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Stem Cells, Vol. 19, No. 1, 59-70, January 2001
© 2001 AlphaMed Press

Steel Factor Sustains SCL Expression and the Survival of Purified CD34+ Bone Marrow Cells in the Absence of Detectable Cell Differentiation

J.R. Caceres-Cortesa,,b,,c, G. Krosla,,b, N. Tessierb, P. Hugod, T. Hoanga,,b,,e,,f,,g

a Laboratory of Hemopoiesis and Leukemia and
b Clinical Research Institute of Montreal, Montreal, Quebec, Canada;
c Research Unit in Cell Differentiation and Cancer, Faculty of Professional Studies-UNAM, FES-Zaragoza, UNAM, Mexico;
d PROCREA BioSciences Inc., Institut de Recherches en Biotechnologie, Montreal, Quebec, Canada; the Departments of
e Pharmacology and
f Biochemistry, and the
g Program of Molecular Biology, University of Montreal, Montreal, Quebec, Canada

Key Words. Apoptosis • Hemopoietic growth factor • Steel • Transcription factor SCL/tal-1 • Differentiation

Julio R. Caceres-Cortes, Ph.D., Research Unit in Cell Differentiation and Cancer, FES-Zaragoza-UNAM, J.C. Bonilla 66 Col. Ejercito de Oriente, Apdo Postal 9-020, Mexico D.F. CP 15000. Telephone: 525-773-4108; Fax: 525-773-4108; e-mail: cortesj{at}servidor.unam.mx


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD34+ cells express the basic helix-loop-helix transcription factor SCL, which is essential for blood cell formation in vivo. In addition, their survival is critically dependent on hemopoietic growth factors. We therefore compared the effects of Steel factor (SF) and GM-CSF on the survival, proliferation, and differentiation of primary human CD34+ cells, as well as the role of SCL during these processes. GM-CSF suppresses apoptosis in CD34+ cells, which proliferate and differentiate into mature granulocytic and monocytic cells (CD34–CD13+) and loose SCL expression. In contrast, SF suppresses apoptosis without a significant increase in cell numbers, and the cells remain CD34+ and SCL+ with a blast-like morphology. Examination of apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) reaction and of the cell cycle status indicated that SF is both a survival factor and a mitogenic factor for CD34+ cells. There was, however, constant cell death in a fraction of the population, which could be rescued by GM-CSF. Co-addition of SF and GM-CSF prevents the downregulation of SCL observed in the presence of GM-CSF by itself, allows for prolonged survival and expansion of CD34+ cells in culture, inhibits monocytic differentiation and impairs granulocytic differentiation. Finally, exposure to an antisense SCL but not a control oligonucleotide decreases SCL protein levels and prevents the suppression of apoptosis by SF without affecting GM-CSF-dependent cell survival. These observations suggest that the hemopoietic transcription factor SCL regulates the survival of CD34+ cells in response to SF.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is central to the regulation of balanced cell numbers both in embryogenesis and in tissues with high cellular turnover in the adult. Thus, withdrawal of growth factors that act to suppress programmed cell death results in apoptosis in hemopoietic cells [1], oligodendrocytes [2], and neuronal cells [3]. The dependence of postmitotic neutrophils on G-CSF [4] or neurons on nerve growth factor [5] illustrates the principle that cell survival can be dissociated from cell proliferation, which was later confirmed by the identification of bcl-2, a cell survival proto-oncogene that does not favor cell cycling [6] and, in, fact slows the cells down at the G1 to S boundary [7]. Recent evidence suggests that apoptosis may be a default pathway initiated by insufficient survival signals from neighboring cells, be it through direct cell-cell interaction or through soluble mediators [2] or by a defective differentiation process [8].

Steel factor (SF) and its receptor c-kit have been shown to control multiple biological processes that include the survival, proliferation, differentiation, and migratory behavior of cells belonging to four seemingly unrelated lineages, hematopoietic, mastocytic, melanocytic, and gametogenic lineages. In melanocytes, SF appears to be involved in cell proliferation and migration [9], while acting as a survival factor for melanoblasts [10, 11]. Interestingly, SF on its own is mainly a survival factor for primordial germ cells [12, 13] but acts in synergy with other cytokines such as leukemia inhibitory factor to support their long-term proliferation. In vitro studies with mastocytes suggest that SF is both a survival and potent mitogenic factor [14, 15].

The importance of SF/c-kit in hemopoiesis was recognized long ago because of the severe anemia observed in Steel and White spotting mice, which were later shown to result from mutations in the loci coding for the ligand or its receptor, respectively [16]. In vitro, SF has invariably been shown to synergize with interleukin 3 (IL-3), GM-CSF, or erythropoietin (Epo) to support colony formation from early erythroid and granulocytic monocytic precursors. Our previous data indicated that SF suppresses apoptosis in several hemopoietic cell lines and primary leukemic myeloblasts [17], whereas another study suggested that SF mainly supports the cycling of normal CD34+ cells [18].

Another biological process, central to the generation of hemopoietic cells, is that of lineage commitment and cell differentiation, which are driven by lineage-specific transcription factors [8]. Because exposure of primary CD34+ cells to GM-CSF and IL-3 results in granulo-monocytic differentiation [19-22] and the presence of Epo with low IL-3 concentrations favors erythroid differentiation [20], it is possible that environmental factors can functionally modulate tissue specific transcription factors [23-25], thereby influencing the differentiation process.

Thus, exposure of murine bone marrow cells to SF and Epo results in erythroid differentiation and upregulates the protein level of the basic helix-loop-helix transcription factor SCL-Tal 1 [25], a gene that is essential for the establishment of the hemopoietic lineage [26-29]. Its function in mediating the cellular response to SF remains nonetheless to be documented. In the erythroid lineage, SCL expression is maintained up to the proerythroblast stage, whereas SCL is not detectable in the monocyte and granulocyte pathway [30-33]. Consistent with this pattern of expression, SCL has been shown to favor erythroid differentiation [32, 34], and in two leukemic cell lines, M1 and TF-1, the downregulation of SCL is required for monocytic differentiation to proceed in response to cytokines [31] or tetradecanoyl phorbol acetate (TPA) [32]. SCL is therefore a positive regulator of erythroid differentiation and a negative regulator of monocyte differentiation.

Although cell lines represent useful tools for the molecular dissection of biochemical pathways, observations with cell lines ultimately need to be complemented by studies with primary cells, which are more complex because of their intrinsic heterogeneity. Furthermore, cell differentiation, cell proliferation, and apoptosis occur simultaneously and cannot be dissociated such that a monitoring of all three processes is important for an understanding of the role of hemopoietic growth factors and transcription factors. In order to reduce the heterogeneity of primary bone marrow cell populations, we chose to study cells that express the surface antigen CD34, which represents a minority of bone marrow cells (0.5%-1%) [35] and includes all multipotent and bipotent hemopoietic precursors of the granulocytic-monocytic lineages (colony-forming units-granulocyte/macrophage [CFU-GM]) or erythroid-megakaryocytic lineages (BFU-E). We therefore probed the survival, cycling, and differentiation of primary CD34+ cells that were purified to homogeneity by comparing the effects of SF and GM-CSF and addressed the function of SCL in these processes.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Monoclonal Antibodies, and Growth Factors
CD34+ cells were purified from normal bone marrow mononuclear cells by a double negative-positive immunomagnetic separation procedure as described previously [36]. Briefly, lineage positive (Lin+) cells were removed by labeling with antibodies against human glycophorin (YTH 89.1, Serotec; Kidlington, Oxford, UK; http://www.serotec.co.uk), CD11b (Mac-1, American Type Culture Collection [ATCC]; Rockville, MD; http://www.atcc.org) and CD72 (CAMPATH-1, Serotec), followed by an incubation with a monoclonal mouse anti-rat (MAR18.5, ATCC) and a goat anti-mouse antibody coupled to magnetic beads (Dynabeads; Cedarlane, Hornby, Ontario; http://www.cedarlanelabs.com). Lineage negative cells were then labeled with a monoclonal anti-CD34 (My-10, ATCC), and subsequently incubated with the Dynabeads. Finally, CD34+ cells were freed of magnetic beads by an overnight incubation at 37°C in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum ([FCS], GIBCO; Grand Island, NJ). The monoclonal anti-CD13 (My-9) was a generous gift of Dr. J.D. Griffin (Dana Farber Cancer Institute; Boston, MA).

The human cell line TF-1 [37], a kind gift of Dr. T. Kitamura (DNAX; Palo Alto, CA), was maintained at 1.5 x 105 cells/ml in IMDM/10% FCS containing human GM-CSF at 5 ng/ml as described previously [18]. The MO7e cell line, generously given by Dr. Avanzi, was maintained at 3 x 105/ml in IMDM/10% FCS containing human IL-3 at 5 U/ml. Both cell lines were passaged three times weekly [17, 38].

Purified recombinant human GM-CSF was a generous gift of Dr. S.C. Clark (Genetics Institute; Cambridge, MA; http://www.genetics.com) and purified recombinant SF was a generous gift from Dr. K. Langley (Amgen; Thousand Oaks, CA; http://www.amgen.com). Recombinant human IL-3 was prepared by transfection into SV40 transformed African green monkey kidney cells (COS) as described previously [38]. Where indicated, serum-free cultures were supplemented with purified bovine serum albumin ([BSA], 10 mg/ml) (Sigma; St Louis, MO; http://www.sigma-aldrich.com) and iron-saturated transferrin (160 µg/ml) (Calbiochem; La Jolla, CA; http://www.calbiochem.com) as described previously [39].

Flow Cytometry Analysis of Cell Surface Markers or of Nuclear SCL
For fluorescence-activated cell sorter (FACS) analysis, cells were labeled with My-10 (anti-CD34, 100 µl of hybridoma culture supernatant for 106 cells) or My-7 (anti-CD13, 1 µg per 106 cells), followed by a goat anti-mouse antibody coupled to fluorescein isothiocyanate (FITC) for My-9 or coupled to phycoerythrin for My-10 (Caltag; San Francisco, CA; http://www.caltag.com), at a final dilution of 1:40. Nonspecific binding was blocked with a mixture of human immunoglobulin (10 µg/ml) in phosphate-buffered saline (PBS) containing 5% FCS and 0.05% Na azide (Sigma) (sample buffer) as well as 10% normal goat serum (Sigma). Negative controls were labeled with an irrelevant isotype-matched monoclonal antibody (MR98).

For nuclear labeling, cells were permeabilized with the Bouin's fixative for 15 min at room temperature, and Triton X-100 (0.1%) for 5 sec. The cells were then labeled with the monoclonal anti-human SCL BTL73 [31] at a 1:10 dilution, washed extensively with PBS, followed by a goat anti-mouse antibody coupled to FITC. The antibody was a generous gift of Dr. Danièle Mathieu-Mahul (Institut de Génétique Moléculaire; Montpellier, France).

Cell Cycle Analysis
Cells were resuspended at a concentration of 1-2 x 106/ml in sample buffer, centrifuged, and resuspended in 100 µl of propidium iodide (PI) staining solution in Krisham buffer (0.5 µg/µl PI, 0.02 mg/ml RNase, 0.1% Na citrate adjusted to pH 7.4) for 30 min at room temperature, prior to flow cytometry analysis on a Coulter XL (Coulter; Miami, FL) using the System II software for acquisition. The cell cycle analysis was done using Multicycle AV software from Phoenix Flow System (San Diego, CA) to discriminate the G1/G0, S, and G2/M phases. DNA check beads (Coulter) were used for calibration.

MTT Reduction Assay
A rapid colorimetric assay for cellular growth has been set up essentially as described by Mosmann [40]. Briefly, cells were incubated for the indicated times and three hours before harvesting, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) was added to the culture medium at a final concentration of 0.135 mg/ml. Cells were centrifuged, and the pellet resuspended in acid isopropanol (0.04 N HCl, 3% SDS in isopropanol). After a few minutes at room temperature, the samples were read at 570 nm on a DU 64 spectrophotometer (Beckman Coulter; Fullerton, CA; http://www.coulter.com).

The TUNEL Reaction
CD34+/Lin– cells were analyzed for apoptosis using a quantitative DNA fragmentation assay, the terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end labeling (TUNEL) reaction [41]. Briefly, cells were fixed with formaldehyde for 5 min at room temperature and permeabilized with 0.1% Triton X-100, 0.1% sodium citrate at 4°C for 2 min. Cells were then incubated for 1 hour at 37°C with 0.3 nmol biotin-16-dUTP (Boehringer-Mannheim GmbH; Mannheim, Germany), 3 nmol dATP (Pharmacia; Uppsala, Sweden; http://www.pnu.com), 20 U TdT (Pharmacia) in TdT buffer (GIBCO) in a total reaction volume of 50 µl. The reaction was stopped by the addition of 0.5 M EDTA. DNA from apoptotic cells that contained free 3'-hydroxy ends was labeled by the TDT with biotinylated dUTP, which was revealed then with streptavidin-FITC (Amersham; Buckinghamshire, England; http://www.apbiotech.com). After washing, the cells were resuspended in PBS for flow cytometry analysis with the FACScan. Ten thousand events were analyzed for each sample.

Double Fluorochrome Staining Assay
A stock solution of dye mix containing 100 µg/ml acridine orange and 100 µg/ml ethidium bromide was prepared in PBS. Cells were suspended at 5 x 105 to 5 x 106 in IMDM medium. One microliter of dye mix was added to 25 µl of cell suspension, and 10 µl of the mixture was placed on a microscope slide for examination by fluorescent microscopy (Leica; Wetzlar, Germany). Cells with bright green chromatin were considered as viable cells whereas those with bright orange chromatin or collapsed chromatin were considered as dead cells. A minimum of 200 cells were counted.

Antisense SCL (as-SCL) Treatment
A 20-mer oligonucleotide was designed in the antisense orientation with regards to the SCL cDNA, which covers position +147 to +166 and includes the first ATG at position +158 (Met 1): (5') CTC CGT CAT CCT GGG GCA TA (3'). The control oligonucleotide PAKL is also a 20 mer and has the following sequence: (5') TTG TTT ACT GCG TCT ACT AT (3'). Both oligonucleotides were phosphorothioate modified. The oligonucleotides were added to the culture every second day at concentrations of 1, 2, and 5 mM. Cells were harvested on days 2 and 5 for Western blotting and apoptosis assays.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Steel Factor Maintains the Viability of CD34+ Cells Without a Significant Increase in Cell Number
Purified CD34+ cells were maintained with or without SF for 9 to 17 days. Cell recovery was assessed by the MTT assay (Fig. 1AGo), and independently by trypan blue exclusion (Fig. 1BGo). In the absence of growth factor, the cells start to die by day 6 of culture (Fig. 1AGo). The presence of SF maintains cells at input levels for up to 17 days (Fig. 1A and BGo), as observed previously for leukemic cells [17]. In contrast, there was a sixfold increase over input levels when CD34+ cells were maintained with GM-CSF, and a 10-fold increase in the presence of both SF and GM-CSF (Fig. 1BGo), consistent with the view that the combined effect of the two factors were additive [42, reviewed in 43]. The fact that cell numbers remain at input levels suggests that SF sustains cell survival in the absence of cell cycling [44], or, alternatively, that SF is both a survival factor [10-17] and a mitogenic factor [18], but its overall effect is counterbalanced by cell death in a fraction of the population. These possibilities were further examined as described below.



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  		Figure 1. Viability and proliferation of CD34+ cells exposed to GM-CSF, Steel factor (SF), or both. Viability and cell proliferation were measured by MTT assay (panel A) and trypan blue exclusion (panel B) on CD34+ cells. For the MTT assay, 2.3 x 104 cells were plated in 100 µl of Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS) in the presence (20 ng/ml) and absence of SF in microtiter 96-well plates. The optical density was expressed relative to that observed on day 0 of culture, which was taken as 1. For cell count, 2 x 105 CD34+ cells were cultured in the presence of SF (20 ng/ml), GM-CSF (6 ng/ml), or both growth factors in IMDM supplemented with 10% FCS in 24-well plates.

 
CD34+ cells were maintained in serum-free medium, in the absence of growth factor, or in the presence of GM-CSF, SF, or both. After three days, cells were harvested and processed for cell cycle analysis with propidium iodide. Data shown in Table 1Go indicate that SF supports DNA synthesis (S phase) and mitosis (G2/M) in CD34+ cells, and its effect is additive to that observed with GM-CSF.


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  		Table 1. GM-CSF and Steel factor (SF) have an additive effect on S phase progression of CD34+ cells
 
Both SF and GM-CSF Suppress Apoptosis in CD34+ Cells
Apoptosis was examined by the TUNEL reaction in cells exposed to the indicated growth factors for five days (Table 2Go and Fig. 2Go). In the absence of growth factor, more than 60% of the cells were apoptotic. In contrast, cell death was reduced to 34% in the presence of SF, 17% in the presence of GM-CSF, and 0.8% when both were added to the cultures. Comparable results were observed in serum-containing medium (Table 2Go), although the presence of FCS significantly increased cell survival under all conditions. The suppression of apoptosis by SF in serum-free medium was much more apparent in two clonal cell lines, TF-1 and MO7e (Fig. 2Go), both of which were previously shown to secrete GM-CSF in response to IL-1 [36] and SF [45], respectively. Together, our observations indicate that both SF and GM-CSF can rescue CD34+ cells from apoptotic death, but the effect of each growth factor on its own was partial. Again in this assay, the effect of GM-CSF was additive or possibly synergistic to that of SF.


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  		Table 2. Apoptosis in CD34+ cells detected by the TUNEL reaction: rescue by Steel factor (SF), GM-CSF, and fetal calf serum (FCS)
 


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  		Figure 2. Suppression of apoptosis in CD34+ cells and MO7e and TF-1 cells by Steel factor (SF), GM-CSF, or both. Apoptosis was determined by the TUNEL reaction and the percentage of positive cells was quantified by flow cytometry. CD34+ cells and MO7e cells were seeded at a concentration of 0.25 x 106/ml (and 0.15 x 106/ml for TF-1 cells) in serum-free medium in the presence of the indicated growth factors for five days. Growth factor concentrations were as described in Figure 1Go. Data shown are typical of two independent experiments for each sample.

 
Because SF on its own can partially suppress apoptosis and support cell division, our results suggest that the apparent absence of cell growth in cultures supported with SF alone is the net result of cell survival, cell proliferation, and constant cell death.

Contrasting Effects of SF and GM-CSF on Granulomonocytic Differentiation
CD34+ cells were maintained with SF or GM-CSF for 10 days, and harvested for analysis of cell surface markers by flow cytometry: CD34 and CD13, a lineage-specific marker that appears on committed CFU-GM and is retained during differentiation up to the stage of segmented neutrophils (Fig. 3Go). The starting population was more than 95% CD34+, albeit with significant heterogeneity in intensity of expression, as assessed by the spread of the peak of fluorescence, and were CD13low. In the presence of GM-CSF, CD34 expression dropped to background levels, whereas that of CD13 increased significantly. In contrast, when cells were maintained for 10 days in the presence of SF, all of the cells remained CD34+ and CD13low. The mean fluorescence intensity with anti-CD34 was, however, lower than that of cells on day 0.



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  		Figure 3. Cell surface phenotype of CD34+ cells maintained with Steel factor (SF) or GM-CSF for 10 days. CD34+ cells were analyzed for surface expression of CD34 (My-10) and CD13 (My-7) by flow cytometry at the initiation of culture and 10 days after exposure to GM-CSF (6 ng/ml) or SF (20 ng/ml). Cells labeled with an irrelevant isotype matched monoclonal antibody served as negative controls (dotted lines). Unlabeled cells were also analyzed for autofluorescence (data not shown). After 10 days in culture, there was an increase in both autofluorescence and nonspecific binding in cells exposed to GM-CSF.

 
Because of the cellular heterogeneity in CD34+ populations, we determined their contents in colony-forming cells prior to and after culture with SF or GM-CSF. Data shown in Table 3Go indicate that both the frequency as well as the distribution of the different types of colonies remained unaltered in cells maintained with SF. Furthermore, the cells retained their capacity to respond to the synergistic effect of SF and GM-CSF. In contrast, no colony-forming cell was recovered from GM-CSF-supported cultures. Thus, despite the shift from CD34high to CD34int observed above, our results indicate that SF efficiently supports the survival of all day 14 colony-forming cells.


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  		Table 3. Frequency of colony-forming cells in cultures of CD34+ cells maintained with Steel factor (SF) or GM-CSF
 
Contrasting SCL Protein Levels in CD34+ Cells Exposed to SF or GM-CSF
SCL is expressed in CD34+ cells [46], in early hemopoietic cells and is downregulated with granulomonocytic differentiation [32, 47, 48]. Because of the contrasting effects of SF and GM-CSF on granulomonocytic differentiation, we evaluated SCL protein level through flow cytometry analysis of CD34+ cells maintained with SF, GM-CSF, or both (Fig. 4Go). Both CD34 and CD13 expression (Fig. 4AGo) and cell morphology were monitored (Fig. 4BGo). We first confirmed that our starting population was more than 95% CD34+. When maintained with SF for 12 h, 69% of the population displayed nuclear staining for SCL and the cells remain blast-like with CD34 staining. After 10 days of exposure to SF, SCL expression was elevated in a similar proportion of the cells. In contrast, in cells maintained with GM-CSF, SCL expression dropped down to background levels as determined with an isotype-matched irrelevant antibody (MR98) and the cells were all differentiated granulocytes and monocytes. Consistent with the fact that cells maintained with both SF and GM-CSF exhibited CD34 expression, significantly reduced granulocytic differentiation and lack of monocytic differentiation (Fig. 4A and BGo), SCL protein levels remained detectable in these (Fig. 4CGo). Together, our results indicate an inverse correlation between SCL expression and granulocyte-monocyte differentiation in response to GM-CSF.



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  		Figure 4. SCL expression in CD34+ cells at the initiation of culture or following long-term exposure to SF and/or GM-CSF. CD34+ cells were maintained with the indicated growth factors, as described in Figure 1Go. Cells were permeabilized with the Bouin's fixative and stained with the monoclonal mouse anti-human SCL, followed by a goat anti-mouse fluorescein isothiocyanate (FITC). Controls were labeled with an irrelevant antibody MR98. In parallel, the purity of the starting population was assessed by labeling intact cells with anti-CD34 (left panels). An average of 10,000 events was analyzed per group, except for SF day 10 and GM day 10 in which cell recovery was much lower, and the number of events was consequently reduced to 4,000 and 1,000, respectively. Cell morphology was evaluated on Wright-Giemsa stained cytosmears.

 
Apoptosis in CD34+ Cells Exposed to as-SCL
In order to examine the potential function of SCL in sustaining the survival of CD34+ cells to either SF or GM-CSF, cells were exposed to modified as-SCL. The feasibility of the approach was first addressed in TF-1 cells, which require either SF [17, 32] or GM-CSF [32, 38] for survival. Cells were exposed continuously to oligonucleotides (as-SCL or control oligonucleotides), which were added to the culture medium every second day during the five-day culture period. Because the control oligonucleotide PAKL was not toxic at concentrations that were below 5 µM (data not shown and Fig. 5Go), we determined the effects of as-SCL on SCL protein levels as well as the survival of TF-1 cells exposed to GM-CSF or SF in the presence or in the absence of 2 and 5 µM as-SCL or PAKL, respectively. Western blotting indicated that a concentration of 2 µM of as-SCL was sufficient to reduce SCL protein levels by half (Fig. 5AGo). In GM-CSF-supported cultures, cell survival was not affected by the presence of as-SCL (Fig. 5BGo). In contrast, in cultures maintained with SF, cell survival was significantly reduced by as-SCL but not the control oligonucleotide (Fig. 5BGo). Unlike parental TF-1 cells, cells expressing bcl-2 were not affected by as-SCL at any concentration tested, suggesting that bcl-2 overexpression overcame the pro-apoptotic effect of as-SCL. We therefore tested the effect of as-SCL on purified CD34+ cells.



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  		Figure 5. Effects of a modified antisense SCL (as-SCL) on SCL protein levels, as well as the survival response of TF-1 cells exposed to Steel factor (SF) or GM-CSF. Western blotting was performed on TF-1 cells exposed to SF or GM-CSF, in the absence or in the presence of 2 µM (+) as-SCL or control oligonucleotide (PAKL) for two days (upper panel). Data shown are typical of two independent experiments. Similar results were observed on day 5 (data not shown). The 42 and 39 kDa protein bands specifically revealed by the antibody correspond to SCL isoforms initiated at Met 1 and Met 26, respectively [33]. Apoptotic cells (lower panel) were detected through staining with the double fluorochrome technique, also shown at 2 µM of oligonucleotide. At least 200 cells were counted. Data shown are the mean of duplicate counts and are typical of two independent experiments. In contrast to parental TF-1 cells, bcl-2 transfectants (MSCV-bcl2) survive equally well under all culture conditions.

 
As with parental TF-1 cells, primary CD34+ cells maintained with GM-CSF were not sensitive to as-SCL over a five-day period, that is, before the onset of overt granulomonocytic differentiation. In contrast, the suppression of apoptosis by SF was specifically and significantly impaired by as-SCL after five days (Fig. 6Go). Together, our observations suggest a requirement in constant SCL levels for the suppression of apoptosis in CD34+ cells by SF. In contrast, high SCL levels may not be essential for their survival in response to GM-CSF. In fact, the cells remained viable even after 10 days of continuous exposure to as-SCL and differentiated normally (data not shown).



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  		Figure 6. Effects of a modified antisense SCL (as-SCL) on the survival of CD34+ cells exposed to Steel factor (SF) or GM-CSF. Purified CD34+ cells were exposed to SF, GM-CSF, or both, in the presence and in the absence of as-SCL (2 µM). Apoptotic cells were examined on days 2 and 5, as described in Figure 5Go.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies indicate that SF suppresses apoptosis in hemopoietic cells in the absence of detectable differentiation [15, 17, 44], whereas IL-3 and GM-CSF may actually favor their terminal differentiation [19-22]. The molecular events underlying these processes remain, however, to be documented. In parallel, the transcription factor SCL was previously shown through immunocytochemistry to be expressed in a subset of CD34+ cells [46]. In the present study, we use purified CD34+ cells to analyze the biologic effects of SF and GM-CSF in these cells. We also optimized flow cytometry techniques to analyze SCL expression in these purified populations and to identify its implication in cell survival in response to cytokines and/or cell differentiation using antisense oligonucleotides.

Our results indicate that either SF or GM-CSF can partially suppress apoptosis in primary CD34+ cells. As observed in cell proliferation and colony formation, the effect of SF and GM-CSF on cell survival was additive, both in serum-free cultures or in serum-containing cultures. Because cell survival was optimal in serum-containing medium, we addressed SCL function under these conditions. Cells maintained with SF retained SCL expression, which is required for their survival response to SF. In contrast, CD34+ cells maintained with GM-CSF underwent differentiation and lost SCL expression. In agreement with this decreased expression, SCL itself does not appear to be essential for GM-CSF-dependent cell survival. Previous work indicated that colony formation supported by GM-CSF in CD34+ cells required PU.1 function [21], although it was not clear whether the absence of colony formation due to disrupted PU.1 function may be attributed to decreased cell proliferation or cell survival in response to GM-CSF. In the present study, we used the TUNEL reaction as well as the pattern of nuclear staining with acridine orange to clearly establish that cells treated with antisense SCL underwent apoptosis on exposure to SF. Together, these observations are consistent with the view that the additive effects of SF and GM-CSF may be due to the convergence of two independent molecular pathways, one requiring SCL function and the other one, possibly, PU.1.

Exposure to SF did not affect the differentiation potential of CD34+ cells, as determined through cell surface marker analysis and replating with later acting factors (data not shown) as well as the capacity of the cells to reconstitute the hemopoietic system of an irradiated host [44]. In contrast, GM-CSF favors granulomonocytic differentiation, an effect that is in part inhibited by co-exposure to SF. Thus, SF and GM-CSF have additive effects on cell proliferation [43], cell cycling, and cell survival, while having opposing effects on cell differentiation (shown herein). It is possible that their opposing effects on cell differentiation are also mediated by SCL. Previous work indicated that SCL is downregulated in differentiating granulomonocytic cells [24, 32, 47]. Ectopic SCL expression in three different hemopoietic cell lines has in fact been shown to inhibit monocytic differentiation in response to TPA, or to cytokines such as leukemia inhibitory factor and oncostatin M [31, 32] as well as granulocytic differentiation in response to retinoic acid [48]. Together, the results suggest that the downregulation of SCL is required for cell differentiation to proceed along the monocytic and, to some extent, the granulocytic pathway. Consistent with this, SF is shown here to sustain SCL expression in primary CD34+ cells and maintain cells in an undifferentiated state, whereas GM-CSF downregulates SCL protein levels and favors granulocyte-monocyte differentiation. Furthermore, comparison of cultures maintained with SF, GM-CSF, or both shows an inverse correlation between SCL expression and monocytic as well as granulocytic differentiation.

A more direct assessment of the role of SCL in monocytic differentiation will entail overexpressing SCL in CD34+ cells. Although work performed on two different cell lines, TF-1 [32] and M1 [31], indicated that ectopic SCL expression prevents their terminal maturation along the monocytic pathway, it was shown that scl–/– embryonic stem cells could contribute to monocyte differentiation when rescued with SCL [27]. It is therefore possible that SCL interferes with some aspects of monocytic differentiation (as observed in isolated cell lines) without antagonizing the whole process itself.

The interrelationship between cell survival and differentiation goes beyond the impact of extracellular signals on apoptotic death. Thus, GATA-1 is a zinc finger transcription factor that is required for terminal maturation in the erythroid lineage. The ablation of GATA-1 gene through homologous recombination in embryonic stem cells results in a block in cell differentiation at the proerythroblast stage. Examination of internucleosomal DNA cleavage sites by the TUNEL reaction revealed that the cells underwent apoptosis [49]. Thus, apoptosis may be linked to insufficient survival signals from the environment, but also to inefficient or abortive cell differentiation [8, 49]. Together, the observations are consistent with the view that the differentiation process requires survival signals that are cell autonomous, as exemplified by the role of GATA-1, or mediated by extracellular signals that include soluble or membrane-bound SF, GM-CSF, and Epo. Evidence available so far suggests that cell survival supported by extracellular growth factors may also be mediated through modulation of lineage-specific transcription factors such as SCL (herein) or GATA-1 [21, reviewed in 50]. Because apoptosis induced by as-SCL can be rescued by ectopic bcl-2 expression, our observations are consistent with the view that SCL performs its survival function upstream of members of the bcl-2 family.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors wish to thank Dr. Martin Gyger (Maisonneuve-Rosemont Hospital, Montreal) for providing access to human bone marrow cells, Nathalie Bouchard for help with the cell separation procedure, Magali Domin and Odile Royer for expert secretarial assistance, and Dr. Guy Sauvageau for critical comments on the manuscript.

This work was supported by grants from the National Cancer Institute of Canada with funds from the Canadian Cancer Society, the Medical Research Council of Canada, and a studentship from the Université de Montréal, Bourse Canada and Bourse Vidéotron (J.R.C.C.).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Williams GT, Smith CA, Spooncer E et al. Haemopoietic colony stimulating factors promote cell survival by suppressing apoptosis. Nature 1990;343:76-79.[CrossRef][Medline]

  2. Barres BA, Hart IK, Coles HS et al. Cell death and control of cell survival in the oligodendrocyte lineage. Cell 1992;70:31-46.[CrossRef][Medline]

  3. Deckwerth TL, Johnson EM Jr. Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor. J Cell Biol 1993;123:1207-1222.[Abstract/Free Full Text]

  4. Begley CG, Lopez AF, Nicola NA et al. Purified colony-stimulating factors enhance the survival of human neutrophils and eosinophils in vitro: a rapid and sensitive microassay for colony-stimulating factors. Blood 1986;68:162-166.[Abstract/Free Full Text]

  5. Kaplan DR, Miller FD. Neutrophin signal transduction in the nervous system. [Review]. Cur Opin Cell Biol 2000;10:381-391.

  6. Hockenbery D, Nunez G, Milliman C et al. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 1990;348:334-336.[CrossRef][Medline]

  7. O'Reilly LA, Huang DC, Strasser A. The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J 1996;15:6979-6990.[Medline]

  8. Linette GP, Korsmeyer SJ. Differentiation and cell death: lessons from the immune system. [Review]. Curr Opin Cell Biol 1994;6:809-815.[CrossRef][Medline]

  9. Nishikawa S, Kusakabe M, Yoshinaga K et al. In utero manipulation of coat color formation by a monoclonal antic-kit antibody: two distinct waves of c-kit-dependency during melanocyte development. EMBO J 1991;10:2111-2118.[Medline]

  10. Lahav R, Lecoin L, Ziller C et al. Effect of the Steel gene product on melanogenesis in avian neural crest cell cultures. Differentiation 1994;58:133-139.[CrossRef][Medline]

  11. Steel KP, Davidson DR, Jackson IJ. TRP-2/DT, a new early melanoblast marker, shows that steel growth factor (c-kit ligand) is a survival factor. Development 1992;115:1111-1119.[Abstract]

  12. Dolci S, Williams DE, Ernst MK et al. Requirement for mast cell growth factor for primordial germ cell survival in culture. Nature 1991;352:809-811.[CrossRef][Medline]

  13. Godin I, Deed R, Cooke J et al. Effects of the steel gene product on mouse primordial germ cells in culture. Nature 1991;352:807-809.[CrossRef][Medline]

  14. Yee NS, Paek I, Besmer P. Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: basis for radiosensitivity of white spotting and steel mutant mice. J Exp Med 1994;179:1777-1787.[Abstract/Free Full Text]

  15. Abrahamson JL, Lee JM, Bernstein A. Regulation of p53-mediated apoptosis and cell cycle arrest by Steel factor. Mol Cell Biol 1995;15:6965-6960.

  16. Morrison-Graham K, Takahashi Y. Steel factor and c-kit receptor: from mutants to a growth factor system. [Review]. Bioessays 1993;15:77-83.[CrossRef][Medline]

  17. Caceres-Cortes J, Rajotte D, Dumouchel J et al. Product of the steel locus suppresses apoptosis in hemopoietic cells. Comparison with pathways activated by granulocyte macrophage colony-stimulating factor. J Biol Chem 1994;269:12084-12091.[Abstract/Free Full Text]

  18. Gore SD, Amin S, Weng LJ et al. Steel factor supports the cycling of isolated human CD34+ cells in the absence of other growth factors. Exp Hematol 1995;23:413-421.[Medline]

  19. Heyworth CM, Dexter TM, Kan O et al. The role of hemopoietic growth factors in self-renewal and differentiation of IL-3-dependent multipotential stem cells. Growth Factors 1990;2:197-211.[Medline]

  20. Sposi NM, Zon LI, Care A et al. Cell cycle-dependent initiation and lineage-dependent abrogation of GATA-1 expression in pure differentiating hematopoietic progenitors. Proc Natl Acad Sci USA 1992;89:6353-6357.[Abstract/Free Full Text]

  21. Voso MT, Burn TC, Wulf G et al. Inhibition of hematopoiesis by competitive binding of transcription factor PU.1. Proc Natl Acad Sci USA 1994;91:7932-7936.[Abstract/Free Full Text]

  22. Yonemura Y, Ku H, Hirayama F et al. Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells. Proc Natl Acad Sci USA 1996;93:4040-4044.[Abstract/Free Full Text]

  23. Blobel GA, Sieff CA, Orkin SH. Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor. Mol Cell Biol 1995;15:3147-3153.[Abstract]

  24. Labbaye C, Valtieri M, Barberi T et al. Differential expression and functional role of GATA-2, NF-E2, and GATA-1 in normal adult hematopoiesis. J Clin Invest 1995;95:2346-2358.

  25. Miller BA, Floros J, Cheung JY et al. Steel factor affects SCL expression during normal erythroid differentiation. Blood 1994;84:2971-2976.[Abstract/Free Full Text]

  26. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 1995;373:432-434.[CrossRef][Medline]

  27. Porcher C, Swat W, Rockwell K et al. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 1996;86:47-57.[CrossRef][Medline]

  28. Robb L, Lyons I, Li R et al. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci USA 1995;92:7075-7079.[Abstract/Free Full Text]

  29. Robb L, Elwood NJ, Elefanty AG et al. The SCL gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J 1996;15:4123-4129.[Medline]

  30. Condorelli G, Vitelli L, Valtieri M et al. Coordinate expression and developmental role of Id2 protein and TAL1/E2A heterodimer in erythroid progenitor differentiation. Blood 1995;86:164-175.[Abstract/Free Full Text]

  31. Tanigawa T, Elwood N, Metcalf D et al. The SCL gene product is regulated by and differentially regulates cytokine responses during myeloid leukemic cell differentiation. Proc Natl Acad Sci USA 1993;90:7864-7868.[Abstract/Free Full Text]

  32. Hoang T, Paradis E, Brady G et al. Opposing effects of the basic helix-loop-helix transcription factor SCL on erythroid and monocytic differentiation. Blood 1996;87:102-111.[Abstract/Free Full Text]

  33. Pulford K, Lecointe N, Leroy-Viard K et al. Expression of TAL-1 proteins in human tissues. Blood 1995;85:675-684.[Abstract/Free Full Text]

  34. Aplan PD, Nakahara K, Orkin SH et al. The SCL gene product: a positive regulator of erythroid differentiation. EMBO J 1992;11:4073-4081.[Medline]

  35. Civin CI, Banquerigo ML, Strauss LC et al. Antigenic analysis of hematopoiesis. VI. Flow cytometric characterization of My-10-positive progenitor cells in normal human bone marrow. Exp Hematol 1987;15:10-17.[Medline]

  36. Rodriguez C, Lacasse C, Hoang T. Interleukin-1 beta suppresses apoptosis in CD34 positive bone marrow cells through activation of the type I IL-1 receptor. J Cell Physiol 1996;166:387-396.[CrossRef][Medline]

  37. Kitamura T, Tange T, Terasawa T et al. Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin. J Cell Physiol 1989;140:323-334.[CrossRef][Medline]

  38. Rajotte D, Haddad P, Haman A et al. Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3. J Biol Chem 1992;267:9980-9987.[Abstract/Free Full Text]

  39. Hoang T, Haman A, Goncalves O et al. Interleukin 1 enhances growth factor-dependent proliferation of the clonogenic cells in acute myeloblastic leukemia and of normal human primitive hemopoietic precursors. J Exp Med 1988;168:463-474.[Abstract/Free Full Text]

  40. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.[CrossRef][Medline]

  41. Sgonc R, Boeck G, Dietrich H et al. Simultaneous determination of cell surface antigens and apoptosis. Trends Genet 1994;10:41-42.[CrossRef][Medline]

  42. Caceres-Cortes JR, Hoang T. Product of the Steel locus can replace leukemic cell interaction. Cancer Res 1992;52:5208-5212.[Abstract/Free Full Text]

  43. Galli SJ, Zsebo KM, Geissler EN. The kit ligand, stem cell factor. [Review]. Adv Immunol 1994;55:1-96.[Medline]

  44. Li CL, Johnson GR. Stem cell factor enhances the survival but not the self-renewal of murine hematopoietic long-term repopulating cells. Blood 1994;84:408-414.[Abstract/Free Full Text]

  45. Kiss C, Cesano A, Zsebo KM et al. Human stem cell factor (c-kit ligand) induces an autocrine loop of growth in a GM-CSF-dependent megakaryocytic leukemia cell line. Leukemia 1993;7:235-240.[Medline]

  46. Mouthon MA, Bernard O, Mitjavila MT et al. Expression of tal-1 and GATA-binding proteins during human hematopoiesis. Blood 1993;81:647-655.[Abstract/Free Full Text]

  47. Brady G, Billia F, Knox J et al. Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Curr Biol 1995;5:909-922.[CrossRef][Medline]

  48. Condorelli GL, Tocci A, Botta R et al. Ectopic TAL-1/SCL expression in phenotypically normal or leukemic myeloid precursors—proliferative and antiapoptotic effects coupled with a differentiation blockade. Mol Cell Biol 1997;17:2954-2969.[Abstract]

  49. Weiss MJ, Orkin SH. Transcription factor GATA-1 permits survival and maturation of erythroid precursors by preventing apoptosis. Proc Natl Acad Sci USA 1995;92:9623-9627.[Abstract/Free Full Text]

  50. Shivdasani RA, Orkin SH. The transcriptional control of hematopoiesis. [Review]. Blood 1996;87:4025-4039.[Free Full Text]

Received September 26, 2000; accepted for publication October 2, 2000.




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