CD34, CD41a, CD61 and CD71 or were indirectly labeled using unconjugated IgG1 mouse monoclonal antibodies detecting CD11b, CD14, CD15, CD16 and CD71. As a secondary antibody for the unlabeled IgG1-antibodies a FITC-conjugated goat anti-mouse IgG1 antibody was used. Data analysis was performed using the CellQuest Software (Becton Dickinson, Heidelberg, Germany).

Quantitative transporter detection by real time PCR

The differentiated cells were harvested and RNA was isolated using peqGold RNA Pure® (peqlab, Biotechnologie GmbH, Erlangen). Afterwards RNA was transcribed using reverse transcriptase (Applied Biosystems, Weiterstadt, Germany). The PCR reactions were performed in triplicate using the ABI Prism 7700 Sequence detector system (Applied Biosystems, Warrington, UK). Real-time PCR for MRP5 was performed as described previously,41 for ABCC4 the following primer and probes were used: 5′-GTCTTCATTTTCCTTATTCTCCTAAACAC (forward), 5′-CCATTTACAGTGACATTTAGCATACTTTGT (reverse) and 5′- (FAM)-CCAGTATGAAAGCCACCAATCTTGAAGCA (probe). The gene expressions of MRP4 and MRP5 were normalized to 18S assessed with standard primers and probes (Applied Biosystems, Warrington, UK). Each experiment was repeated at least 3 times.

Immunoblot analysis

Membrane fractions that were isolated by centrifugation at 100,000 × g were loaded onto a 7.5% sodium-dodecylsulfate polyacrylamide gel after incubation with sample buffer at 95°C for 10 min. Immunoblotting was performed using a tank blotting system (Biorad, Hercules, CA) and an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Freiburg, Germany). The primary antibodies against MRP4 and MRP5 SNG and AMF were diluted 1:1000 in TBS containing 0.05% Tween and 1% bovine serum albumin. The secondary antibody was goat-anti-rabbit HRP-conjugated (Biorad, Hercules, CA) and diluted 1:2000 in TBS containing 0.05% Tween and 1% bovine serum albumin.

Detection of transporter localization by immunofluorescence microscopy

Cells cultivated on slides or air dried bone marrow aspirates were fixed for ten minutes in ethanol at −20°C. Then they were washed in PBS and incubated for five minutes in 0.1% TRITON-X100 (Serva, Heidelberg, Germany) for permeabilization. Slides were blocked with 5% FCS, washed again and incubated with the primary antibody overnight (MRP4 and MRP5 1:100 in dilution, LAMP-2 1:10 in dilution). After subsequent washing steps and blocking with 5% FCS for 30 min, slides were incubated with Alexa Fluor® 488 (goat anti-rabbit) for MRP4 and MRP5 or Alexa Fluor® 568 (goat anti-mouse) (Molecular Probes/MoBiTec, Göttingen, Germany) for LAMP-2 for 1 hr. After following washings, cells were incubated with TOTO®-3-iodide in a 1:2000 dilution with DAKO® Fluorescent Mounting Medium (DakoCytomation, Carpinteria, CA) for nuclei-staining. Fluorescence micrographs were taken with confocal laser scanning microscope (Chromaphor Analysen Technik, Duisburg, Germany). Samples were observed with a Nikon inverted microscope and a 100× oil-immersion objective. A CCD camera and VoxCell scan software from VisiTech International (Sunderland, UK) were used for analysis.

Cellular viability assay

To investigate cytotoxicity exhibited by cytostatic drugs the alamar blue™ assay (Biosource, Camarillo, CA) was used. This assay indirectly determines the cellular vitality by measuring the mitochondrial metabolic activity of viable cells. Proliferating cells are able to reduce the alamar blue™ into a red, fluorescent dye. This reduction can be quantified photometrically at 560 and 595 nm by a microplate reader (Victor2 1420 Multilabel Counter, Wallac, Boston, MA). HL60 and U937 cells were differentiated as described previously. Harvested cells were seeded in a 96-well plate at 75,000 cells per well in triplicate. In case of the M-07e cell line 50,000 cells per well were used. Then either 100 μM 6-mercaptopurine or, as a control, respective volumes of its 0.1 M sodium hydroxide solution were added to each well. After 3 days 10% alamar blue was added and incubated for about 3.5 hr at 37°C.

Statistical analysis

Statistical significance between 2 treatment conditions was assessed by Student's two-tailed, paired t-test, Mann Whitney U or Wilcoxon signed rank tests where applicable and as indicated. Multiple testing of more than 2 groups was performed by One-way repeated measures ANOVA with Bonferroni's multiple comparison test. p less than 0.05 was considered statistically significant. The software package Graph Pad Prism Version 3.02 (GraphPad Software Inc., San Diego, CA) was used.

Results

Expression and localization of MRP4 and MRP5 in CD34+ progenitor cells differentiated toward megakaryocytes

For the megakaryocytic/thrombocytic lineage CD34+ progenitor cells were isolated from human cord blood and cultivated in the presence of 100 nM TPO as differentiating factor for 14 days. During this time morphological changes occurred in that cellular volumes increased and the cells tended to grow in clusters rather than as single cell suspension. For further proof of the megakaryocytic differentiation an antibody detecting CD41 (glycoprotein IIb of the glycoprotein IIbIIIa complex), which is early expressed in megakaryopoiesis, was used. Under megakaryocytic differentiation conditions, we found cells positive for CD41a in a range from 23% to 36% in the different preparations with a median of 29% (Fig. 1a). Antibodies against CD34 and CD16 (FcγIII-receptor), a marker for the myelomonocytic lineage, were used to assess heterogeneous differentiation, and a maximum of 2% positive cells was found. Cultivation of cells for longer than 14 days did not result in a significantly improved differentiation (data not shown).

Figure 1.

Expression of MRP4 in CD34+ cells differentiated toward megakaryocytes. (a) State of differentiation was determined by labeling CD34+ cells differentiated toward megakaryocytes for two weeks in the presence of 100 nM thrombopoietin for CD41 (black curve; transparent curve represents IgG1 isotype control). (b) mRNA expression of MRP4 in freshly isolated CD34+ progenitor cells (CD34+, n = 5) and progenitors differentiated toward megakaryocytes in the presence of 100 nM thrombopoietin for 14 days (plus TPO, n = 7) depicted as copy numbers per ng total RNA (*p < 0.05, one-sided Mann-Whitney U test).

The mRNA levels of MRP4 and MRP5 were measured using real time-PCR and the cellular localization of MRP4 was investigated using confocal laser scanning microscopy. Within megakaryocytic preparations MRP4 mRNA expression varied substantially from 2.7 × 103 to 2.4 × 105 copies per nanograms of total RNA with a median of 5.8 × 104 copies of MRP4 mRNA per nanograms of total RNA (n = 7). A comparison with undifferentiated CD34+ progenitor cells, where a median MRP4 mRNA expression of 7.9 × 103 copies per nanograms of total RNA (range 530 to 2.4 × 104, n = 5) was found, revealed that megakaryocytic differentiation is accompanied by a statistically significant increase in MRP4 mRNA expression (p < 0.05; Fig. 1b). MRP5 mRNA was expressed to a lower extent as compared with MRP4, and MRP5 mRNA expression was not significantly affected by differentiation (data not shown). Immunolocalization analyses of MRP4 revealed that in addition to membrane staining, which is characteristic for progenitor cells, a signal was also found intracellularly after differentiation toward the megakaryocytic lineage (Figs. 2a and 2b). For identification of the intracellular structures, double-staining preparations were performed with LAMP-2, a marker for dense granules and lysosomes of mature platelets. This staining revealed a partial colocalization of LAMP-2 and MRP4 after megakaryocytic differentiation (Fig. 2b). Finally, MRP4 expression was investigated in human bone marrow aspirates and could be localized in megacaryocytes in plasma membrane and in granular structures within the cytosol (Figs. 2c and 2d). Cellular localization was also investigated for MRP5, which was found to a lesser extent in the plasma membrane of CD34+ and megakaryocytic cells (data not shown).

Figure 2.

Localization of MRP4 in CD34+ cells differentiated toward megakaryocytes and bone marrow megakaryocytes. For immunolocalization, freshly isolated (a), in the presence of 100 nM thrombopoietin differentiated CD34+ cells (b) and bone marrow aspirates (c,d) were incubated with antibodies against MRP4 (ad) or LAMP-2 (b,d). Bars represent 20 μm (a) or 10 μm (bd). Arrow heads: megakaryocytes; arrows: erythrocytes.

Expression and localization of MRP4 and MRP5 in CD34+ progenitor cells differentiated toward myelomonocytes

CD34+ cells were isolated from leukapheresis and cord blood samples and cultivated in the presence of SCF, IL-3 and G-CSF for myelomonocytic differentiation. After 14 days of cultivation 2 different cell populations developed with one population consisting of adherent cells and the other one of cells in suspension. To further characterize cellular differentiation, cell populations were investigated for the expression of the lineage specific cell surface proteins CD11b (complement receptor 3), CD15 (LewisX) and CD16 (Fcγ-III-receptor) by cell surface labeling using specific antibodies and flow cytometry. Figure 3 depicts results of a cell surface labeling experiment of adherent cells after myelomonocytic differentiation, which is representative for at least 3 independent differentiation experiments, and reveals 53.3% of the cells positive for CD11b (Fig. 3a) and 23.8% positive for CD16 (Fig. 3b), whereas only 7.6% of the cells were found CD15 positive and less than 2% CD34 positive. In the suspension fraction 44.6% of cells were found positive for CD11b, 9.6% for CD16, 5.6% for CD15 and 0.5% for CD34. Expression of CD11b and CD16 as markers for the myelomonocytic lineage suggests that the progenitor cells were successfully differentiated towards monocytes and neutrophiles and that differentiation was advanced in the adherent cell population.

Figure 3.

Expression of MRP4 in CD34+ cells differentiated toward monocytes and peripheral mononuclear subpopulations. CD34+ cells were labeled using antibodies against (a) CD11b, (b) CD15 (gray curve) and CD16 (black curve; transparent curve represents IgG1 isotype control). Results are representative for at least 3 independent differentiation experiments. (c) MRP4 mRNA expression of freshly isolated and differentiated CD34+ cells; quantities were normalized to 18S rRNA and values represent independent experiments with their median (*p < 0.05, one-sided Wilcoxon signed rank test), lines indicate corresponding values of respective experiments. (d) MRP4 mRNA expression in CD34+ cells, monocytes (MC) as well as granulocytes (GC); quantities were normalized to 18S rRNA and values represent mean ± SD (n = 5; *p < 0.05; one-way ANOVA).

When expression of MRP4 mRNA was investigated in the adherent fraction of independently differentiated CD34+ progenitor populations, each executed experiment revealed a reduction in MRP4 mRNA expression by cells isolated from leukapheresis samples or cord blood and differentiated toward the myelomonocytic lineage as compared with control CD34+ progenitor cells of the respective preparation (Fig. 3c, n = 5). Although each preparation varied in the basal expression of MRP4 mRNA, the reduction in the median expression was significant (5.2 × 10−3 relative MRP4 mRNA/18S rRNA expression for CD34+ cells vs. 3.5 × 10−3 for CD34+ in the presence of G-CSF, p < 0.05). As seen for megakaryocytic differentiation, MRP5 mRNA expression was not affected under differentiation conditions (data not shown). In addition, transporter expression was investigated in CD34+ progenitor cells freshly isolated from different samples of human cord blood (n = 5) and monocytes and granulocytes isolated from healthy volunteers (n = 5). Here, MRP4 was found to be significantly lower in monocytes than in granulocytes (MRP4 mRNA/18S rRNA ratios: 8.1 ± 5.4 × 10−5 vs 2.8 ± 1.6 × 10−4, respectively; p < 0.05) and both were presenting significantly lower amounts of this transporter compared with the cord blood-derived CD34+ progenitors (1.2 ± 0.7 × 10−3; p < 0.05). Relative MRP4 mRNA expression of these cells normalized to 18S rRNA is shown in Figure 3d. Again, MRP5 mRNA expression was not different among the various cell populations.

Expression and function of MRP4 and MRP5 in the megakaryoblastic cell line M-07e differentiated with TPO

When M-07e cells are incubated with TPO, they undergo differentiation toward megakaryocytes.35 This is demonstrated in an experiment representative for at least 3 independent differentiation procedures by a shift in the geometrical mean of fluorescence intensity when cells were labeled for CD61 (glycoprotein IIIa) from 2.9 to 8.6 (negative control: 1.9; Fig. 4a). In contrast, intensity of CD71 labeling (transferrin-receptor) was reduced from 66.1 to 11.8 (negative control: 1.8) in the presence of 50 ng/ml TPO (Fig. 4b).

Figure 4.

Expression and function of MRP4 in the M-07e cell line differentiated in the presence of thrombopoietin. M-07e cells were stained with antibodies against (a) CD61 and (b) CD71 after cultivation in growth medium (gray curve) or in the presence of 50 ng/ml thrombopoietin for three days (black curve). For negative control, cells were incubated with normal mouse IgG1 instead of a primary antibody (transparent curve) or assessed without any staining (broken transparent curve). Results are representative for at least 3 independent differentiation experiments. (c) MRP4 mRNA expression normalized to 18S rRNA (values represent mean ± SD, n = 3; p < 0.05, Student's t-test). (d) Immunoblotting and relative optical density of MRP4 expression (values represent mean ± min/max, n = 2). (e) Differentiated cells were incubated with either 100 μM 6-mercaptopurine (black column) or 0.1 mM sodium hydroxide solution (white column) and cell viability was assessed as described in Material and Methods. Values represent mean ± SD (n = 3; p < 0.01; Student's t-test).

Expression of MRP4 mRNA was found to be significantly increased in TPO-treated cells. In detail, transporter expression of M-07e cells shifted from MRP4 mRNA/18S rRNA ratios of 5.4 ± 3.8 × 10−4 to 2.7 ± 0.9 × 10−3 in cells cultivated in the presence of TPO (p < 0.05; n = 3; Fig. 4c). MRP5 mRNA was expressed at a lower level (MRP5 mRNA/18S rRNA ratio 9.4 ± 5.5 × 10−5) and was not significantly altered upon treatment with TPO (data not shown). On protein level incubation of the megakaryocytic leukemic cell line with TPO resulted in a 5.5-fold increase of MRP4 expression (p < 0.05; Fig. 4d). Moreover, TPO-treated M-07e cells were significantly less sensitive to 100 μM 6-mercaptopurine than untreated cells. In fact, a shift in cellular viability from 49 ± 3% in native M-07e cells to 91 ± 5% in differentiated cells was observed (p < 0.05; Fig. 4e).

Expression and function of MRP4 and MRP5 in the promyeloblastic cell line HL60 differentiated with vitamin D3 and arsenic trioxide

HL60 cells differentiate to monocytoid or granulocytoid cells in the presence of vitamin D3 or arsenic trioxide, respectively.36, 37 When cells were incubated with 1 μM vitamin D3 and labeled for CD11b a shift of fluorescence intensity occurred from 4.7 to 17.8 (negative control 1.8; Fig. 5a). In the case of treatment with 0.5 μM arsenic trioxide fluorescence intensity shifted from 2.3 to 4.9 (negative control 1.4; Fig. 5b). Results are representative for at least 3 independent differentiation experiments.

Figure 5.

Expression and function of MRP4 in the HL60 cell line differentiated in the presence of vitamin D3 and arsenic trioxide. HL60 cells were cultivated in growth medium (gray curve) or in the presence of the differentiating agents (black curve) vitamin D3 (a) or arsenic trioxide (b), then they were stained with antibodies against CD11b. For negative control cells were incubated with normal mouse IgG1 instead of a specific primary antibody (transparent curve) or assessed without staining (broken transparent curve). Results are representative for at least 3 independent differentiation experiments. (c) mRNA expression of MRP4 normalized to 18S rRNA (values represent mean ± SD, n = 3, ***p < 0.01; one-way ANOVA). (d) Differentiated cells were incubated with either 100 μM 6-mercaptopurine (black column) or 0.1 mM sodium hydroxide solution (white column) and cell viability was assessed. Values represent mean ± SD (n = 3; ***p < 0.001, **p < 0.01; one-way ANOVA).

MRP4 mRNA levels significantly decreased under differentiation conditions (p < 0.05). In more detail, untreated HL60 cells showed a MRP4 mRNA/18S rRNA ratio of 2.0 ± 0.5 × 10−3, which was reduced to 0.26 ± 0.21 × 10−3 with vitamin D3 and to 0.26 ± 0.37 × 10−3 with arsenic trioxide (Fig. 5c). MRP5 mRNA was expressed at a lower level (MRP5 mRNA/18S rRNA ratio 3.1 ± 1.1 × 10−5) and showed no significant change under differentiating conditions (data not shown). Furthermore, a change in cell sensitivity toward 6-mercaptopurine (6-MP) was observed after incubation with differentiation agents. 67 ± 4% of the undifferentiated HL60 cells were viable in the presence of 100 μM 6-MP, but only 41 ± 1% of the vitamin D3 treated cells and 47 ± 5% of the arsenic trioxide treated cells (p < 0.01; Fig. 5d).

Expression and function of MRP4 and MRP5 in the myelomonocytic cell line U937 differentiated with vitamin D3 and PMA

U937 cells differentiate toward monocyte/macrophage-like cells in the presence of vitamin D3 or PMA, respectively.38–40 When cells were incubated with 1 μM vitamin D3 and labeled for CD11b a shift of fluorescence intensity occurred from 6.4 to 26.1(negative control 1.3; Fig. 6a). In the case of treatment with 50 nM PMA fluorescence intensity shifted from 6.5 to 18.1 (negative control 1.3; Fig. 6b). Treatment with vitamin D3 and labeling for CD14 caused a change of fluorescence intensity from 1.6 to 4.9 (negative control 1.3; Fig. 6c). PMA-treated cells labeled for CD14 showed an increase in fluorescence intensity from 2.8 to 5.8 (negative control 1.3; Fig. 6d). These results are representative for at least 3 independent differentiation experiments.

Figure 6.

Expression and function of MRP4 in the U937 cell line differentiated in the presence of vitamin D3 and PMA. U937 cells were cultivated in growth medium (gray curve) or in the presence of the differentiating agents (black curve) vitamin D3 (a,c) or PMA (b,d) and stained with antibodies against CD11b (a,b) and CD14 (c,d). For negative control cells were incubated with normal mouse IgG1 instead of a specific primary antibody (transparent curve) or assessed without staining (broken transparent curve). Cells were cultivated in the presence of 1 μM vitamin D3 (a, c) or 50 nM PMA (b, d). Results are representative for at least 3 independent differentiation experiments. (e) mRNA expression of MRP4, quantities were normalized to 18S rRNA (values represent mean ± SD, n = 3, *p < 0.05; one-way ANOVA). (f) For viability assays differentiated cells were incubated with either 100 μM 6-mercaptopurine (black columns) or 0.1 mM sodium hydroxide solution (white columns) and cell viability was assessed. Values represent mean ± SD (n = 3; **p < 0.01; *p < 0.05; one-way ANOVA).

Under both differentiation conditions MRP4 mRNA levels were found to be significantly reduced as compared with undifferentiated U937 cells (p < 0.05; Fig. 6e). More precisely, native U937 cells expressed MRP4 at a MRP4 mRNA/18S rRNA ratio of 8.5 ± 0.7 × 10−5, which was decreased in the presence of vitamin D3 to 3.6 ± 1.3 × 10−5 and of PMA to 2.6 ± 1.5 × 10−5. MRP5 mRNA expression was not altered by differentiation and remained at 3.7 ± 0.9 × 10−5. Exposure to 100 μM 6-mercapopurine resulted in a significantly decreased cellular viability of the differentiated cells (Fig. 6f). 74 ± 3% of the undifferentiated cells were insensitive to the cytostatic drug, whereas only 59 ± 3% of vitamin D3-treated cells (p < 0.01) and 65 ± 4% of PMA-treated cells (p < 0.05) remained viable.

To exclude a direct transporter-modulating effect induced by PMA, we incubated HepG2 cells, a hepatoblastoma cell line with stable MRP4 expression, with PMA but found no change in transporter expression (data not shown).

Discussion

ABC-type transport proteins have been identified in almost all types of mature peripheral blood cells.6 The critical role played by platelets in hemostasis and thrombosis is related to their function as exocytotic cells that store effector molecules such as ADP in special compartments and release them at the site of vascular injury.42 The high abundance of MRP4 in human platelets and in vitro evidence for its involvement in transport of transmitter molecules including ADP23 indicates an important role of this protein in platelets. Furthermore, cyclic nucleotides, substrates of MRP4 and MRP5,20 have been shown to modulate differentiation of megakaryoblastic and leukemia cells.27–30 Regulation of these proteins during hematopoietic differentiation has not been studied.

In search of hematopoietic stem cells various sources for cells expressing the cell surface marker CD34 have been used, including bone marrow, fetal liver, peripheral blood and umbilical cord blood.43–45 Although in peripheral blood CD34+ cells occur in less than 0.01% of the cells, cord blood is highly enriched in progenitor cells.44 These progenitors have been found to be slightly more mature (have less self-renewal activity) than their adult marrow counterparts, but have a higher short-term proliferative capacity.46 Using different cytokine combinations, it is possible to differentiate these cells towards megakaryocytic progenitor cells or, alternatively, toward myelomonocytic cells.46–48 In this study, we used TPO for differentiation toward the megakaryocytic lineage and SCF, IL-3 and G-CSF for differentiation toward the monocytic lineage. FACS staining using lineage specific surface markers revealed the successful differentiation into the megakaryocytic (CD 41+ cells) and into the myelomonocytic phenotype (CD11b/CD16+ cells).

To further explore the effect of differentiation on expression of MRP4 and MRP5, additional cell model systems were used. These included the megakaryoblastic cell line M-07e, the promyelocytic leukemia HL-60 cells and the myelomonocytic cell line U937. These are pluripotent cells capable of undergoing differentiation along lineage specific pathways in response to a variety of agents.35–40 M-07e cells were differentiated toward megakaryocytes with TPO, proven by CD61 expression and reduction of CD71. HL60 cells were differentiated towards monocytoid or granulocytoid cells in the presence of vitamin D3 or arsenic trioxide, respectively, and U937 cells differentiate toward monocyte/macrophage-like cells in the presence of vitamin D3 or PMA. Both were indicated by CD11b and CD14 expression, marker proteins for myelomonocytic differentiation.

We could demonstrate in this study that MRP4 expression increases significantly during the CD34+ cell differentiation toward megakaryocytes. The same effect was observed in M-07e cells and MRP4 was also detected in mature megakaryocytes in a bone marrow biopsy. The transition of megakaryocytes to released platelets includes a systematic series of events such as nuclear endomitosis, organelle synthesis, cytoplasmic maturation and expansion, formation of proplatelets and finally platelet amplification and release.42 Their hematopoietic function is sustained by a variety of storage organelles that release their contents to the extracellular surroundings. Dense granules for example contain the vasoconstrictive agent serotonin, the nonmetabolic pool of ATP and ADP as well as calcium and magnesium ions.42 MRP4, which is highly expressed in these granules, was suggested to facilitate storage by actively influxing one of these compounds, ADP, into the granules.23 Dense granules are formed at the early stage of megakaryocyte maturation within multivesicular bodies.24 These granules originate from a dual mechanism: direct targeting from the Golgi complex as well as endocytosis from the plasma membrane.25 Our findings that (a) MRP4 is already expressed in the plasma membrane of CD34+ progenitor cells, (b) MRP4 expression increases during megakaryocytic differentiation, (c) MRP4 is located in intracellular structures and partly colocalized with LAMP-2, a marker for dense granules, in CD34+ cells differentiated to megakaryocytes and (d) MRP4 is expressed in bone marrow megakaryocytes supports the notion that MRP4 is integrated into dense granular structures and probably fulfills important functions in mediator storage at very early stages of megakaryocyte maturation.

In contrast, expression of MRP4 was found to decrease during differentiation processes to mature leukocytes. The presence of transport proteins such as MDR1-P-glycoprotein or breast cancer resistance protein (BCRP, ABCG2) in primitive CD34+ hematopoietic progenitor cells has been reported49, 50 and related to the side population phenotype in hematopoietic stem cells.51 This is of importance since leukemic stem cells are thought to overlap with hematopoietic stem cell side populations, hence carry the multidrug resistance phenotype and give rise to disease relapses when treated with chemotherapy. Transport proteins have been identified in blast cells from acute myeloid leukemia as well. Unequivocal data confirm MDR1 as an independent adverse prognostic factor for response and survival in de novo AML.52–54 Recent studies suggest that MDR1 and BCRP are coexpressed in blasts of AML patients and that this represents a robust resistant AML phenotype.55–57 In addition, MRP1 emerged as a relevant player in predicting treatment outcome in de novo AML patients when its efflux activity was taken into account, and the predictive value was even stronger, if activities of MRP1 and MDR1 were combined.53, 54 Among the other members of the ABCC drug transporter family, MRP2, MRP3, MRP4 and MRP5 are expressed in AML blast cells and high expression of MRP3 was significantly correlated with a poor prognosis in childhood.58 In our own investigations, we found MRP4 and MRP5 mRNA expressed in blast cells of adult AML patients with considerable variability. Interestingly, when the different AML subtypes were compared, we found that the least differentiated subtypes expressed the highest levels of MRP4 and MRP5.59 This supports our finding here that differentiating conditions lead to a downregulation of these drug transporters. Moreover, both proteins confer resistance to cytotoxic thiopurine nucleotides and their expression may therefore affect sensibility toward these antineoplastic drugs in the way that it increases with de-differentiation. Chemotherapy with agents inducing terminal differentiation has thus far been exerted only on patients suffering from acute promyelocytic leukemia. Recently, low doses of 6-mercatopurine and methotrexate were added to the treatment regimen for maintenance therapy, based on data suggesting improved disease-free survival.60 A combination of conventional with differentiating agents was also used in a small study with AML patients not eligible for intensive chemotherapy because of age, poor clinical conditions or treatment refusal. Treatment consisted of retinoic acid, vitamin D3, as well as low doses of 6-thioguanine and cytarabine and responsive patients showed a clear survival advantage over nonresponders and a historical series of patients treated with supportive care only.61

In conclusion, the differentiation of hematopoietic stem cells affects the expression of membrane proteins transporting endogenous signaling molecules as well as drugs. MRP4 expression is increased significantly with the CD34+ cell differentiation to megakaryocytes in accordance with the development of cell specific functions such as the storage and release of mediators comprising cGMP, eicosanoids and ADP. In the in vitro differentiation of leukemia cell lines, MRP4 expression increased with dedifferentiation. MRP4 confers resistance to cytotoxic thiopurine nucleotides and its expression may therefore affect chemotherapy.

Acknowledgements

The anti-MRP4 and MRP5 antisera were provided by the Deutsches Krebsforschungszentrum (DKFZ), Division of Tumor Biochemistry headed by Prof. D. Keppler.

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