STEM CELLS AND DEVELOPMENT 13:183–191 (2004) © Mary Ann Liebert, Inc.
Cutting Edge Communication Flt3/Flk-2 Ligand in Combination with Thrombopoietin Decreases Apoptosis in Megakaryocyte Development OLAFUR EYSTEINN SIGURJONSSON,1,5 KRISTBJORN ORRI GUDMUNDSSON,1 VILHELMINA HARALDSDOTTIR,2 THORUNN RAFNAR,3 BJARNI A. AGNARSSON,4 and SVEINN GUDMUNDSSON1
ABSTRACT The growth factors thrombopoietin (TPO) and Flt3/Flk-2-ligand (FL), either independently or in combination, modulate megakaryocyte development. Our results show that bone marrow CD341 cells cultured with TPO and FL differentiate at a slower rate into CD411 cells and are delayed in apoptosis at the later stages of the cultures compared to cells cultured with TPO alone. Our data also show that FL in synergy with TPO may inhibit apoptosis in megakaryocyte development by up-regulating bcl-2 and inducing conformational changes of p53, in MK progenitors. FL in combination with TPO slows down maturation and consequently delays apoptosis of MK progenitor cells. INTRODUCTION
M
EGAKARYOCYTES , PRECURSORS OF BLOOD PLATELETS ,
differentiate from hematopoietic stem cells (HSC) in the bone marrow (BM) (1). Following myeloablative chemotherapy, this process is often critically impaired, rendering patients thrombocytopenic for several weeks (2–4). HSC transplantation to reconstitute hematopoiesis may require additional platelet transfusions, with a potential increase in the risk of infections and at considerable costs (5). This has motivated the supplementation of conventional autografts with ex vivo-expanded megakaryocyte (MK)-rich products to enhance in vivo platelet production and shorten the period of thrombocytopenia (3,6,7). A number of cytokines, either independently or in combination, modulate megakaryocytopoiesis. These include thrombopoietin (TPO) (8–10) and Flt3/Flk-2-ligand (FL), which is known to be a potent co-stimulator for the expansion of HSC in
synergy with TPO (11,12). Alone, TPO has a limited proliferative effect on human CD341 cells in culture, and the addition of early-acting cytokines, e.g., stem cell factor (SCF) and FL, is required to increase total cell proliferation and MK expansion (13,14). It has been demonstrated that TPO can prevent apoptosis in MKs, MK progenitor cells, and HSC both in vitro and in vivo (15,16). It has also been shown that immature MKs undergo apoptosis in the absence of TPO, whereas mature MKs do not (17). Furthermore, TPO suppresses apoptosis and acts as a survival factor in the megakaryoblastic cell line MO7e (18,19). The mechanisms regulating apoptosis in HSC are not fully elucidated, but members of the Bcl-2 family of apoptosis regulators have been shown to have an important effect on the viability of hematopoietic progenitor cells (20,21). In megakaryocytopoiesis, Bcl-xL is up-regulated during MK differentiation of CD341 HSC, but is not detectable in senescent MKs (22). Bcl-xL has also
1 The
Blood Bank, Department of Basic Research, Landspitali-University Hospital, 121 Reykjavik, Iceland. of Hematology and Oncology, Landspitali-University Hospital, 121 Reykjavik, Iceland. 3 Iceland Genomics Corporation, 105 Reykjavik, Iceland. 4 Department of Pathology, Landspitali-University Hospital, 121 Reykjavik, Iceland. 5 Present address: Immunologisk Institutt, Rikshospitalet, 0027 Oslo, Norway. 2 Department
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SIGURJONSSON ET AL. been shown to be strongly expressed in different kinds of cell lines with MK properties, whereas Bcl-2 is relatively weakly expressed (23). On the other hand Bcl-2 was shown to be expressed more extensively in mature myeloid cell lines (23). p53 is a conformationally flexible transcription factor that can function either as a proapoptotic protein or a survival protein, depending on its protein conformation (24). TPO can induce conformational changes in the p53 protein, which alters the protein from its pro-apoptotic, anti-proliferative phenotype (suppressor) to a conformation that has a diminished ability to arrest cell cycle progression and induces apoptosis (promoter) (25). Using our serum-free MK culture model (26), we investigated the effect of FL on apoptosis and expression of p53 and Bcl-2 in TPO-induced megakaryocyte development.
MATERIALS AND METHODS Bone marrow Femoral BM samples were collected from patients undergoing hip prosthesis surgery (total hip arthroplasty) in the normal process of the operation. The BM was suspended in isotonic NaCl solution (Icelandic Pharmaceuticals Ltd, Reykjavík, Iceland) containing 1000 U ml21 EDTA (Merck, Darmstadt, Germany). All samples were anonymous.
Isolation of bone marrow mononuclear cells and CD341 cells Low-density mononuclear cells (MNC) and CD341 cells were isolated as previously described (26). In brief, MNC were isolated by centrifugation with HISTOPAQUE-1077 solution (density, 1.077g ml21) (Sigma Chemical Company, St. Louis, MO) and washed in RPMI-1640 medium (Life Technologies Ltd, Paisley, UK). Adherent cells were discarded after 12 h of incubation at 37°C and 5% CO2 in RPMI-1640 containing 10% fetal bovine serum (FBS) (Life Technologies Ltd) supplemented with 100 U ml21 penicillin (Life Technologies Ltd) and 100 U ml21 streptomycin (Life Technologies Ltd). The nonadherent MNC were used for further isolation. CD341 cells were isolated using the Dynal CD34 Progenitor Cell Selection System (Dynal, Oslo, Norway) according to the manufacturer’s instructions. The CD341 cells were collected and purity was assessed by flow cytometry after staining with CD34 antibody (HPCA-2-PE, Becton Dickinson, San Diego, CA), using a Becton Dickinson FACSCalibur flow cytometer equipped with an argon ion laser. A minimum of 10,000 events were acquired in list mode and analyzed with CellQuest software 3.1f. (Becton Dickinson).
Cell cultures Cells were cultured as previously described (26). Briefly, CD341 cells (5 3 104 ml21 ) were cultured in 24-well plates (Becton Dickinson, San Diego, CA) for a maximum of 21 days at 37°C and 5% CO2 in Stemspan serum-free medium (Stem Cell Inc, Vancouver, BC, Canada) supplemented with 100 U ml21 penicillin and 100 U ml21 streptomycin. Cultures were initiated with TPO (50 ng ml21) (R&D Systems Inc, Minneapolis, MN) and/or FL (50 ng ml21) (R&D). Every 5 days, one-half of the culture volume was removed and replaced with fresh medium and growth factors, bringing the total volume to 1 ml.
Growth factor withdrawal cell cultures CD341 cells (5 3 104 ml21 ) were cultured in 24-well plates for up to 21 days at 37°C and 5% CO2 in Stemspan serum-free medium, supplemented with 100 U ml21 penicillin and 100 U ml21 streptomycin, TPO (50 ng ml21 ), and/or FL (50 ng ml21). On day 5 or 7, TPO and/or FL were removed by washing the cells in Stemspan serum-free medium. The cells were resuspended in fresh medium containing the appropriate growth factors, bringing the total volume to 1 ml. Withdrawal at days 5–7 was selected because the number of MK progenitor cells in the cultures was highest at that time and polyploidy was in its initial stages (26).
Phenotypic and ploidy analysis Differentiation of CD341 cells toward the MK lineage was monitored by measuring the expression of CD41a (HIP8-FITC, Pharmingen, San Diego, CA) and ploidy during the culture period. To monitor differentiation to monocytes, cells were stained with antibody against CD14 (MOP9-PE, Becton Dickinson). Cells were harvested and washed twice in a phosphate-buffered saline solution (PBS). The cells were then incubated with 10 ml of CD41a FITC and subsequently fixed in 70% ethanol and stored at 220°C for 3 h. The ethanol was discarded by centrifugation. To stain the DNA, the cells were incubated for 15 min at 37°C with propidium iodide (PI) (1 mg ml21 ; Sigma) in PBS containing RNase A (200 mg ml21 ; Sigma) and 0.1% Tween 20 (Merck). A minimum of 10,000 events were acquired in list mode and analyzed with CellQuest software 3.1f.
Apoptosis analysis Apoptosis was measured using the Annexin V-FITC Apoptosis Detection Kit I (Pharmingen) according to the manufacturer’s instructions. In brief, cells were suspended in binding buffer (Pharmingen) and incubated with Annexin-V FITC (Pharmingen) and PI for 15 min
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TPO AND FL MODULATION OF MK PROGENITOR CELLS at room temperature. The cells were then resuspended in 200 ml of binding buffer and analyzed on a flow cytometer within 1 h. A minimum of 10,000 events were acquired in list mode and analyzed with the CellQuest software 3.1f. Annexin-V-/PI- cells were considered viable, Annexin-V1/PI- were considered apoptotic and Annexin-V1/PI1 were considered dead. Unlabeled cells in binding buffer were used as negative controls.
as described above. On days 1, 5, 10, 15, and 20, 4 mg ml21 of agonistic anti-Fas antibody (CH11, mouse IgM; Beckman Coulter, Fullerton, CA) with or without 4 mg ml21 blocking anti-Fas antibody (ZB4; BeckmanCoulter) or 4 mg ml21 purified mouse IgG1, kappa (MOPC21; Pharmingen) were added to the cultures for 12 h. The cells were harvested and analyzed for apoptosis by flow cytometry as described above.
Apoptosis and CD41 expression
Statistical analysis
In some experiments, apoptosis and CD41a expression were measured simultaneously. Cells were suspended in binding buffer and incubated with Annexin-V FITC, VIA-PROBE (7-Amino-Actinomycin-D; Pharmingen) and CD41a PE (HIP8, PE; Pharmingen) or isotypematched control mouse IgG1 (X40, PE, Becton Dickinson). The cells were resuspended in binding buffer and analyzed on a flow cytometer within 1 h. Annexin-VVIA-PR OBE-/Annexin-V1VIA-PROB E-c ells (viable/apoptotic cells) were gated and CD41a and Annexin-V expression analyzed within that population. A minimum of 10,000 events were acquired in list mode and analyzed with the CellQuest software 3.1f. Unlabeled cells in binding buffer were used as negative controls.
Statistical calculations were made using GraphPad Prism version 3.00 for Windows, (GraphPad Software, San Diego, CA). Significance tests were performed using a paired Student’s t-test. The difference was considered significant if p , 0.05. The results are presented as mean 6 standard error of the mean.
Bcl-2 and p53 analysis Intracellular expression of Bcl-2 and p53 was measured by flow cytometry, using Cytofix/Cytoperm Plus Intracellular Staining Kit (Pharmingen) according to the manufacturer’s instructions. In brief, cells were harvested and Fc receptors blocked with unconjugated irrelevant mouse IgG 1. The cells were then washed and fixed and incubated with either PE-conjugated Bcl-2 antibody (6C8; Pharmingen), PE-conjugated p53 (D07, Pharmingen), FITC-conjugated p53 (Pab240; Research Diagnostic Inc, Flanders, NJ), or isotype-matched control antibodies (PE-conjugated mouse IgG2b, PE-conjugated mouse IgG1, or FITC-conjugated mouse IgG1 ; all from Pharmingen). The cells were then resuspended in PBS and analyzed on a flow cytometer as described above.
CD95 expression and induction of CD95-mediated apoptosis CD95 expression was measured by flow cytometry in separate, identical cultures. Cells were incubated with anti-CD95 antibody (DX2, PE; Pharmingen) or an isotype-matched control antibody (IgG1 PE; Becton Dickinson) washed 23 in PBS and resuspended in 400 ml of 1% paraformaldehyde solution. A minimum of 10,000 events were acquired in list mode and analyzed with CellQuest software 3.1f. For induction of apoptosis, CD341 cells were cultured
RESULTS Apoptosis in TPO-induced megakaryocyte development We have already described a serum-free model for culturing MKs from bone marrow CD341 cells in vitro (26). Using that model, we investigated the effect of FL on apoptosis and expression of p53 and Bcl-2 in TPO-induced megakaryocyte development. CD341 cells were cultured with TPO alone or with TPO and FL. The proportion of apoptotic cells remained low from day 0 to 7, with a slight increase in apoptosis on day 1 (Fig. 1A,B). On day 14, the proportion of apoptotic cells started to increase, peaking at day 21 (Fig. 1A,B). A significant difference in apoptosis was not found between CD341 cells cultured with TPO alone or TPO and FL on days 0–14. On day 21, cultures containing both TPO and FL showed significantly decreased proportions of apoptotic cells compared to cultures containing TPO alone (p 5 0.0013). Subdiploid (apoptotic) (27,32) CD411 cells started to increase on days 14–21 (Fig. 1C). Cells cultured with TPO and FL showed a significantly lower proportion of subdiploid CD411 cells on days 14 (p 5 0.009) and 21 (p 5 0.005) compared to cultures with TPO alone.Therefore, adding FL to the cultures decreases apoptosis of the cells.
Apoptosis and growth factor withdrawal To analyze further the role of TPO and FL on apoptosis in MK development, we examined the effect of growth factor withdrawal on the cultures. When CD341 cells were cultured with TPO alone, the increase in apoptosis on days 10 and 14 was significantly higher in cultures where TPO had been withdrawn compared to cul-
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FIG. 1. Proportion of apoptotic cells in cultures. (A) CD341 cells cultured with thrombopoietin (TPO) (j ) or TPO and Flt3/Flk2 ligand (FL) ( u ). Proportion of Annexin-V1/PI- cells (n 5 8). A significant difference was found between CD341 cells cultured with TPO and TPO/FL on day 21 (p , 0.05). (B) Representative dot plots showing changes in CD41 expression and ploidy. CD341 cells were cultured with TPO or TPO and FL and analyzed on days 1 and 14. Ploidy was analyzed by PI staining. (C) Representative dot plots for the kinetics of apoptosis in cultures. Annexin-V1/PI- cells were considered apoptotic. (D) Proportion of apoptotic (Annexin-V1/Propidium Iodide (PI)-) cells in withdrawal cultures. CD341 cells cultured with TPO or TPO and FL. On day 7, cells cultured with TPO were washed and resuspended in fresh medium with TPO (j ) or without TPO ( u ) (top). Significant difference was found between cultures containing TPO and cultures without TPO on days 10 and 14 (p , 0.05). Also on day 7, cells cultured with TPO and FL were washed and resuspended in fresh medium with TPO/FL ( u ), without TPO ( ), without FL (j ), or without TPO/FL ( ) (bottom). Significant difference was found between cultures containing TPO and FL and cultures without TPO and FL on day 10 (p , 0.05). Data are presented as mean 6 SEM, n 5 3.
tures where TPO had not been withdrawn (p 5 0.034 and p 5 0.024, respectively) (Fig. 1D). When CD341 cells were cultured with TPO and FL, withdrawing TPO or FL or both growth factors had the effects of increasing apoptosis on day 10, but the difference was only significant when TPO and FL had both been withdrawn (p 5 0.0162) (Fig. 1D). On day 14, withdrawing TPO or FL or both TPO and FL showed a trend toward an increase in apoptosis, but this increase was not statistically significant (p . 0.05) (Fig. 1D). Taken together, these data suggest that culturing CD341 cells with
FL in addition to TPO may make the cells more resistant to apoptosis upon subsequent growth factor withdrawal at days 5–7.
Apoptosis and CD41 expression To test whether CD411 cells become apoptotic, cultured cells were analyzed by flow cytometry after Annexin-V FITC, CD41 PE, and VIA-PROBE staining. Apoptotic cells expressing the CD41 molecule increased during the culture period, both when CD341 cells were
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TPO AND FL MODULATION OF MK PROGENITOR CELLS cultured with TPO alone or TPO and FL (Fig. 2A,B). CD341 cells cultured with TPO alone showed a greater increase in the proportion of CD411 apoptotic cells compared to cells cultured with TPO and FL. This increase was significant on days 15 (p 5 0.007) and 20 (p , 0.001) (Fig. 2A,B), suggesting that FL delays both maturation and apopotosis in CD411 cells. To see the pattern of CD41 expression in nonapoptotic cells (Annexin-V-CD411 ), CD341 cells were cultured with TPO or TPO and FL. When cultured with either TPO or TPO and FL, the proportion of CD411 cells in the nonapoptotic fraction increased from days 1 to day 10. After day 10, the proportion of CD411 cells in the nonapoptotic fraction decreased rapidly when cultured with TPO alone but stayed unchanged when cultured with TPO and FL (Fig. 2A,B). The proportion of CD411 cells in the nonapoptotic fraction was significantly higher in cultures with TPO, on days 5 (p , 0.001) and 10 (p , 0.001), and significantly lower on days 15 (p 5 0.025) and 20 (p 5 0.021) compared to cells cultured with TPO and FL, again indicating that FL may delay the onset of apoptosis in this cell population.
can either induce or inhibit apoptosis, depending on its conformation (23,25). Bcl-2 expression decreased slightly from days 0 to 3 and then peaked on days 5 and 7 (Fig. 3A,B). On day 14, Bcl-2 expression decreased, being slightly higher when cells were cultured with both TPO and FL (Fig. 3A,B). No significant difference was found in Bcl-2 expression between cultures with TPO or TPO and FL (p . 0.05). Next we examined the effect of growth factor withdrawal on Bcl-2 expression. CD341 cells were cultured with TPO for 5 days and then washed and resuspended in fresh medium with or without TPO. In separate experiments, CD341 cells were cultured with TPO and FL for 5 days and then washed and resuspended in fresh medium with either TPO and FL, FL, TPO, or without TPO or FL. When CD341 cells were cultured with TPO alone, there was no difference in Bcl-2 expression where TPO had been withdrawn compared to cultures where TPO had not been withdrawn (p . 0.05) (Fig. 3C), indicating that TPO is not directly connected to Bcl-2 expression. When CD341 cells were cultured with TPO and FL, Bcl-2 expression decreased at first with all combinations of cytokines (Fig. 3C). This decrease continued for all cytokine combinations, except where FL alone was included in the medium. In this case, Bcl-2 expression increased steadily and was significantly higher on day 20 compared to the other growth factor combinations (p , 0.05) (Fig. 3C). These results indicate that FL has an antiapoptotic effect that is overridden by TPO induced maturation.
Expression of Bcl-2 and p53 in megakaryocyte development Intracellular expression of the apoptosis-associated proteins p53 and Bcl-2 was analyzed during megakaryocyte development. Bcl-2 is known to inhibit apoptosis by binding the pro-apoptotic protein Bax, whereas p53
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FIG. 2. CD41 expression on apoptotic cells and nonapoptotic cells. CD341 cells cultured with thrombopoietin (TPO) (j ) or TPO and Flt3/Flk-2 ligand (FL) ( u ). (A) A significantly higher proportion of apoptotic CD411 cells was detected in cultures with TPO on days 10 through 20 (p , 0.05). The proportion of nonapoptotic CD411 cells was significantly higher on days 5 and 10 in cultures with TPO but significantly lower on days 15 and 20 (p , 0.05). Data are presented as mean 6 SEM, n 5 3. (B) Representative dot plots for expression of Annexin-V and CD41. Annexin-V-VIA-PROBE-/Annexin-V1VIA-PROBE- cells (viable/apoptotic cells) were gated and CD41 and Annexin-V expression analyzed within that population on days 1, 7, 14, and 21.
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FIG. 3. Bcl-2 expression in megakaryocyte development. (A) CD341 cells cultured with thrombopoietin (TPO) (j ) or TPO and Flt3/Flk-2 ligand (FL) ( u ). Data are presented as mean 6 SEM, n 5 5. Bcl-2 expression is presented as mean fluorescence index (MFI); calculated as the ratio of sample mean channel:control mean channel. A significant difference was not found between cultures with TPO or TPO and FL on the indicated time points. (B) Representative histograms for Bcl-2 expression in megakaryocyte development. Light-gray-shaded lines are isotype specific controls. (C) Bcl-2 expression in megakaryocyte development after growth factor withdrawal. CD341 cells cultured with TPO or TPO and TPO/FL. On day 5 the cells were washed and resuspended in fresh medium with TPO (j ) or without TPO (m ) (left). No significant difference in Bcl-2 expression was found between cultures with TPO or without TPO (p . 0.05). Also on day 5, the cells were washed and resuspended in fresh medium with TPO/FL (j ), with TPO (d ), with FL (m ), or without TPO/FL (r ) (right). On day 10, cells resuspended with TPO (p 5 0.002) or FL (p 5 0.023) alone showed a significantly lower expression of Bcl-2 compared to cells cultured with TPO and FL or without both growth factors. On day 20, the cells showed a significantly higher expression of Bcl-2 (p , 0.05) where TPO was withdrawn. Bcl-2 expression is presented as mean fluorescence index (MFI); calculated as the ratio of sample mean channel:control mean channel. Data is presented as mean 6 SEM, n 5 3.
Our earlier results show that Bcl-2 is more higly expressed earlier in MK development. Withdrawing TPO from cultures with TPO and FL increased the expression of Bcl-2 later in the cultures, whereas withdrawing FL or both TPO and FL did not have a similar effect. Intracellular expression of p53 was analyzed using antibodies to two different forms of the p53 protein: (1) clone DO7, which recognizes normal p53, and (2) clone Pab240, which recognizes a special conformation of p53 that inhibits apoptosis. CD341 cells were cultured with TPO or TPO and FL for 21 days and p53 expression analyzed by flow cytometry. When p53 expression was analyzed using the DO7 antibody, no expression was detected until day 21 in cultures with TPO or TPO and FL (Fig. 4A,B). Significant difference was not found in p53 expression (DO7) between cultures with TPO or TPO and FL (p . 0.05). Expression of the promoter conformation of p53 (Pab240) was high-
est during the first 7 days of the culture period (Fig. 4A,B). There was a slight decrease in p53 (Pab240) expression on day 3, both when cultured with TPO or TPO and FL. On days 14 and 21, the p53 (Pab240) expression had decreased somewhat. A significant difference was not found in p53 (Pab240) expression between cultures with TPO or TPO and FL (p . 0.05). No expression of p53 (Pab240) was detected at day 0 (data not shown).
CD95 expression and induction of CD95-mediated apoptosis No significant difference was found in CD95 (Fas) expression on CD341 cells cultured with TPO or TPO and FL during the culture period (p . 0.05) (Fig. 4C). CD95 expression was relatively high during MK development (60–90%), being lower in the early stages of the cultures. Incubating the cells with anti-CD95 antibody (CH11) did
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FIG. 4. Expression of two different forms of p53 in megakaryocyte development. (A) CD341 cells cultured with thrombopoietin (TPO) (j ) or TPO and Flt3/Flk-2 ligand (FL) ( u ) and labeled with normal p53 (DO7) or promoter p53 (Pab240). Significant difference was not found between cultures with TPO or TPO and FL on the indicated time points, p53 expression is presented as mean fluorescence index (MFI); calculated as the ratio of sample mean channel:control mean channel, n 5 3. Data are presented as mean 6 SEM. (B) A representative histogram for the expression of two different forms of the p53 protein in megakaryocyte development. Light-gray-shaded lines are isotype specific controls. (C) CD95 expression in megakaryocyte development. CD341 cells were cultured with TPO (j ) or TPO and FL ( u ). A significant difference was not found in CD95 expression between cultures with TPO or TPO and FL on the indicated time points. Data are presented as mean 6 SEM, n 5 3.
not induce apoptosis in MKs or MK progenitor cells (data not shown), suggesting that these cells are not susceptible to CD95-mediated apoptosis.
DISCUSSION MK progenitor cells go through a strictly regulated maturation process in which the cells become polyploid before they start shedding platelets. This unique cellular process must involve active suppression of apoptosis while the cell collects many times its normal content of chromosomes. Additionally, limited caspase activation has been directly implicated in pro-platelet formation, and overexpression of Bcl-xL leads to impaired platelet
fragmentation (34). It has been demonstrated that there is an association between apoptosis, senescence, and proplatelet formation in MKs and that they propably end their lifespan by entering apoptosis after proplatelet formation (28–31,33,34). An increase in apoptosis has been detected in mature MKs compared to MK progenitor cells derived from CD341 cells, suggesting that the terminal phase of the MK lifespan is characterized by the onset of apoptosis (28). Previously, we have established a serum-free culture system where we showed that TPO in synergy with FL increases expansion and slows down megakaryocyte development (26). In this paper, we show that FL in combination with TPO may also prevent apoptosis in MK development, and therefore play a part in slowing down the
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SIGURJONSSON ET AL. developmental process and preventing the cells to form pro-platelets. Our results show that MK progenitor cells are relatively insensitive to apoptosis under both conditions, and, at later stages, the protective effect of TPO and FL is additive. Thus, FL seems to play a pivotal role in regulating apoptosis during MK development. An increase in the proportion of cells expressing CD41 was followed by an increase in apoptosis of the cells at later stages of the cultures. Apoptosis was related to down-regulation of Bcl-2, decrease in ploidy, and up-regulation of p53. Conversely, protection from apoptosis was related to up-regulation of Bcl-2, increase in ploidy, and down-regulation of p53 (conformationl changes). These cells are derived from immature MKs and MK progenitor cells that most likely were protected from apoptosis, more extensively in cultures containing both TPO and FL. Growth factor withdrawal-induced apoptosis probably plays an important role in the regulation of hematopoiesis and hemostasis (24). Our study shows that FL in combination with TPO slows down maturation and consequently delays apoptosis of MK progenitor cells and MKs cultured from bone marrow CD341 cells in vitro. It is our belief that apoptosis is suppressed during the developmental phase when the MK is becoming polyploid and preparing to form pro-platelets. However, after the formation of pro-platelets, what is left of the cell undergoes apoptosis and gets disposed of by macrophages in the bone marrow. Another theory might be that the platelets are formed by apoptosis that needs to be suppressed until the appropriate time in development (35). We think that FL in synergy with TPO is one of the growth factors that suppresses apoptosis during MK development, perhaps by inducing expansion of the cell pool of the progenitor cells and/or preventing polyploid cells from entering apoptosis.
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ACKNOWLEDGMENTS The investigators thank Halldor Jonsson, Jr., Rikardur Sigfusson, Svavar Haraldsson, and Ingibjorg Sigurvinsdottir for their valuable assistance in bone marrow procurement.
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Address reprint requests to: Dr. Olafur Eysteinn Sigurjonsson Immunologisk Institutt Rikshospitalet Sognsvannveien 20 0027 Oslo, Norway E-mail:
[email protected] Received January 10, 2004; accepted February 13, 2004.
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