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Stem Cells, Vol. 15, No. 1, 63-72, January 1997
© 1997 AlphaMed Press

Raf-1 Protein is Required for Growth Factor-Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines

Karen W. Muszynskia, Francis W. Ruscettib, John M. Gooyaa, Diana M. Linnekinb, Jonathan R. Kellera

a Intramural Research Support Program, SAIC Frederick and
b Laboratory of Leukocyte Biology, Biological Response Modifiers Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, USA

Key Words. Raf-1 • Synergy • Hematopoiesis • Stem cells

Dr. Jonathan Keller, P.O. Box B, NCI-FCRDC, Frederick, MD 21702-1201, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Raf-1 is a serine/threonine kinase that has been identified as a component of growth factor-activated signal transduction pathways, and is required for growth factor-induced proliferation of leukemic cell lines and colony formation of hematopoietic progenitors stimulated with single colony-stimulating factors, which promote the growth of committed hematopoietic progenitor cells. However, it is known that the most primitive progenitors in the bone marrow require stimulation with multiple cytokines to promote cell growth. We have determined that c-raf antisense oligonucleotides inhibit the growth of murine lineage-negative progenitors stimulated with two-, three- and four-factor combinations of growth factors, including GM-CSF + interleukin (IL)- 1, IL-3 + steel factor (SLF), IL-3 + IL-11 + SLF and IL-3 + IL-11 + SLF + G-CSF. In addition, c-raf antisense oligonucleotides inhibit the synergistic response of the MO7e human progenitor cell line induced to proliferate with IL-3 + SLF (99%) or GM-CSF + SLF (99%). In contrast, c-raf antisense oligonucleotides only partially inhibited day 14 colony formation of CD34+ human progenitors stimulated with IL-3 + SLF (50%) or GM-CSF + SLF (55%) but completely inhibited day 7 colony formation. However, pulsing CD34+ cells with additional oligonucleotides on day 7 of the colony assay further inhibited day 14 colony formation (70%-80%). Furthermore, a comparison of the effect of c-raf antisense oligonucleotides on the synergistic response of normal human fetal liver cells in [3H]thymidine incorporation assays and colony assays showed strong inhibition in short-term proliferation assays and partial inhibition in 14-day colony assays. Taken together, these results demonstrate that partial inhibition of colony formation of primitive human progenitors stimulated with multiple growth factors is a result of the length (14 days) of the human colony assay and does not represent a differential requirement of primitive progenitors for Raf-1. Thus Raf-1 is required for the proliferation and differentiation of primitive hematopoietic progenitor cells stimulated with synergistic combinations of cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Raf-1 is a 74 kDa serine/threonine kinase that is located in the cell cytoplasm and is activated by phosphorylation following mitogen or growth factor stimulation of a variety of cell types [1]. Biochemical studies have determined that in hematopoietic cells, Raf-1 is activated by phosphorylation following growth factor stimulation of factor-dependent cell lines with interleukin (IL)-2, IL-3, IL-4, GM-CSF, erythropoietin (EPO), G-CSF, colony-stimulating factor (CSF-1) and steel factor (SLF) [2-7]. In addition, experiments using c-raf antisense oligonucleotides to inhibit Raf-1 expression have determined that Raf-1 is required for growth factor-induced proliferation of murine progenitor cell lines stimulated with IL-2, IL-3, GM-CSF, G-CSF, CSF-1, EPO, IL-4, IL-6, leukemia inhibitory factor (LIF) or SLF, as well as colony formation of murine and human bone marrow (BM) cells stimulated with single growth factors (IL-3 or GM-CSF) [4, 7, 8].

While IL-3 and GM-CSF alone induce the growth of committed hematopoietic progenitors in vitro, growth of more primitive hematopoietic progenitors requires stimulation with multiple cytokines [9-13]. For example, it has been demonstrated that cytokines such as SLF, IL-1, IL-4, IL-6 and LIF, which alone have little or no effect on colony formation, can synergize with CSFs such as IL-3 or GM-CSF to enhance both the size and number of colonies of BM cells enriched for primitive progenitors [13-20]. In this regard, it is unknown if Raf-1 is required for the proliferation and differentiation of more primitive progenitors in the BM. Therefore, to determine the role of Raf-1 in growth factor-induced synergistic responses, we used c-raf antisense oligonucleotides to inhibit Raf-1 expression in several different populations of hematopoietic cells enriched for primitive progenitors that respond to synergistic combinations of cytokines including, murine lineage-negative (Lin–) progenitors, CD34+ purified human progenitors, human fetal liver cells and the growth factor-dependent human MO7e leukemic cell line. The results demonstrate that Raf-1 is required for the in vitro growth of both human and murine primitive progenitors stimulated with multiple combinations of growth factors. Furthermore, our experiments demonstrate a limitation of the antisense approach in the 14-day human colony assay and indicate that partial inhibition of colony formation by antisense oligonucleotides must be evaluated in the context of potential degradation of the oligonucleotides.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
BM Cells
Normal murine BM cells were aspirated from BALB/c mouse femurs with Iscove's modified Dulbecco's medium (IMDM) and layered on lymphocyte separation medium (Organon Teknika Corporation; Durham, NC) to obtain light density cells. Animal care was provided in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985). Lin– cells were selected using previously published techniques [9]. Briefly, unseparated murine BM cells were incubated with RA3-6B2 (B220 antigen), RA3-8C5 (both gifts of R. Coffman, DNAX Corporation; Palo Alto, CA), Mac-1 (Boehringer-Mannheim; Indianapolis, IN), Lyt-2 (CD8), and L3T4 (CD4) (Becton Dickinson; Rochelle Park, NJ) antibodies which recognize myeloid and lymphoid-specific cell surface antigens for 30 min at 4°C. Cells were then washed twice and incubated with antirat IgG-coated magnetic beads (Dynal Corporation; Oslo, Norway) at a bead-to-cell ratio of 40:1 for 30 min at 4°C. Cells were then separated with a magnetic particle concentrator (Dynal) and the Lin– cells were recovered in the supernatant and resuspended in IMDM supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 U/ml), and 3 mg/ml L-glutamine. Lin–/Kit+ cells were purified by fluorescence-activated cell-sorting using an anti-c-kit polyclonal antibody (Pharmingen; San Diego, CA). Human BM cells were obtained by aspiration from healthy donors after informed consent. The low density mononuclear cells were isolated from the interphase after Ficoll-Hypaque (Pharmacia Biotech; Piscatawa, NJ) gradient centrifugation, washed twice and suspended in IMDM supplemented with 25% FCS, 1% detoxified bovine serum albumin, penicillin (100 U/ml), streptomycin (100 U/ml) and 3 mg/ml L-glutamine. CD34+ cells were obtained by positive selection using previously published techniques [21]. Briefly, magnetic beads (Dynabead M-450 CD34, Dynal) with CD34-specific BL-3C5 monoclonal antibodies attached to them were added to cells at a bead-to-cell ratio of 1:1 and anti-Fab antiserum (10 µl/1 x 107 cells) (Detachabead, Dynal) was used for detachment of beads from positively selected cells. CD34+ cells were recovered by magnetically separating the beads using a magnetic particle concentrator (Dynal) and resuspended in IMDM with supplements.

Human Fetal Liver Cells and Cell Lines
Human fetal liver tissue retrieved from products of conception following induced abortions at 13-18 weeks gestation and obtained with informed consent, were supplied by Advanced Bioscience Resources, Inc. (Alameda, CA). Liver samples were homogenized through a 16-gauge hypodermic needle and low density mononuclear cells were isolated from the interphase after Ficoll-Hypaque (Pharmacia Biotech) gradient centrifugation. Cells were washed twice and suspended in QBS-58 serum-free medium (Quality Biologicals, Gaithersburg, MD) supplemented with penicillin (100 U/ml), streptomycin (100 U/ml) and 3 mg/ml L-glutamine. The MO7e growth factor-dependent human cell line was originally derived from the peripheral blood of an infant with acute megakaryocytic leukemia [22]. MO7e cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS, penicillin (100 U/ml), streptomycin (100 U/ml), and 3 mg/ml L-glutamine and GM-CSF (50 ng/ml).

Growth Factors
Purified recombinant murine (rMu)IL-3 and rMuGM-CSF were obtained from Peprotech (Rocky Hill, NJ). Purified recombinant human (rHu)G-CSF was supplied by L. Souza, Amgen Corporation (Thousand Oaks, CA). SLF and rHuIL-3 were a gift of S. Gillis, Immunex Corporation (Seattle, WA). rHuGM-CSF was a gift from I. McNiece, Amgen Corporation. Human EPO was obtained from the Biological Response Modifiers Program, Frederick Cancer Research and Development Center (FCRDC) (Frederick, MD) and was originally purchased from Ortho Biotech (Raritan, NJ). Human SLF and human IL-11 were purchased from Peprotech and purified human IL-1{alpha} was supplied by Hoffmann-LaRoche (Nutley, NJ).

Synthesis of Oligodeoxyribonucleotides
Phosphorothioate antisense (5'-TCCCTGTATGT GCTC CAT-3'), sense (5'-ATGGAGCACATACAGGGA-3') and nonsense (5'-TTTTTGCACCAGCTTGCC-3') oligonucleotides synthesized by the H-phosphonate method were gel purified using electrophoresis and thin-layer chromatography [23, 24]. Additional oligonucleotides with phosphorothioate linkages at the second 5' and the last 3' base [25] were synthesized by the phosphoramadite method using tetraethylthiuram disulfide and standard procedures (Applied Biosystems User Bulletin No. 58) on an automated synthesizer (model 380-B; Applied Biosystems; Foster City, CA) and purified using oligonucleotide purification cartridges (Applied Biosystems). Purified oligomers dissolved in ammoniated water were concentrated in a Speed Vac concentrator and resuspended at a 100 µM concentration in sterile H2O.

[3H]thymidine Incorporation Assay
Normal murine BM cells (1 x 105), fetal liver cells (1 x 104), or MO7e cells (5 x 103) were seeded in 100 µl of their usual media in 96-well plates and preincubated overnight (12-15 h) with one-half the final concentration of oligonucleotides at 37°C, 5% CO2. Cells growing in media alone or in media plus growth factors were used as controls. The following day, the remaining aliquot of oligonucleotides was added and cells were stimulated with IL-3 (30 ng/ml), GM-CSF (50 ng/ml), SLF (50 ng/ml) or EPO (5 U/ml) as indicated. Fetal liver and MO7e cells were grown for 96 h and normal murine BM cells were grown for 72 h at 37°C, 5% CO2. Before harvesting, BM and fetal liver cells were pulsed overnight (12-14 h) and MO7e cells were pulsed for 6-8 h with 1 µCi [3H]thymidine (6.7 Ci/mmol, New England Nuclear; Boston, MA). Cells were harvested onto glass filters and [3H]thymidine incorporation was measured by scintillation counting.

Colony Assay
A modification of the method of Stanley et al. [26] was used to measure colony formation of BM cells. Purified murine Lin– cells (5 x 103) or Lin–/kit+ cells (5 x 102), unseparated human BM cells (3 x 105), CD34+ purified human cells (3 x 104) or human fetal liver cells (5 x 104) were suspended in 100 µl/plate of their usual media supplemented with growth factors in 15 ml tubes. Murine BM cells were stimulated with rMuIL-3 (30 ng/ml), GM-CSF (50 ng/ml), or SLF (50 ng/ml), and human BM and fetal liver cells were stimulated with rHuIL-3 (30 ng/ml), GM-CSF (50 ng/ml) or SLF (50 ng/ml). One-half the final concentration of oligonucleotides was added to each tube and cells were incubated overnight for 12-15 h at 37°C, 5% CO2. The following day, the remaining oligonucleotides were added to each 15 ml tube and cells were allowed to sit at room temperature for 30 min. Media supplemented with growth factors at previously indicated concentrations was added to each 15 ml tube to give a final volume of 1 ml/plate, and cells were plated in 0.5% Seaplaque agarose in 35-mm Lux petri dishes. Cell viability was assessed by trypan blue exclusion counting before plating and all samples contained >90% viable cells. Plates were incubated in a fully humidified incubator at 37°C, 5% CO2. In some experiments using unseparated or CD34+ human BM cells, an additional aliquot of oligonucleotides in 100 µl of medium was added directly to the agar plates on day 7. Colonies containing >50 cells (unless otherwise indicated) were scored on day 7 for unseparated and Lin– purified murine BM cells, and on day 14 for unseparated or CD34+ purified human BM cells and fetal liver cells.

Western Blot Analysis
Fetal liver cells, seeded at 1 x 105 cells/ml in a 24-well plate, were treated with 10 µM concentrations of sense or antisense oligonucleotides as described for the [3H]thymidine assay and stimulated with SLF (50 ng/ml) plus IL-3 (30 ng/ml). Cells were harvested 72 h after growth factor stimulation and lysed in 100 µl RIPA buffer (50 mM Tris-HCL, pH 7.3, 150 mM NaCl, 1% Triton X-l00, 0.5% deoxycholate, 0.1% SDS, 5 mM EDTA, 1 mM sodium orthovanadate, 25 mM sodium fluoride, 10 mM sodium pyrophosphate, 25 mM glycerophosphate). Insoluble material was removed by centrifugation at 4°C for 30 min at 12,000 g. Protein lysates were resolved by SDS-PAGE and transfered electrophoretically to nitrocellulose membranes. The blots were blocked with 5% nonfat dry milk in TBST buffer (50 mM Tris-HCL, pH 7.3, 150 mM NaCl, 0.5% Triton X-100) for 30 min and then incubated overnight at 4°C with anti-c-Raf mouse monoclonal antibody (Transduction Laboratories; Lexington, KY). After incubation with the antibody, blots were washed three times for 10 min with TBST buffer and then incubated with a secondary antibody (goat antirabbit IgG) conjugated to alkaline phosphatase for 30 min at room temperature. Protein bands were detected using the ECL system (Amersham Corp.; Arlington Heights, IL). The filter was then washed two times in TBST and reprobed with a mouse monoclonal anti-MAP kinase antibody (Zymed Laboratories; San Francisco, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Effect of c-raf Antisense Oligonucleotides on Normal Murine and Human BM Cell Colony Formation Induced by Synergistic Combinations of CSFs
It has been previously demonstrated that phosphorothioate oligonucleotides containing base pair antisense sequences complementary to the translation start site of the murine and human c-raf genes specifically inhibit expression of both c-raf mRNA [8] and Raf-1 protein [4, 7] in a variety of hematopoietic cell types. Therefore, to determine the requirement for Raf-1 in mediating the synergistic effects of multiple cytokines on cell growth, we evaluated the effect of c-raf antisense oligonucleotides on colony formation of primitive progenitors in the BM.

We first examined the dose response of c-raf oligonucleotides on IL-3-induced proliferation of unseparated normal murine BM cells using a [3H]thymidine incorporation assay. c-raf antisense oligonucleotides inhibited IL-3-induced proliferation of normal murine BM cells in a dose-dependent manner (Fig. 1Go). Maximum inhibition of proliferation (99%) was achieved at a 7.0 µM concentration of c-raf antisense oligonucleotides, while sense or nonsense oligonucleotides with the same purine:pyrimidine ration as the antisense oligonucleotides had little or no effect on proliferation at this concentration (Fig. 1Go).



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  		Figure 1. Dose-dependence of c-raf antisense oligonucleotide inhibition of proliferation of normal murine BM cells. Unseparated murine BM cells (1 x 105) were treated with c-raf sense, nonsense or antisense oligonucleotides at the concentrations indicated and stimulated with IL-3 (30 ng/ml). Cells were cultured for 72 h and assayed for [3H]thymidine incorporation as described in Materials and Methods. The results are the mean of triplicate samples from a single experiment.

 
To determine the requirement for Raf-1 in mediating the synergistic effects of multiple cytokines on the growth of primitive progenitors in the BM, we first evaluated the effect of c-raf antisense oligonucleotides on colony formation of murine Lin– cells stimulated with synergistic combinations of cytokines. Stimulation of Lin– progenitor cells with IL-1, IL-11 or G-CSF alone has no effect on colony formation, while stimulation with SLF alone induces formation of clusters of less than 20 cells (data not shown). The combination of GM-CSF + IL-1-induced formation of a greater number of colonies than GM-CSF alone and a synergistic effect on colony formation was also observed when Lin– cells were stimulated with IL-3 + SLF (Table 1Go). Addition of IL-11 or G-CSF enhanced the size of colonies induced by IL-3 + SLF but did not affect colony number (Table 1Go). In addition, there is a linear relationship between the number of Lin– cells seeded and the number of colonies induced by synergistic factors (data not shown).


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  		Table 1. Effect of c-raf oligonucleotides on colony formation of Lin– progenitors stimulated with synergistic combinations of cytokines
 
c-raf antisense oligonucleotides inhibited colony formation of Lin– cells stimulated with IL-3 or GM-CSF alone by 97% and 94%, respectively, and also inhibited colony formation induced by synergistic combinations of GM-CSF + IL-1 (84%) and IL-3 + SLF (84%). In addition, c-raf antisense oligonucleotides inhibited colony formation induced by the three-factor combination of IL-3 + SLF + IL-11 (88%), and the four-factor combination of IL-3 + SLF + IL-11 + G-CSF (90%) (Table 1Go). To evaluate the effect of c-raf antisense oligonucleotides on colony formation of a more highly enriched population of primitive progenitors, Lin– cells were further purified by selection of Lin–/Kit+ cells. Similar to their effect on Lin– cells, c-raf antisense oligonucleotides inhibited formation of Lin–/Kit+ cells stimulated with IL-3 or IL-3 + SLF by 88% and 93%, respectively (Table 2Go). Thus, c-raf antisense oligonucleotides inhibited colony formation of murine Lin– cells and Lin–/Kit– cells stimulated with a variety of synergistic combinations of cytokines.


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  		Table 2. Effect of c-raf oligonucleotides on colony formation of Lin–/Kit+ progenitors stimulated with synergistic combinations of cytokines
 
Effect of c-raf Antisense Oligonucleotides on Colony Formation of CD34+ Purified Human BM-Derived Progenitors Induced by Synergistic Cytokines
We next determined the effect of c-raf antisense oligonucleotides on colony formation of CD34+ purified human BM-derived progenitors stimulated with multiple cytokines. Colony formation of CD34+ cells stimulated with IL-3 + SLF or GM-CSF + SLF showed a >100% increase over the number of colonies induced by IL-3 or GM-CSF alone (Table 3Go). As expected, c-raf antisense oligonucleotides inhibited IL-3- or GM-CSF-induced colony formation of CD34+ cells by 90% and 97%, respectively [7]; however, the antisense oligonucleotides only partially inhibited colony formation of CD34+ cells stimulated with the synergistic combinations of IL-3 + SLF (50%) or GM-CSF + SLF (55%) (Table 3Go). Partial inhibition of colony formation was also observed when CD34+ cells were stimulated with other synergistic combinations of cytokines, including G-CSF + SLF and the four-factor combination of G-CSF + GM-CSF + SLF + IL-6 (data not shown). Morphological analysis by Wright-Giemsa staining of the colonies induced by IL-3 + SLF or GM-CSF + SLF indicated there was no significant difference in the distribution of colony types between untreated, sense- or antisense-treated cells. GM-CSF + SLF induced formation of granulocyte (47%), macrophage (15%) and mixed granulocyte/macrophage (36%) colonies of sense-treated cells and also induced formation of granulocyte (63%), macrophage (9%) and granulocyte/macrophage (27%) colonies of antisense-treated cells. Similarly, IL-3 + SLF stimulation of sense-treated cells induced formation of granulocyte (50%), macrophage (9%) and granulocyte/macrophage (40%) colonies and induced formation of granulocyte (45%), macrophage (10%) and granulocyte/macrophage (46%) colonies of antisense-treated cells. This result suggested that partial inhibition of colony formation by c-raf antisense oligonucleotides did not reflect differential inhibition of progenitors for different cell types. In addition, day 14 colonies of antisense-treated cells were smaller (100-200 cells) than colonies of sense-treated cells, which ranged in size from 100 to >=1,000 cells. Since optimum colony formation of human BM cells requires a 14-day incubation period, we considered the possibility that the concentration of oligonucleotides might be insufficient for complete inhibition of colony formation of human BM cells. However, increasing the concentration of oligonucleotides did not result in an increase in the inhibitory effects of the antisense oligonucleotides on colony formation (data not shown).


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  		Table 3. Effect of c-raf oligonucleotides on colony formation of CD34+ BM-derived progenitorsa
 
Partial Inhibition of Colony Formation of Human BM Cells by c-raf Antisense Oligonucleotides is Related to Degradation of the Oligonucleotides in the 14-Day Colony Assay
It has been suggested that the stability of oligonucleotides in tissue culture cells is less than three days [27, 28]. Therefore, since we evaluated colony formation of human progenitors after 14 days, degradation of the antisense oligonucleotides could account for partial inhibition of colony formation of human BM cells. To evaluate this possibility, the effect of c-raf oligonucleotides on colony formation of unseparated and CD34+ human BM cells stimulated with GM-CSF + SLF was evaluated on both day 7 and day 14 (Table 4Go). c-raf antisense oligonucleotides inhibited day 7 colony formation of unseparated BM cells by 89% and colony formation of CD34+ cells by 93% (Table 3Go). In contrast, c-raf antisense inhibition of day 14 colony formation of unseparated BM cells and CD34+ cells was partially inhibited by 39% and 40%, respectively (Table 4Go). Thus, the inhibitory effects of the antisense oligonucleotides were almost complete for day 7 colony formation, while day 14 colony formation was only partially inhibited.


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  		Table 4. c-raf antisense oligonucleotide inhibition of day 7 and day 14 colony formation of human BM cells stimulated with GM-CSF + SLFa
 
We next determined if we could increase the inhibitory effects of the antisense oligonucleotides on day 14 by pulsing the cultures with an additional dose of oligonucleotides on day 7 (Fig. 2Go). As expected, c-raf antisense oligonucleotides inhibited day 14 colony formation of unseparated and CD34+ BM cells stimulated with GM-CSF + SLF by 41% and 50%, respectively (Fig. 2Go). However, in parallel plates, which were pulsed with an additional dose of oligonucleotides on day 7, c-raf antisense oligonucleotides inhibited day 14 colony formation of unseparated BM cells by 74% and inhibited colony formation of CD34+ cells by 70%, while the addition of sense oligonucleotides on day 7 had little or no effect on colony formation (Fig. 2Go). Thus, the addition of oligonucleotides at multiple time points results in an increase in c-raf antisense inhibition of day 14 colony formation.



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  		Figure 2. The effect of multiple additions of c-raf oligonucleotides on colony formation of human BM cells stimulated with GM-CSF + SLF. (A) Unseparated human BM cells (3 x 105) or (B) CD34+ purified progenitors (3 x 104) were treated with 10 or 5 µM concentrations of oligonucleotides, respectively, in the presence of GM-CSF + SLF and plated in soft agar. An additional dose of oligonucleotides (10 or 5 µM in 100 µl of complete media) was subsequently added to the plates on day 7 and colony formation was evaluated on day 14. The results are the mean ± SE for duplicate plates from a single experiment and are representative of three separate experiments.

 
Effect of c-raf Antisense Oligonucleotides on Proliferation of MO7e Cells Stimulated with Single or Synergistic Combinations of Growth Factors
The increased inhibitory effects of multiple additions of antisense oligonucleotides on day 14 colony formation suggested that the partial effect of c-raf on synergistic responses was due to degradation of the oligonucleotides. However, complete inhibition of day 14 colony formation of cells stimulated with either single (unseparated BM) or synergistic factors (unseparated and CD34+) was not observed in the colony assay regardless of the protocol used to deliver the oligonucleotides. Consequently, we could not determine if all of the partial effects of c-raf antisense oligonucleotides on colony formation were related to limitations in the colony assay due to oligonucleotide instability. Therefore, we further evaluated the effect of c-raf oligonucleotides on the growth of hematopoietic progenitors stimulated with synergistic combinations of growth factors in short-term proliferation assays.

Since CD34+ cells could not be obtained in sufficient quantities for use in the proliferation assays, we evaluated the effect of the oligonucleotides on growth factor-induced proliferation of a human hematopoietic progenitor cell line, MO7e, which maximally proliferates in response to synergistic combinations of growth factors. While MO7e cells proliferate in response to IL-3, GM-CSF and SLF alone, the proliferative response of MO7e cells stimulated with IL-3 + SLF or GM-CSF + SLF is greater than the additive effect of either factor alone (i.e., synergistic) (Fig. 3Go). c-raf antisense oligonucleotides completely inhibited IL-3- (99%), GM-CSF- (99%), or SLF-induced (99%) proliferation of MO7e cells. Furthermore, c-raf antisense oligonucleotides completely inhibited proliferation induced by the combinations of IL-3 + SLF (99%) and GM-CSF + SLF (99%), while c-raf sense oligonucleotides had no significant effect on proliferation (Fig. 3Go). Thus, Raf-1 is required for growth factor-induced proliferation of MO7e cells stimulated with either single or synergistic combinations of growth factors.



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  		Figure 3. c-raf antisense oligonucleotides inhibit growth factor-induced proliferation of MO7e cells stilated with synergistic combinations of growth factors. Factor-dependent MO7e cells (5 x 103) responsive to synergistic combinations of cytokines were treated with 10 µM concentrations of c-raf sense or antisense oligonucleotides in the presence of IL-3 (30 ng/ml), GM-CSF (50 ng/ml) or SLF (50 ng/ml) alone or in combinations as indicated. Cells cultured in media alone or in media + growth factors were used as controls. Cells were assayed for [3H]thymidine incorporation 96 h after the addition of growth factor as described in Materials and Methods. The results are the mean ± SE for triplicate samples from a single experiment and are representative of three separate experiments.

 
The Effect of c-raf Antisense Oligonucleotides on the Proliferation of Fetal Liver Cells Stimulated with Single or Synergistic Combinations of Growth Factors
Human fetal liver cells represent a population of normal hematopoietic cells enriched for progenitors that respond to synergistic combinations of factors in both the colony assay and five-day proliferation assays [29, 30]. Therefore, we compared the effect of the antisense oligonucleotides on the growth of fetal liver cells in the five-day proliferation assay to their effect on colony formation in the 14-day colony assay. In the proliferation assay, c-raf antisense oligonucleotides inhibited the proliferation of fetal liver cells stimulated with SLF (99%), IL-3 (99%), GM-CSF (92%) and EPO (99%) (Fig. 4Go). In addition, c-raf antisense oligonucleotides inhibited the synergistic proliferative response induced by IL-3 + SLF (93%), GM-CSF + SLF (97%), EPO + SLF (94%), IL-3 + EPO (88%), and the five-factor combination of IL-3 + EPO + SLF + G-CSF + IL-6 (86%) (Fig. 4Go).



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  		Figure 4. c-raf antisense oligonucleotides inhibit proliferation of human fetal liver cells stimulated with single or synergistic combinations of growth factors. Human fetal liver cells (1 x 104) were treated with 10 µM concentrations of c-raf sense or antisense oligonucleotides in the presence of IL-3 (30 ng/ml), GM-CSF (50 ng/ml), EPO (5 U/ml), SLF (50 ng/ml), G-CSF (50 ng/ml), IL-6 (50 ng/ml) alone or in combinations of these growth factors as indicated. Cells growing in media alone or in media + growth factors were used as controls. Cells were cultured for 96 h and assayed for [3H]thymidine incorporation as described in Materials and Methods.

 
In the colony assay, IL-3-, GM-CSF-, and EPO-induced colony formation of fetal liver cells increased 10-fold when cells were stimulated with synergistic combinations of growth factors including IL-3 + SLF, GM-CSF + SLF, EPO + SLF and IL-3 + EPO (Table 5Go). In contrast to the results of the proliferation assay, c-raf antisense oligonucleotides partially inhibited colony formation of fetal liver cells stimulated with either single factors including IL-3 (74%), GM-CSF (71%) and EPO (69%) or the synergistic combinations of IL-3 + SLF (55%), GM-CSF + SLF (71%), EPO + SLF (64%) or EPO + IL-3 (61%) (Table 5Go). These results demonstrate that c-raf antisense oligonucleotides completely inhibit the growth factor-induced synergistic response of fetal liver cells in the proliferation assay but only partially inhibit colony formation of fetal liver cells stimulated with synergistic combinations of cytokines.


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  		Table 5. Effect of c-raf oligonucleotides on CSF-induced colony formation of human fetal liver cellsa
 
c-raf Antisense Oligonucleotides Inhibit Raf-1 Protein Expression in Fetal Liver-Derived Primitive Progenitor Cells
It has been demonstrated that c-raf antisense oligonucleotides specifically inhibit Raf-1 protein expression in FDCP-1 murine myeloid cells [7] and in the MO7e human cell line [8]. However, the effect of c-raf antisense oligonucleotides on Raf-1 protein expression has not been previously evaluated in a normal human cell population. To determine if c-raf antisense oligonucleotides specifically inhibit c-raf gene expression in a normal population of primitive progenitor cells, we evaluated the effect of the antisense oligonucleotides on Raf-1 protein expression in human fetal liver cells stimulated with IL-3 + SLF by Western blot analysis (Fig. 5Go). Whole cell lysates of untreated and sense or antisense oligonucleotide-treated fetal liver cells were probed with an anti-Raf-1 monoclonal antibody which recognizes the 74 kDa Raf-1 protein. Raf-1 expression was completely inhibited in antisense-treated cells in contrast to sense-treated cells, in which the oligonucleotides had little or no effect on expression of Raf-1 (Fig. 5Go). In comparison, neither sense nor antisense oligonucleotides inhibited expression of MAP kinase (Fig. 5Go), a 44 kDa serine/threonine kinase located downstream in the Raf-1 regulated signal transduction pathway [31, 32]. The decrease in the level of MAP kinase expression observed in the antisense-treated cells reflects a difference in the number of cells present in the media control (6.2 x 105), sense- (5.4 x 105) and antisense-treated (3.2 x 105) cultures prior to lysis. These results demonstrate that c-raf antisense oligonucleotides specifically inhibit Raf-1 protein expression in a normal population of primitive progenitor cells.



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  		Figure 5. c-raf antisense oligonucleotides inhibit Raf-1 protein expression in primitive progenitor cells. Fetal liver cells (1 x 105) treated with c-raf sense or antisense oligonucleotides as previously described, were stimulated with SLF (50 ng/ml) + IL-3 (30 ng/ml). Untreated fetal liver cells stimulated with IL-3 + SLF were used as controls. Cells were lysed 72 h after the addition of growth factors and cell lysates were analyzed for Raf-1 expression by Western blot analysis. An anti-c-Raf mouse monoclonal antibody detected the 74 kDa Raf-1 protein band in the untreated and sense-treated control lanes but not in the antisense-treated cell lysates. The filter was reprobed with an anti-MAP kinase antibody which detected the 44 kDa MAP kinase protein band in both antisense- and sense-treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
Previous experiments have established that c-raf antisense oligonucleotides specifically inhibit Raf-1 protein expression and that Raf-1 is required for colony formation of normal murine and human BM cells stimulated with single cytokines [4, 7, 8]. Similar to their effect on colony formation of murine Lin– cells stimulated with single cytokines, c-raf antisense oligonucleotides also inhibited colony formation of murine Lin– (84%-99%) or Lin–/Kit+ (88%) cells stimulated with synergistic combinations of cytokines. Furthermore, while c-raf antisense oligonucleotides completely inhibited (>99%) colony formation of human CD34+ cells stimulated with single cytokines, complete inhibition of CSF-induced colony formation of CD34+ cells stimulated with synergistic combinations of growth factors could not be achieved. This suggested several possibilities: A) that not all human BM-derived progenitors required Raf-1 for CSF-induced colony formation; B) that stimulation with synergistic factors could override the requirement for Raf-1, or C) that differences between the seven-day murine and 14-day human colony assay might account for the reduced effectiveness of the antisense oligonucleotides in inhibition of colony formation of human BM cells.

Comparing the effects of c-raf antisense oligonucleotides on day 7 versus day 14 colony formation of human BM cells stimulated with IL-3 + SLF revealed that, similar to the result with murine BM cells, the c-raf antisense oligonucleotides almost completely inhibited colony formation of human BM cells on day 7. However, day 14 colony formation was only partially inhibited (39%-40%). Day 7 colonies arise from more mature, committed progenitors in the BM [33-36], while day 14 colonies arise from primitive progenitors that require stimulation by multiple cytokines to enter the cell cycle [33]. Thus, partial inhibition could represent a differential sensitivity of committed versus primitive progenitors to the c-raf antisense oligonucleotides. Alternatively, oligonucleotide instability could account for partial inhibition of day 14 colony formation. In this regard, the addition of a second dose of oligonucleotide on day 7 increased the inhibitory effects of the antisense oligonucleotides on day 14 colony formation, suggesting that degradation of the oligonucleotides was responsible for the observed partial inhibition. In support of this conclusion and consistent with the results of a previous study [8], c-raf antisense oligonucleotides inhibited >99% of MO7e cell proliferation induced by synergistic combinations of cytokines [8].

Furthermore, c-raf antisense oligonucleotides completely inhibited (86%-99%) the proliferation of fetal liver cells stimulated with either single or synergistic combinations of cytokines ([3H]thymidine assays). Interestingly, colony formation of fetal liver cells was only partially inhibited (56%-75%) in the 14-day colony assay. Taken together, the results of [3H]thymidine and colony-forming assays using fetal liver cells suggest that partial rather than complete inhibition of CSF-induced colony-formation of human cells by c-raf antisense oligonucleotides is primarily the result of the instability of the oligonucleotides in the colony assay. However, these results do not rule out the possibility that colony formation of some primitive cells in the BM is partially Raf-1-independent. Our experiments demonstrate that the significance of partial inhibition of colony formation by antisense oligonucleotides must be evaluated in the context of potential degradation of the oligonucleotides in the human colony assay.


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
c-raf antisense inhibition of growth factor-induced proliferative responses of both normal and leukemic hematopoietic cells stimulated with synergistic combinations of cytokines demonstrates that stimulation with synergistic combinations of cytokines is not sufficient to override the requirement for Raf-1 in growth factor-induced proliferation. In addition, these results demonstrate that expression of Raf-1 is required for the proliferation and differentiation of the primitive hematopoietic progenitors which respond to synergistic combinations of cytokines. These results are consistent with those of previous studies suggesting that Raf-1 functions as a shared second messenger that integrates cell surface signals for the multiplicity of ligand/receptor systems involved in the induction of proliferation [1].


   Acknowledgments
 
We thank Kathy Mood for synthesis of the oligonucleotides and Dr. Dan Longo for review of the manuscript. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. Government. The work upon which this publication is based was performed pursuant to Contract #NO1-CO-56000 with the National Cancer Institute, Department of Health and Human Services. The U.S. Government retains a nonexclusive, royalty-free license to use or duplicate this article in any manner and for any purpose whatsoever, and to have or permit others to do so.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

  1. Rapp UR. Role of raf-1 serine/threonine protein kinase in growth factor signal transduction. Oncogene 1991;6:495-500.[Medline]

  2. Turner B, Rapp U, App H et al. Interleukin 2 induces tyrosine phosphorylation and activation of p72-74 raf-1 kinase in a T-cell line. Proc Natl Acad Sci USA 1991;88:1227-1231.[Abstract/Free Full Text]

  3. Miyazawa K, Hendrie PC, Mantel C et al. Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of raf-1, GTPase-activating protein and mitogen-activated protein kinase. Exp Hematol 1991;19:1110-1123.[Medline]

  4. Carroll MP, Spivak JL, McMahon M et al. Erythropoietin induces raf-1 activation and raf-1 is required for erythropoietin-mediated proliferation. J Biol Chem 1991;266:14964-14969.[Abstract/Free Full Text]

  5. Carroll MP, Clark-Lewis I, Rapp UR et al. Interleukin-3 and granulocyte-macrophage colony-stimulating factor mediate rapid phosphorylation and activation of cytostolic c-raf. J Biol Chem 1990;265:19812-19817.[Abstract/Free Full Text]

  6. Baccarini M, Sabatini DM, App H et al. Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the raf-1 proto-oncogene product. EMBO J 1990;9:3649-3657.[Medline]

  7. Muszynski KW, Ruscetti FW, Heidecker G et al. Raf-1 protein is required for growth factor-induced proliferation of hematopoietic cells. J Exp Med 1994;181:2189-2199.[Abstract/Free Full Text]

  8. Brennscheidt U, Riedel D, Kolch W et al. Raf-1 is a necessary component of the mitogenic response of the human megakaryoblastic leukemia cell line M07 to human stem cell factor, granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 9. Cell Growth Differ 1994;5:367-371.[Abstract]

  9. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science 1988;241:58-62.[Abstract/Free Full Text]

  10. Metcalf D. The Hematopoietic Colony-Stimulating Factors. Amsterdam: Elsevier Science Publishers, 1984:55-92.

  11. Metcalf D. The molecular control of cell division differentiation commitment and maturation in haemopoietic cells. Nature 1989;339:27-30.[Medline]

  12. Nicola NA. Hematopoietic cell growth factors and their receptors. Annu Rev Biochem 1989;58:45-77.[Medline]

  13. Heyworth CM, Whetton AD, Nicholis S et al. Stem cell factor directly stimulates the development of enriched granulocyte-macrophage colony-forming cells and promotes the effects of other colony-stimulating factors. Blood 1992;80:2230-2236.[Abstract/Free Full Text]

  14. Jacobsen SEW, Ruscetti FW, Dubois CM et al. Induction of colony-stimulating factor receptor expression on hematopoietic progenitor cells: proposed mechanism for growth factor synergism. Blood 1992;80:1-10.[Free Full Text]

  15. Ruscetti FW, Dubois CM, Jacobsen SEW et al. Transforming growth factor ß and interleukin-1. In: Lord B, Dexter TM, eds. Growth Factors in Haemopoiesis. London: W.B. Saunders Company, 1991:703-721.

  16. de Vries P, Brasel KA, Eisenman JR et al. The effect of recombinant mast cell growth factor on purified murine hematopoietic stem cells. J Exp Med 1991;173:1205-1211.[Abstract/Free Full Text]

  17. Broxmeyer HE, Maze R, Miyazawa K et al. The kit receptor and its ligand, steel factor, as regulators of hemopoiesis. Cancer Cells 1991;3:480-487.[Medline]

  18. Bartelmez SH, Bradley TR, Bertoncello I et al. Interleukin 1 + interleukin 3 + colony-stimulating factor 1 are essential for clonal proliferation of primitive myeloid BM cells. Exp Hematol 1989;17:240-249.[Medline]

  19. Leary A, Wong G, Clark SC et al. Leukemia inhibitory factor differentiation inhibiting activity/human interleukin for DA cells augments proliferation of human hematopoietic stem cells. Blood 1990;75:1960-1965.[Abstract/Free Full Text]

  20. Rennick D, Hudak S, Yang G et al. Regulation of hemopoietic cell development by interleukins 4, 5 and 6. Immunol Res 1989;8:215-225.[Medline]

  21. Smeland EB, Funderud S, Kvalheim G et al. Isolation and characterization of human hematopoietic progenitor cells: an effective method for positive selection of CD34+ cells. Leukemia 1992;6:845-852.[Medline]

  22. Avanzi GC, Lista P, Giovinazszo B et al. Selective growth response to IL-3 of a human leukaemic cell line with megakaryoblastic features. Br J Haematol 1988;69:359-363.[Medline]

  23. Stec WJ, Zon G, Egan W et al. Automated solid-phase synthesis, separation, and stereochemistry of phosphorothioate analogues of oligodeoxynucleotides. J Am Chem Soc 1984;106:6077-6079.

  24. Froehler BC, Ng PG, Matteucci MD. Synthesis of DNA deoxynucleotide H-phosphonate intermediate. Nucleic Acids Res 1986;14:5399-5407.[Abstract/Free Full Text]

  25. Temsamani J, Tang JY, Padmapriya M et al. Pharmacokinetics, biodistribution, and stability of capped oligodeoxynucleotide phosphorothioates in mice. Antisense Res Dev 1993;3:277-284.[Medline]

  26. Stanley ER, Bartocci A, Patinkin D et al. Regulation of very primitive multipotent, hemopoietic cells by hemopoietin-1. Cell 1986;45:667-671.[Medline]

  27. Marti G, Egan W, Noguchi P et al. Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells. Antisense Res Dev 1992;2:27-39.[Medline]

  28. Hawley P, Gibson I. The detection of oligodeoxynucleotide molecules following uptake into mammalian cells. Antisense Res Dev 1992;2:119-127.[Medline]

  29. Hann IM, Bodger MP, Hoffbrand AV. Development of pluripotent hematopoietic progenitor cells in the human fetus. Blood 1983;62:118-123.[Abstract/Free Full Text]

  30. Papayannopoulou T, Brice M, Broudy VC et al. Isolation of c-kit receptor-expression cells from BM, peripheral blood, and fetal liver: functional properties and composite antigenic profile. Blood 1991;78:1403-1412.[Abstract/Free Full Text]

  31. Kyriakis JM, App H, Zhang X et al. Raf-1 activates MAP kinase-kinase. Nature 1992;358:417-420.[Medline]

  32. Howe LR, Leevers SJ, Gomez N et al. Activation of the MAP kinase pathway by the protein kinase raf. Cell 1992;71:335-342.[Medline]

  33. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood 1993;81:2844-2853.[Abstract/Free Full Text]

  34. Leary AG, Zeng HQ, Clark SC et al. Growth factor requirements for survival in G0 and entry into the cell cycle of primitive human hemopoietic progenitors. Proc Natl Acad Sci USA 1992;89:4013-4017.[Abstract/Free Full Text]

  35. Suda T, Suda J, Ogawa M. Proliferative kinetics and differentiation of murine blast cell colonies in culture: evidence for variable G periods and constant doubling rates of early pluripotent hemopoietic progenitors. J Cell Physiol 1983;117:308-318.[Medline]

  36. Lajtha LG. Stem cell concepts. Differentiation 1979;14:23-28.[Medline]

Received June 5, 1996; accepted for publication September 11, 1996.



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