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Stem Cells, Vol. 18, No. 4, 261-272, July 2000
© 2000 AlphaMed Press

Differential Expression of Bcl-2 Homologs in Human CD34+ Hematopoietic Progenitor Cells Induced to Differentiate into Erythroid or Granulocytic Cells

Dag Josefsena, June H. Myklebusta, Jon Lømoa, Mouldy Siouda, Heidi K. Blomhoffb, Erlend B. Smelanda

a Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway;
b Institute of Medical Biochemistry, The University of Oslo, Oslo, Norway

Key Words. Hematopoiesis • Human • CD34+ cells • Erythropoiesis • Bcl-2 • Bcl-xL • Apoptosis

Dag Josefsen, M.D., Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway. Telephone: 47-22-93-45-69; Fax: 47-22-50-07-30; e-mail: dag.josefsen{at}labmed.uio.no


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34+ human hematopoietic progenitor cells. CD34+ cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-xL protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-xL protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells.

The role of the distinct upregulation of Bcl-xL in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-xL. Addition of Bcl-xL ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34+ cells dependent on the cytokine stimulation given. The use of Bcl-xL-specific ribozymes suggested that Bcl-xL is important for survival but not for differentiation of erythroid progenitor cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis plays an important role in regulation of normal tissue homeostasis in various tissues [1]. Recent research has revealed the complex intracellular machinery involved in the regulation of apoptosis. This involves processes important in the initiation of apoptosis as well as proteases responsible for the execution of apoptosis. The still-growing Bcl-2 family of proteins is an important upstream intracellular regulator of this process [2, 3]. The prototype of this family, the product of the bcl-2 gene, was first discovered at the chromosomal breakpoint t (14;18) usually found in follicular lymphoma [4]. This rearrangement of bcl-2 leads to an aberrant regulation of Bcl-2 protein expression, and deregulation of Bcl-2 expression is probably involved in tumorigenesis of this disease. Some members of the Bcl-2 family, such as Bcl-2, Bcl-xL, and Mcl-1, function as antiapoptotic proteins, whereas Bax, Bad, Bak, Bik and Bid work as cell death promoters [2]. Members of this family share varying degrees of homology at four conserved domains identified in Bcl-2, BH1-BH4 (BH = Bcl-2 homology domain). BH1 and BH2 seem to be important for antiapoptotic effects, whereas BH3 is important for proapoptotic effects [2, 5]. A subgroup of proapoptotic proteins, the BH3 subfamily, only shows homology at this domain, further supporting the important role for BH3 in the induction of apoptosis [6, 7]. Several of the Bcl-2 family proteins are associated with intracellular membranes like the outer membrane of mitochondria, the nuclear envelope, and the endoplasmatic reticulum [2]. Increasing evidence shows that localization of Bcl-2 homologs at mitochondrial membranes plays a key role in the regulation of apoptosis [8]. Bcl-2 homologs can dimerize, either as homodimers or as heterodimers with other family members, and thereby affect the balance between apoptosis and cell survival [9]. Therefore, a shift in the ratio between proapoptotic and antiapoptotic Bcl-2 homologs is important for the regulatory functions of apoptosis by these proteins. Bcl-xL has also been shown to associate with proteins such as Apaf-1 not belonging to the Bcl-2 family, and thereby inhibits caspase activation [10, 11]. Structural studies of Bcl-x have shown that it has similarities to diphtheria toxin and the colicins capable of pore formation [12]. Thus, recent findings have demonstrated that Bcl-xL can inhibit apoptosis in a heterodimerization-independent way, affecting mitochondrial pore formation [13].

Knockout studies of Bcl-2 in mice have shown that all lineages of nonlymphoid hematopoietic cells develop normally in healthy mice [14]. Moreover, a recent study where Bcl-2 was transfected into erythroid progenitor cells delayed but did not prevent apoptosis [15]. In addition, normal erythropoietic development was not disrupted by overexpression of Bcl-2. In contrast, Bcl-x deficient mice died at the embryonic stage, and analysis of hematopoietic cells of the fetal liver demonstrated a massive apoptosis of these cells [16]. In agreement with these findings, studies on human CD34+ hematopoietic progenitor cells have shown that both Bcl-2 and Bcl-xL are widely expressed, whereas in CD34+CD38– cells, which are enriched for the most immature hematopoietic progenitor cells, Bcl-xL is highly expressed while only a few cells express Bcl-2 [17]. Thus, Bcl-xL seems to be more important for the survival of the most immature hematopoietic progenitor cells than Bcl-2.

A recent report demonstrated that Bcl-xL was downregulated during differentiation of CD34+ cells along the granulocyte lineage but not along the monocyte/macrophage lineage [18]. These findings are in agreement with previous findings demonstrating that Bcl-xL as well as Mcl-1 and Bcl-2 are downregulated in fully matured neutrophils [19]. Moreover, data from erythroid cell lines as well as human erythroid progenitor cells have shown that at least Bcl-xL is important for the survival of erythroid cells [20, 21].

In the present work, we have studied regulation of various Bcl-2 homologs in human CD34+ hematopoietic progenitors during early erythroid or granulocytic differentiation using different growth factors. We found that in erythroid progenitor cells the level of Bcl-xL was increased, whereas Bax and Bcl-2 were downregulated relative to cells cultured in medium alone. In contrast, in granulocytic progenitor cells we observed a decrease in Bcl-xL and Bcl-2 protein levels, whereas the level of Bax remained unaltered or was slightly increased. Interestingly, our data demonstrated a biphasic regulation of Mcl-1 in both erythroid and granulocytic progenitor cells with an initial increase in protein expression in erythroid progenitor cells, whereas we observed an initial decrease in Mcl-1 protein level of granulocytic cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hematopoietic Growth Factors
Recombinant human (rHu) G-CSF was a gift from Roche (Basel, Switzerland). rHu erythropoietin (rHuEpo) was purchased from Boehringer Mannheim (Mannheim, Germany), and rHu stem cell factor (rHuSCF) was purchased from R&D Systems (Abingdon, UK; http://www.rndsystems.com). The prefixes designating human growth factors are hereafter deleted. Unless indicated, growth factors were used at the following predetermined optimal concentrations: G-CSF (50 ng/ml), SCF (50 ng/ml), and Epo (5 IU/ml).

Cell Separation
Bone marrow cells were obtained by iliac crest aspiration from normal adult volunteers with informed consent and approval by the Regional Ethics Committee. Mononuclear cells were separated by Ficoll-Hypaque gradient centrifugation (Lymphoprep, Nycomed; Oslo, Norway; http://www.amersham.co.uk). CD34+ cells were isolated by positive selection according to previously described methods [22]. Briefly, bone marrow mononuclear cells were rosetted with Dynabeads® M-450 directly coated with the CD34 monoclonal antibody (mAb) 561 (Dynal; Oslo, Norway; http://www.dynal.no) for 30 min at 4°C with tilting and gentle rotation. The bead-to-cell ratio was 1:1. Rosetted cells were attracted to a samarium cobalt magnet (Dynal), and nonrosetting cells were removed by pipetting and washed (x5). Positive selected cells were detached from beads by incubation with anti-Fab antiserum (DETACHaBEAD, Dynal) at a concentration of 34 mg/ml for 45 min at room temperature with tilting and gentle rotation. Isolated cells free of beads were washed and counted. The purity of CD34+ cells isolated by this method was reproducibly >95% as determined by immunostaining and flow cytometric analyses.

Immunophenotyping
Freshly isolated CD34+ cells and cells cultured for 4, 8, 14, and 21 days (5 x 105 cells/2 ml) in RPMI 1640 (GIBCO; Grand Island, NJ) supplemented with 10% fetal calf serum ([FCS]; Sera-lab; Sussex, UK), with or without Epo and SCF or G-CSF and SCF were incubated with the following mAbs: phycoerythrin (PE)-labeled CD34 mAb (HPCA-2) purchased from Becton Dickinson (San Jose, CA; http://www.bd.com). PE-labeled CD19 and CD14, and fluorescein isothiocyanate (FITC)-labeled CD13, CD15, and CD33 were purchased from DAKO (Copenhagen, Denmark; http://www.dako.dk). Cells cultured for 14 and 21 days were supplied weekly with new medium containing growth factors. Irrelevant isotype-matched FITC- or PE-labeled mAbs (DAKO) served as negative controls. The samples were run on a FACSCalibur flow cytometer, and analyses were performed with CELLQuest software (BD).

Cell Morphology
Freshly isolated CD34+ cells, or cells cultured for eight days as previously described, were spun down on microscopic slides by cytocentrifugation (5 x 104 cells/0.05 ml), air-dried overnight, and stained with Giemsa. The slides were evaluated by light microscope.

Cell Culture and Identification of Cell Death
CD34+ cells (104 cells/0.2 ml) were cultured in RPMI 1640 (GIBCO) containing 10% FCS with or without G-CSF, SCF, or Epo in 96-well round bottom microtiter plates. Cell death was demonstrated by staining cells directly with propidium iodide ([PI]; Calbiochem Corp.; La Jolla, CA; 5 mg/ml; http://www.calbiochem.com) for 5-30 min on ice. At least 1,000 cells per sample were run on a BD FACSCalibur flow cytometer with an argon laser tuned at 488 nm. PI-positive cells were gated, and the results were analyzed by CELLQuest software (BD).

Apoptosis was confirmed by using the TUNEL assay (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling). This is a method for detection of single-strand DNA fragmentation [23]. CD34+ cells (1-2 x 105 cells/ml) were cultured for four days in RPMI containing 10% FCS with or without G-CSF, SCF, or Epo in 24-well plates. The cells were then prepared and labeled using the In Situ Cell Death Detection Kit from Boehringer Mannheim (Mannheim, Germany) according to the manufacturer's protocol. The cells were thereafter run on a BD FACSCalibur flow cytometer. TUNEL positive cells were gated, and the results were analyzed by CELLQuest software (BD).

The percentage of cells displaying DNA fragmentation was also determined by the Nicoletti technique, as previously described [24]. Briefly, 2 x 105 cells cultured for six days under the same conditions as for the TUNEL assay, were fixed in 70% ethanol, washed once in Hanks' buffered salt solution (HBSS), resuspended in 1 ml HBSS and 1 ml phosphate citric acid buffer (0.2 M Na2PO4 and 0.004 M citric acid, pH 7.8), and incubated at room temperature for 5 min. After centrifugation, cells were resuspended in 1 mL HBSS containing 20 mg/ml PI and 10 Kunitz units of DNase free RNase A and analyzed on a BD FACSCalibur flow cytometer with CELLQuest software (BD). Due to fragmentation of DNA, apoptotic cells appear as hypodiploid in the DNA histogram.

Treatment of CD34+ Cells with Bcl-xL Ribozymes
CD34+ cells were seeded at 30,000 cells per well in 0.1 ml X-VIVO 15 complete medium, in flat-bottomed 96-well plates, and then incubated with ribozymes complexed with DOTAP at 5 µg/ml as described by the manufacturer. After 24 h of incubation, Epo and SCF were added to the cells, cultured at 37°C for another four days, and assessed for cell death, differentiation, and the level of Bcl-xL protein expression. Ribozymes used in this study were created in vitro as previously described [25]. The Bcl-xL sequence is 5'-GGUUGCUCUGACUGAUGAGGCCGAAAGGCCGAAACAUUUUUA-3'.

Western Blot Analysis
Freshly isolated CD34+ cells (>=5.0 x 105 cells) or CD34+ cells cultured with or without Epo, G-CSF, or SCF in RPMI containing 10% FCS were washed twice in PBS, resuspended in at least 25 µl sample buffer (10% glycerol, 5% mercaptoethanol, 0.0625 M Tris-HCl [pH 6.8], 2.5% SDS) and boiled for 15 min. Thereafter, 5 µg of total protein from each sample were run on 12% SDS polyacrylamide gels and blotted onto nitrocellulose filters. The filters were then pretreated with TBS containing 0.1% Tween-20 and 5% dry milk and incubated at room temperature 1-2 h in TBS buffer (TBS plus 0.1% Tween-20, 1% dry milk and 0.1% FCS) containing 0.5% (v/v) anti-Bcl-2 mAb (Santa Cruz Biotechnology; Heidelberg, Germany), or 0.5% rabbit antiserum against Bax and Bcl-x (Santa Cruz Biotechnology), or 0.1% rabbit antiserum against Mcl-1 (Pharmingen; San Diego, CA; http://www.pharmingen.com). After washing, the blots were incubated for 1 h with 0.033% (v/v) goat anti-mouse IgG (Bcl-2) or 0.025% goat anti-rabbit IgG (Mcl-1, Bcl-x, and Bax) antibodies conjugated to horseradish peroxidase (Biorad; Hercules, CA), and the respective proteins were visualized using ECL + Western blotting kit (RPN 2106; Amersham; Buckinghamshire, UK; http://www.amersham.co.uk). To control protein loading of the gels, the Western blots were stained with Ponceau S to visualize equal amounts of proteins in each experiment (data not shown).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Lineage Markers on CD34+ Cells Stimulated with Different Growth Factor Combinations
The CD34+ cell population can be induced to differentiate into various lineages dependent on the growth factor combinations used, and more or less unilineage outgrowth can be obtained in vitro [26]. We used mAbs to several lineage-associated cell surface antigens to determine the relative proportion of granulocytic and erythroid cells after stimulation with selected cytokine combinations. In analogy with previous data [22, 27], we found that glycophorin A (GPA), which is expressed on cells restricted to the erythroid lineage, was not detected on freshly isolated CD34+ cells, while the granulocytic marker CD15 was weakly expressed on 22% of the cells, and CD13 was expressed on 42% of the cells (n = 3, data not shown). Table 1Go summarizes the expression of lineage-dependent surface markers on cells incubated for four or eight days in medium alone or with Epo and SCF or G-CSF and SCF. In cells cultured with Epo and SCF, GPA was expressed on 46% of the cells after four days and on 70% of the cells after eight days (mean of five experiments, Table 1Go). Transferrin receptor (CD71), which is expressed on erythroid cells and activated cells, was expressed on 79% of the cells after four days and 89% of the cells after eight days (n = 5, Table 1Go). The granulocytic marker CD15 was expressed on 24% of Epo- and SCF-stimulated cells after four days, and on 16% of the cells after eight days of culture (n = 4, Table 1Go). Other myeloid markers such as CD13 and CD14 were expressed on 41% and 16% after four days respectively, and on 20% and 12% after eight days of culture.


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  		Table 1. Phenotyping of CD34+ cells cultured for four or eight days in medium alone or with Epo + SCF or G-CSF + SCF
 
In contrast, in cells incubated with G-CSF and SCF, there was an upregulation of the myeloid markers. CD13 was expressed on 88% after four days of culture and 89% after eight days (n = 4, Table 1Go). The monocytic marker CD14 was expressed on 22% and 49% after four and eight days of culture, respectively. The granulocytic marker CD15 was found to be strongly expressed on 77% and 80% of the cells after four and eight days, while few or no cells expressed GPA after four and eight days of culture (Fig. 1Go and Table 1Go). Thus, G-CSF and SCF stimulation primarily induced granulocytic progenitor cells, which strongly expressed CD15 and CD13, and partly CD14. In contrast, we observed predominantly GPA- and CD71-positive erythroid progenitor cells in Epo- and SCF-stimulated cells. Moreover, examination of CD34+ cells cultured under the same conditions for 14 or 21 days revealed that GPA was expressed on more than 90% of cells cultured with Epo and SCF, and CD15 was expressed on 97% of cells cultured in the presence of G-CSF and SCF (Figs. 1A and 1BGo, one representative experiment of three).




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  		Figure 1. Immunophenotypical examination by two-color flow cytometry of freshly isolated CD34+ cells, or cells cultured for 4, 8, 14, and 21 days in the presence of either Epo and SCF (A), or G-CSF and SCF (B) as described in Materials and Methods. The cells (5 x 104 cells) were then stained with either PE- or FITC-conjugated antibodies against lineage restricted surface markers as shown in the figure. Irrelevant isotype matched mAbs served as negative controls. One representative experiment of four is presented.

 
We have also tested other cytokine combinations known to induce erythroid differentiation of CD34+ cells. Cells were cultured with a multifactor combination consisting of Epo, interleukin 3 (IL-3), IL-9, and SCF, or a combination of Epo and low doses of IL-3 and GM-CSF. These combinations have been used by others to enhance erythroid cell formation from hematopoietic progenitor cells [28]. However, the combination we used with Epo and SCF gave results similar to the multifactor combination, and it was markedly better than Epo combined with low doses of IL-3 and GM-CSF (data not shown).

To confirm the immunophenotypical findings, we examined microscopic slides of cytospin specimens of freshly isolated CD34+ cells, cells cultured with Epo and SCF, G-CSF and SCF, or in medium alone for eight days. The morphological examination demonstrated that after eight days of culture, there were predominantly erythroid precursor cells in cultures stimulated with Epo and SCF (Fig. 2Go). In cells cultured with G-CSF and SCF there were mainly cells with a granulocytic morphologic appearance (Fig. 2Go). Taken together, these results verified that we obtained populations of predominantly erythroid or granulocytic progenitor cells dependent on the growth factor combinations given.



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  		Figure 2. Morphologic examination of freshly isolated CD34+ cells, or cells cultured for eight days (5 x 104 cells/1 ml) in the presence of Epo and SCF, or G-CSF and SCF, as described in Materials and Methods. The cells were then spun down to microscopic slides by cytocentrifugation, stained with Giemsa, and then evaluated by microscopy. One representative experiment of three is shown.

 
Regulation of Bcl-2 Homologs in Erythroid and Granulocytic Progenitor Cells
The expression of different Bcl-2 homologs in hematopoietic progenitor cells seems to vary dependent on the maturation stage [17, 19, 29]. We therefore wanted to examine the expression of various Bcl-2 family proteins in CD34+ cells stimulated to differentiate into granulocytic versus erythroid cells. Interestingly, we observed marked differences in the protein expression of several Bcl-2 homologs between early erythroid and granulocytic progenitor cells. In CD34+ cells stimulated with Epo and SCF, we found a gradual upregulation in the levels of both Bcl-xL and Mcl-1 at days 4 and 8, whereas the level of Bcl-2 was downregulated relative to cells cultured in medium alone (Fig. 3Go). In most experiments, we observed in addition to the 31 kDa Bcl-xL, an additional band with 33-34 kDa. This band is probably due to unspecific binding of the primary antibody, as has been described by others [30]. Moreover, the expression of Bax was slightly downregulated after four days of culture in cells stimulated with Epo and SCF, whereas after eight days, the Bax level was similar to that of cells cultured in medium alone. There were only minor changes in expression of the different Bcl-2 homologs in cells cultured in medium for four and eight days versus that in unstimulated cells. Equal protein loading was controlled by Ponceau S staining of the nitrocellulose filters.



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  		Figure 3. Relative expression of Mcl-1, Bcl-xL, Bcl-2, and Bax proteins at days 4 (A) and 8 (B). CD34+ cells were cultured for four or eight days in the presence of Epo and SCF, G-CSF and SCF. Control cells were cultured in medium alone, and relative protein expression was determined by Western blotting as described in Materials and Methods. One representative experiment of six (A) or three (B) are shown. Equal protein loading was controlled by Ponceau S staining of the nitrocellulose filters (not shown).

 
In contrast, in G-CSF- and SCF-stimulated cells, we found that Bcl-2, Bcl-xL, and Mcl-1 were downregulated, whereas Bax was unaltered or slightly upregulated as compared with control cells cultured in medium alone (Figs. 3A and 3BGo). We also examined the effects after stimulation with single factors. Epo as well as G-CSF stimulation alone gave the same effects on regulation of Bcl-2 homologs in CD34+ cells as when combined with SCF, while SCF given alone followed a pattern similar to that in cells cultured in medium alone (data not shown).

The extent of protein regulation was estimated by using densitometry (Fig. 4Go). Compared with CD34+ cells cultured in medium, Bcl-xL levels were increased more than threefold (n = 6, p < 0.05) in Epo- and SCF-stimulated cells after four days of culture, while in G-CSF- and SCF-stimulated cells, Bcl-xL levels were reduced by 50% (n = 6, p < 0.05) (Fig. 4AGo). In contrast, the expression of Bax protein in Epo- and SCF-stimulated cells was reduced to 60% as compared with the level in cells cultured in medium alone (n = 6, p < 0.05), whereas in G-CSF- and SCF-stimulated cells, the level of Bax was not significantly altered (Fig. 4BGo). Moreover, we found an approximate threefold increase in Mcl-1 levels in Epo- and SCF-stimulated cells (n = 6, p < 0.05, Fig. 4CGo), while Mcl-1 levels remained relatively unchanged in G-CSF- and SCF-stimulated cells as compared with control cells (n = 6, Fig. 4CGo). Finally, Bcl-2 expression was reduced to 30% of control levels at day 4 for both Epo and SCF and G-CSF-and SCF-stimulated cells (n = 6, p < 0.05, Fig. 4DGo). The same pattern of regulation of these Bcl-2 homologs was also seen after eight days of culture (data not shown).



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  		Figure 4. Densitometric analysis of Western blots of (A) Bcl-xL, (B) Bax, (C) Mcl-1, and (D) Bcl-2. The expression of proteins in Epo and SCF versus G-CSF- and SCF-stimulated CD34+ cells after four days of culture relative to the expression in control cells (medium) is shown. Data are presented as mean ± SE of six independent experiments. * denotes statistical significance (p < .05) using the paired Wilcoxon test.

 
These findings demonstrated marked regulation of several Bcl-2 family members in early differentiation of CD34+ cells into different cell lineages. In order to examine regulation of these proteins during later stages of erythroid and granulocytic differentiation of CD34+ cells, we examined the expression of Bcl-2 family members after three weeks of culture in the presence of either Epo and SCF or G-CSF and SCF. In agreement with the findings during early differentiation, we observed that CD34+ cells cultured with Epo and SCF abundantly expressed Bcl-xL but not Bax or Bcl-2, whereas cells stimulated with G-CSF and SCF expressed Bax, but not Bcl-xL or Bcl-2 at detectable levels (Fig. 5Go). Surprisingly, the level of Mcl-1 expression was decreased in CD34+ cells stimulated with Epo and SCF (Fig. 5Go), whereas CD34+ cells cultured in the presence of G-CSF and SCF strongly expressed Mcl-1. Mcl-1 expression thus shows a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decrease in granulocytic progenitor cells and increase in erythroid progenitor cells.



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  		Figure 5. Relative expression of Bcl-xL (5A), Bax (5A), and Mcl-1 (5B) proteins after 21 days of culture. CD34+ cells were cultured in the presence of Epo and SCF or G-CSF and SCF, and relative protein expression was determined by Western blotting as described in Materials and Methods. Fig. B: one representative experiment of three is shown.

 
Taken together, the expression of the various Bcl-2 homologs examined here shows a complex and differential regulation upon stimulation of CD34+ cells with Epo and SCF versus G-CSF and SCF.

Regulation of Cell Viability
As we found marked differences in regulation of Bcl-xL and Bax in erythroid progenitor cells compared with that in myeloid progenitor cells, it was important to explore whether these differences influenced the level of apoptosis in the two cell populations. The level of apoptotic cells was demonstrated by the TUNEL assay, showing a slight reduction of apoptotic cells from 22% in medium to 15% in Epo- and SCF-stimulated cells and 17% in G-CSF- and SCF-stimulated cells at day 4 (n = 6, Fig. 6AGo). Similar findings were obtained using the Nicoletti technique, where fragmented DNA is displayed as a subdiploid peak in DNA histograms. Using this method, we found 9% apoptotic cells in Epo- and SCF-stimulated cells, and 10% apoptotic cells in G-CSF- and SCF-stimulated cells (n = 3, Fig. 6BGo, one representative experiment is shown). Since the effect on apoptosis could be influenced by the degree of proliferation, we also counted the cells after incubation. However, in both Epo and SCF and G-CSF- and SCF-stimulated cells, we found a four- to five-fold increase in the number of cells after four days of culture (n = 3, data not shown). Taken together, in spite of differences in regulation of Bcl-2 homologs, we did not observe statistically significant differences in the levels of apoptosis during the study period.




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  		Figure 6. Effects on apoptosis of CD34+ cells, as shown by TUNEL staining (6A) or by the Nicoletti technique (6B) after stimulating the cells with Epo ± SCF or G-CSF ± SCF. Control cells were cultured in medium alone. Cells were cultured for four days (>2.0 x 105 cells/1ml) in 48-well plates with Epo ± SCF, G-CSF ± SCF or in medium alone as described in Materials and Methods. Fig 6AGo) Extent of DNA fragmentation in cells was determined by the TUNEL assay. Mean (±SE) of six independent experiments is shown. The differences in the levels of apoptosis were not statistically significant. Fig 6BGo) DNA content was determined by the Nicoletti technique, demonstrating a loss in the cellular content of DNA in apoptotic cells The percentage of apoptotic cells in the subdiploid peak is indicated by the M1 gate. The results are from one representative experiment of three.

 
Effects of Bcl-x Ribozymes on Viability and Erythroid Differentiation of CD34+ Cells
The prominent upregulation of Bcl-xL expression after stimulation of CD34+ cells with Epo and SCF led us to further examine the role of Bcl-xL in early erythroid cells. Ribozymes against Bcl-xL mRNA were created in vitro, as previously described [25]. The ribozyme was introduced into the cells by the use of liposomes (DOTAP) at predetermined optimal noncytotoxic concentrations [31]. CD34+ cells were preincubated with either medium or DOTAP alone, or Bcl-xL ribozyme complexed with DOTAP. After 24 h of culture, Epo and SCF were added to all the wells. As shown in Figure 7AGo, there was a marked increase in cell death in Epo- and SCF-stimulated cells receiving Bcl-xL ribozymes as compared with cells cultured in the presence of DOTAP alone (Bcl-xL ribozymes, 51% cell death; DOTAP, 20% cell death, n = 3). However, erythroid differentiation of CD34+ cells, as assessed by expression of GPA and CD71 (transferrin receptor), was not affected by addition of the Bcl-xL ribozyme (data not shown). Thus, our present data demonstrate the important role of Bcl-xL in survival of erythroid progenitor cells. In addition, the Bcl-xL ribozyme did not prevent erythroid differentiation.




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  		Figure 7. Effects on cell death of CD34+ cells, as shown by propidium iodide (PI) staining (7A) and regulation of Bcl-xL protein expression (7B) after preincubation of CD34+ cells with Bcl-xL ribozyme or DOTAP alone for 24 h, followed by stimulation with Epo and SCF for three days, as described in Materials and Methods. Figure 7AGo shows a marked increase in cell death after preincubation with ribozyme (% cell death ± SE, n = 3). Figure 7BGo demonstrates a partial downregulation of Bcl-xL protein in the presence of ribozyme. One experiment of two is presented.

 
To examine whether Bcl-xL ribozyme resulted in downregulation of its target molecule, we assessed the level of Bcl-xL protein expression. We observed a partial downregulation of Bcl-xL in ribozyme-treated cells (Fig. 7BGo). Protein loading was controlled by PKC-ß2 expression. Taken together, our results demonstrate an important role of Bcl-xL in survival of CD34+ erythroid progenitor cells, while partial downregulation of Bcl-xL does not affect erythroid differentiation.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper we show that the expression of various Bcl-2 homologs is differently regulated during early differentiation of CD34+ hematopoietic progenitor cells in the erythroid versus the granulocytic direction. In CD34+ cells stimulated with Epo and SCF, which promotes erythroid differentiation, we found a significant increase in the expression of the apoptosis-suppressing proteins Bcl-xL and Mcl-1, whereas the level of Bcl-2 was markedly downregulated. However, toward late stages of differentiation, the level of Mcl-1 protein was downregulated, thus demonstrating a biphasic regulation of Mcl-1 in these cells. To our knowledge, this upregulation of Mcl-1 protein during early erythroid differentiation has not been demonstrated before. In cells stimulated with G-CSF and SCF, antiapoptotic Bcl-2 family members including Bcl-xL, Mcl-1 and Bcl-2 were downregulated, while Bax expression was maintained at a high level, thus leaving the cells in a more proapoptotic condition. Moreover, in spite of marked differences in the regulation of Bcl-2 homologs examined here, the level of apoptosis was similar in CD34+ cells stimulated with either a combination of Epo and SCF, or G-CSF and SCF.

The upregulation of Bcl-xL during early erythropoiesis is in agreement with previous data indicating that Epo induces Bcl-xL expression in erythroid cells. It was recently demonstrated that deprivation of Epo in the erythroid-dependent mouse cell line HCD57 resulted in a decrease in the expression of Bcl-xL within 48 h [21]. If Epo thereafter was added to the cultures, the level of Bcl-xL was restored to the same level as in cells maintained in Epo [21]. The increased cell death promoted by a Bcl-xL-specific ribozyme in cell cultures receiving Epo and SCF (Fig. 7BGo) indicated that Bcl-xL plays an important role in the regulation of apoptosis in erythroid progenitor cells. This is in line with several recent observations. Thus, in HEL and K562 leukemia cells undergoing erythroid differentiation, there was an increase in cell death accompanied by diminished expression of bcl-x mRNA and protein [20], suggesting an important role of Bcl-xL in survival of erythroid progenitor cells. Moreover, Motoyama et al. recently demonstrated using Bcl-x null mouse embryonic stem (ES) cells that bcl-x prevents apoptotic cell death of both primitive and definitive erythrocytes at the end of maturation [32]. A role for Bcl-xL is further supported by the findings of Gregoli et al., demonstrating that Bcl-xL at this late stage of differentiation plays a central role for survival and thus the final maturation [33]. Taken together, the data therefore point to an important role for Bcl-xL in regulation of apoptosis during erythropoiesis.

Interestingly, the partial downregulation of Bcl-xL protein expression obtained by use of Bcl-xL-specific ribozymes did not prevent erythroid differentiation induced by Epo and SCF. These findings are in agreement with previous findings by others showing that increased expression of Bcl-xL in HEL and K562 cells inhibited apoptosis, whereas erythroid differentiation was not affected [20]. Similar results have been shown for Bcl-2 in HL60 cells. Overexpression of Bcl-2 in these cells inhibited apoptosis, while differentiation and maturation were unaffected [34]. Transfection of either Bcl-2 or A1 into FDC-Pmix or 32D c13 myeloid cell lines, respectively, further supports this notion [35, 36]. Both of these cell lines survived and differentiated normally in the absence of growth factors. Moreover, transfection of Bcl-2 into either IL-7-deficient mice, or mice lacking monocyte colony-stimulating factor (M-CSF), allowed survival as well as differentiation into mature T lymphocytes or monocytes, respectively [37-39]. Therefore, current knowledge suggests that Bcl-2 and Bcl-xL are not directly involved in regulation of differentiation of hematopoietic progenitor cells.

In granulocytic progenitor cells, we found a gradual downregulation of the apoptosis-suppressing proteins Bcl-xL, and Bcl-2, while the level of Bax was unaltered or slightly upregulated. Our results are in general agreement with previous findings showing that Bcl-2 and Bcl-xL are downregulated in mature human neutrophil granulocytes, while Bax is abundantly expressed [19]. Sanz and coworkers also recently demonstrated that mobilized human CD34+ cells from peripheral blood stimulated with a growth factor combination including G-CSF lost the expression of Bcl-xL when fully matured [18], which fits well with our data. However, they observed a transient increase in the level of Bcl-xL at day 6 before a marked decline, whereas in our experiments, we found a downregulation of Bcl-xL at days 4, 8, and 21. The discrepancy in the initial regulation of Bcl-xL in these studies could be due to differences in cytokine combinations and/or the rate and mode of differentiation in the cell populations under study. Sanz et al. used a growth factor combination containing SCF, G-CSF, IL-3, and IL-6, and at day 6, 70% of the cells were still CD34+, whereas in our experiments only 31% and 15% were CD34+ after four or eight days of culture, respectively (Table 1Go).

The expression of Mcl-1 in G-CSF- and SCF-stimulated cells was biphasic with an initial decline observed at days 4 and 8, and an upregulation at day 21. In agreement with our findings of increased expression of Mcl-1 in late-differentiating granulocytic cells, Moulding and coworkers recently demonstrated that Mcl-1 could be upregulated in mature neutrophils in the presence of GM-CSF followed by a delay in apoptosis of these cells [30]. Thus, stimulation with different growth factors can upregulate at least Mcl-1 and thereby affect the apoptotic fate of these cells. Importantly, Zhou and coworkers recently demonstrated that Mcl-1 had moderate viability-enhancing effects in a spectrum of hematopoietic cells in transgenic mice, including myeloid cells of both immature and mature stages of maturation, further implicating an important role of Mcl-1 in regulation of apoptosis of myeloid cells [40].

Taken together, our data demonstrate a complex and differential regulation of Bcl-2 family members during erythroid and granulocytic differentiation of CD34+ cells. Moreover, our findings suggest that Bcl-xL plays an important role in survival of erythroid progenitor cells, whereas it does not seem to be important for normal differentiation of these cells.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to Nina Måsvær and Lise Forfang for expert technical assistance, to Leiv Rusten for helpful advice in morphologic examination of cytospin slides, and to Birgitte Boye for reviewing the manuscript.

This work was supported by The Norwegian Cancer Society.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received May 8, 2000; accepted for publication May 9, 2000.




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