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HEMATOPOIESIS
From the Section of Neonatal-Perinatal Medicine,
Department of Pediatrics, Herman B Wells Center for Pediatric Research,
Indiana University School of Medicine, Indianapolis, IN; and
the Division of Experimental Hematology, Cincinnati Children's
Hospital Medical Center, Cincinnati, OH.
Two alternatively spliced stem cell factor (SCF) transcripts encode
protein products, which differ in the duration of membrane presentation. One form, soluble SCF (S-SCF) gets rapidly processed to
yield predominantly secreted protein. The other form,
membrane-associated SCF (MA-SCF) lacks the primary proteolytic
cleavage site but is cleaved slowly from an alternate site, and thus
represents a more stable membrane form of SCF. Mutants of SCF that lack
the expression of MA-SCF (Steel-dickie) or possess a defect
in its presentation (Steel17H) manifest
deficiencies in erythroid cell development. In this study, we have
compared the consequence(s) of activating Kit, the receptor for SCF by
MA-SCF with S-SCF, and an obligate membrane-restricted (MR)
form of SCF (MR-SCF) on erythroid cell survival, proliferation, cell
cycle progression, and the activation of p38 and ERK MAP kinase
pathways. Activation of Kit by MR-SCF was associated with a
significantly lower incidence of apoptosis and cell death in erythroid cells compared to either other isoform. MR- or
MA-SCF-induced stimulation of erythroid cells resulted in similar and
significantly greater proliferation and cell cycle progression compared
to soluble SCF. The increase in proliferation and cell cycle
progression via MA- or MR-SCF stimulation correlated with sustained and
enhanced activation of p38 and ERK MAP kinase pathways. In addition,
MR- or MA-SCF-induced proliferation was more sensitive to the
inhibitory effects of ERK inhibitor compared to S-SCF-induced
proliferation. In contrast, soluble SCF-induced proliferation was more
sensitive to the inhibitory effects of p38 inhibitor compared with MR-
or MA-SCF. These results suggest that different isoforms of SCF may use different biochemical pathways in stimulation of survival and/or proliferation of erythroid cells.
(Blood. 2002;100:1287-1293) Mice mutant at either the Steel
(Sl) or dominant White spotting
(W) loci display similar phenotypic abnormalities,
including defects in hematopoiesis, gametogenesis, and
melanogenesis.1 The receptor tyrosine kinase Kit and the
ligand of Kit, stem cell factor (SCF), are encoded at the W
and Sl loci, respectively.2 Sl
encodes a membrane protein with extracellular, membrane-spanning, and
cytoplasmic domains. Two alternatively spliced mRNA
transcripts encode SCF protein products, soluble (S)-SCF and a
membrane-associated (MA)-SCF, that differ in sequences amino-terminal
of the transmembrane segment.3-7
S-SCF is processed rapidly and efficiently by proteolytic
cleavage at a site in exon 6 to produce secreted protein of 164 amino
acids. As a consequence, very low levels of S-SCF are detected on the
surface of hematopoietic microenvironment (HM)-derived stromal
cells.5-9 In contrast, MA-SCF lacks the major proteolytic cleavage site encoded by exon 6 that is responsible for the
generation of secreted protein, and thus represents a more stable
membrane form of the protein.5-9 Ultimately, in some cells
MA-SCF is also processed to produce secreted protein using a secondary
cleavage site encoded by exon 7 sequences. This cleavage occurs at a
slower rate.5-7 Our laboratory has previously shown using
site-directed mutagenesis that loss of proteolytic cleavage sites in
both exons 6 and 7 results in the generation of an obligate
membrane-restricted (MR) and biologically active form of
SCF.7-9
Important insights into the respective role(s) of MA- and S-SCF have
come from the characterization of Sl mutants, transgenic and
knockout mice that express only the slowly secreted form of SCF
(MA-SCF).3,4,8-12 Two alleles at the Sl locus,
Steel-dickie (Sld) and
Steel17H (Sl17H), are of
particular interest with regard to the function of the MA form of SCF.
Sld encodes a biologically active but obligately
secreted SCF protein as a result of an intragenic deletion of sequences
encoding the membrane-spanning domain and cytoplasmic tail of the
protein.3,4 Because of the severity of the
phenotypes of Sld mice, the membrane form of SCF
would appear to be critical for normal function in the affected
lineages.1 The Sl17H allele, as a
result of a frame shift mutation, encodes a protein in which the
cytoplasmic tail is replaced by a novel polypeptide chain of similar
length.10 We and others have shown that this frame shift
mutation results in a significant reduction in the expression of the MA
form of SCF on HM-derived stromal cells.9,12 These animals
also demonstrate defective hematopoiesis.
One of the major consequences of the lack of membrane expression
(Sld) or defective presentation
(Sl17H) of SCF by the stromal cells of the HM is
manifested in the cells of erythroid lineage.1,3,4,8,9
Both mutants demonstrate lifelong macrocytic anemia in spite of the
presence of secreted SCF. Studies performed on transgenic mice that
express MR-SCF in Sld or
Sl17H mutant background have demonstrated a
significant correction in both hematocrits and total red cell numbers
compared with mutants expressing the S-SCF as a
transgene.8,9 Further studies performed in mice expressing
only the MA-SCF have also suggested an essential role for membrane
presentation of SCF in normal erythroid development.11
In spite of the in vivo studies described above, it remains unclear
whether an obligate membrane-restricted form of SCF alone is sufficient
for normal erythroid proliferation and survival or whether different
isoforms of SCF activate differing signaling pathways in Kit-positive
cells. Therefore, to more clearly delineate the role of membrane
presentation of SCF and soluble SCF in erythropoiesis, we have compared
survival, proliferation, cell cycle progression, and the activation of
p38 and ERK MAP kinase pathways in response to stimulation of erythroid
cells by HM-derived stromal cells engineered to express either MR-SCF
alone, MA-SCF, or S-SCF.
In the present study, the role of ERK and p38 MAP kinases were examined
because various hematopoietic cytokines, interleukins, and
colony-stimulating factors that regulate hematopoietic cell growth,
survival, and differentiation have been found to activate these kinase
pathways.13-19 However, the respective role(s) of these
distinct MAP kinases in erythropoiesis has not yet been fully
elucidated. Our results demonstrate that activation of Kit by membrane
isoforms of SCF is associated with significantly lower incidence
of apoptosis and cell death of erythroid cells compared with
the soluble isoform. Further, activation of Kit by MR-SCF results in
proliferation, cell cycle progression, and activation of both p38 and
ERK MAP kinase at levels similar to that observed via MA-SCF. In
contrast, stimulation by S-SCF results in relatively less
proliferation, cell cycle progression, and activation of p38
and ERK MAP kinase. Interestingly, MR- or MA-SCF-induced proliferation was significantly more sensitive to the inhibitory effects of ERK MAP
kinase inhibitor compared with S-SCF, whereas S-SCF-induced proliferation was significantly more sensitive to the inhibitory effects of p38 MAP kinase inhibitor compared with MR- or MA-SCF. These
data suggest that MR-SCF can substitute for MA form in mediating functional and biochemical responses via Kit in erythroid cells. Further, the extent of use of different signaling biochemical pathways
downstream of Kit may depend upon the nature of presentation of the SCF ligand.
Cell line
Proliferation assays
Apoptosis/survival assays The effect of different SCF isoforms on G1E-ER2 cell survival/death (apoptosis and necrosis) was assayed by staining the cells with annexin V and propidium iodide (PI). On the day before assay, parental Sl/Sl4 stromal cells and stromal cells expressing various isoforms of SCF were prepared as above and seeded at 1 × 105 cells/well in 0.1% gelatin-coated 24-well plates. The cultures were incubated in DMEM, 10% CS at 37°C. After 24 hours, 5 × 105 G1E-ER2 cells were added to each well either in the presence or the absence of indicated concentrations of the MEK inhibitor or p38 inhibitor and cultured for 48 hours. Thereafter, cells were harvested and washed once with PBS and resuspended in 100 uL binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells were then stained with 5 uL annexin V (Pharmingen, San Diego, CA) and 500 ng PI (Calbiochem, La Jolla, CA) and incubated at room temperature for 15 minutes in the dark. Then 400 uL binding buffer was added and the cells were analyzed using flow cytometric analysis on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).Expression of SCF in stromal cell transfectants and activation of MAP kinase pathways in G1E-ER2 cells Expression of SCF isoforms in stromal cell transfectants was determined by loading equal amounts of protein (2.4 µg per lane) and performing Western blot analysis using an anti-mouse SCF polyclonal antibody (Peprotech, Rocky Hill, NJ). Activation of p38 and ERKs was determined by using phospho-specific p38 and ERK MAP kinase antibodies (Cell Signaling Technology, Beverly, MA). These antibodies detect p38 and ERK-1 and ERK-2 MAP kinase only when these proteins are catalytically activated by phosphorylation at threonine (Thr) 180/tyrosine (Tyr) 182 for p38 and Thr202 and Tyr204 for ERK-1 and ERK-2. Briefly, Sl/Sl4 cells expressing various SCF isoforms were prepared as described above. The cells were washed and plated on 6-well gelatin-coated plates (1 × 106/well) and cultured for 36 to 48 hours. G1E-ER2 cells were factor-starved for 6 to 8 hours in IMDM, then 5 to 6 × 106 cells were loaded onto stromal cells and further cocultured for various time points at 37°C. Thereafter, cells were harvested and lysed in lysis buffer (10 mM K2HPO4, 1 mL EDTA, 5 mM EGTA, 10 mM MgCl2, 1 mM Na2VO4, 50 mM beta-glycerol-phosphate, 10 ug/mL leupeptin, 1 ug/mL pepstatin, and 10 µg/mL aprotinin) at 4°C for 30 minutes. Cell lysates were clarified by centrifugation for 30 minutes at 10 000g at 4°C. Western blot analysis was performed according to the manufacturer's instructions (New England Biolabs, Beverly, MA). Phosphorylation of ERK and p38 MAP kinase was quantitated by performing densitometry on Western blots using the Eagle Eye II system (Stratagene, La Jolla, CA). Data are presented as relative phosphorylation of ERK and p38, with highest phosphorylation taken as 100.
Activation of Kit by MA-SCF or MR-SCF stimulates enhanced proliferation and cell cycle progression in erythroid cells compared with S-SCF stimulation We have previously described stromal cells derived from mice deficient in the expression of endogenous SCF that have been engineered to express different forms of SCF (Sl/Sl4).7-9 To compare proliferation, survival, and cell cycle progression of erythroid cells in response to stimulation by soluble, MR-, and MA-SCF, we used an SCF-responsive erythrocytic progenitor cell line, G1E-ER2.9,20-22 To closely mimic the events associated with erythroid cell proliferation, cell cycle progression, and survival within the HM, a coculture assay system was used that allows direct interaction of stromal elements with target erythroid cells. Stromal cell transfectants expressing comparable levels of soluble, MA, or the MR forms of SCF were used in these studies (Figure 1A). Concentration of secreted SCF protein in S-SCF stromal cell cultures was estimated by performing Western blot analysis and comparing the amount of secreted SCF protein in conditioned medium with known amounts of Escherichia coli-derived recombinant SCF. As shown in Figure 1B (lane 1), approximately 1 ng/mL SCF protein is secreted in these stromal cell cultures. This level is similar to that noted in our previously published observations.6 Analysis of SCF-induced proliferation of G1E-ER2 cells was performed using a thymidine incorporation assay. G1E-ER2 cells were cocultivated with mitomycin C-treated stromal cell transfectants expressing the soluble, MA, or the MR form of SCF. After 48 hours of coculture, [3H]thymidine was added for 6 hours, and [3H] incorporation was determined. As expected and as previously shown,9 coculturing G1E-ER2 cells with untransfected parental Sl/Sl4 stromal cells (which lack SCF expression) induced only low levels of proliferation (Figure 2A). In contrast, a significant increase in proliferation of G1E-ER2 cells was noted in response to cocultivation on stromal cells expressing either soluble, MA-, or MR-SCF over untransfected parental Sl/Sl4 cells. A similar level of proliferation of G1E-ER2 cells was seen in response to stimulation by both MA- and MR-SCF isoforms (Figure 2A). Consistent with the inability of S-SCF to sustain the proliferation of hematopoietic stem and progenitor cells as described previously,6 stimulation of G1E-ER2 cells by S-SCF resulted in significantly less proliferation over a 48-hour coculture period compared to MA- or MR-SCF (Figure 2A). Also, an increase in the entry of cells in the "S" phase of cell cycle was noted when the cells were cocultivated on stroma expressing MA or MR forms of SCF (Figure 2B). These data suggest that the slow secreting and the obligate membrane-restricted form of SCF induce similar levels of proliferation and cell cycle progression. Thus, MR-SCF may substitute for the MA isoform in inducing proliferation of erythroid cells, whereas S-SCF appears to induce quantitatively fewer proliferative signals than isoforms with longer membrane presentation.
SCF is an important survival factor for Kit-positive cells and has
previously been shown to protect some cells from growth factor
withdrawal-induced apoptosis.23 To determine the
survival-promoting effects of soluble, MA, and MR isoforms of SCF on
G1E-ER2 cells, we compared apoptosis in G1E-ER2 cells cocultured on
stromal cells expressing SCF isoforms using a combination of propidium
iodide (PI) and annexin V staining. G1E-ER2 cells were factor-starved for 4 to 6 hours and then cocultured for 48 hours on mitomycin C-treated parental Sl/Sl4 cells or stromal cells
expressing soluble, MA, or MR-SCF. Cells were harvested, stained with
annexin V and PI, and scored for the percentage of apoptotic (annexin V+/PI
Sustained and enhanced activation of MAP kinase (ERK) in G1E-ER2 cells is induced by MA and MR forms of SCF We have previously demonstrated sustained activation of Kit in response to stromal cell presentation of MA- and MR-SCF, and transient activation of Kit in response to soluble SCF.8,24 To examine the consequence(s) of SCF presentation on intracellular kinase signaling pathways associated with MAP kinases in G1E-ER2 cells, we examined the activation of ERK and p38 MAP kinases. Activation of ERK (ERK-1 and ERK-2) was determined by examining the phosphorylation of ERK-1 and ERK-2 using phospho-specific antibodies. G1E-ER2 cells were cocultured on stromal cells expressing S-SCF, MA-SCF, or MR-SCF protein for various time points, and cellular lysates were analyzed. As shown in Figure 4, coculture of G1E-ER2 cells on stroma expressing MA-SCF and MR-SCF resulted in both enhanced and sustained activation of ERK (ERK-1 and ERK-2) over a period of 120 minutes. Coculturing G1E-ER2 cells on stroma expressing S-SCF also resulted in sustained ERK activation, however, at significantly reduced levels.
To further characterize the extent of involvement of the ERK MAP kinase
pathways in SCF-induced proliferation of G1E-ER2 cells, we used
specific pharmacologic inhibitors of the MAP kinase ERK PD98059 and p38
SB203580 pathways. First, we determined the specificity of PD98059 and
SB203580. G1E-ER2 cells were treated with the 2 inhibitors, and the
effect of each of these inhibitors on the activation of the other MAP
kinase pathway was examined in response to membrane SCF stimulation. As
shown in Figure 5A (lane 3), treatment of
G1E-ER2 cells with the ERK MAP kinase inhibitor PD98059 (12.5 µM) had
no effect on the activation of p38 MAP kinase. Conversely, treatment of
G1E-ER2 cells with the p38 inhibitor SB203580 (12.5 uM) had no effect
on the activation of the ERK MAP kinase (Figure 5B, lane 3). Similar
results were obtained in the presence of 25 and 50 µM SB203580 (data
not shown). These data demonstrate that the 2 inhibitors used in these
studies do not inhibit the activation of each other.
Next, we cocultured G1E-ER2 cells on stromal cells expressing various
isoforms of SCF in the presence or absence of indicated concentrations
of PD98059 (Figures 6A). As an additional positive control, we first
performed these experiments with soluble recombinant rat (rr) SCF.
After 48 hours of culture, [3H]thymidine was added for 6 hours, and [3H] incorporation was determined. Under these
conditions, a dose-dependent inhibition in proliferation of G1E-ER2
cells was observed in the presence of rrSCF and concentrations of
PD98059 ranging from 12.5 to 50 µM (data not shown). A dose-dependent
inhibition in proliferation of G1E-ER2 cells was also observed when
G1E-ER2 cells were stimulated by each isoform of SCF (Figure
6A). However, sustained and enhanced activation of ERK in G1E-ER2 cells via MA- or MR-SCF correlated with
statistically greater inhibition of proliferation in the presence of
PD98059 compared with soluble SCF (Figure 6A). This increase in the
inhibition of proliferation induced via PD98059 in G1E-ER2 cells
stimulated with MA- or MR-SCF was more apparent at lower concentrations
of the inhibitor (Figure 6A). Consistent with these observations, a
lower dose of PD98059 (12.5 µM) completely inhibited ERK MAP kinase
activation in G1E-ER2 cells cocultivated on MR-SCF, but not the soluble
form (Figure 6B, lanes 6 and 3, respectively). These data suggest that
enhanced and sustained activation of ERK in G1E-ER2 cells by stromal
cells expressing MA- or MR-SCF correlates with greater dependency of
these cells on the ERK MAP kinase pathway for proliferation compared to
soluble SCF-induced proliferation. Further, these data also
demonstrate that MR-SCF resembles MA-SCF with regard to the level and
kinetics of ERK activation.
Sustained and enhanced activation of p38 MAP kinase in G1E-ER2 cells by MA and MR forms of SCF In addition to ERK MAP kinases, p38 MAP kinase also has been implicated in some cells in cytokine-induced proliferation. To investigate the effect of SCF isoforms on activation of p38 in G1E-ER2 cells, we performed coculture experiments as described above and analyzed activation of p38. Activation of p38 was determined by examining the phosphorylation of p38 using an antibody that specifically recognizes the activated form of p38. As shown in Figure 7, coculture of G1E-ER2 cells on stroma expressing MA- and MR-SCF resulted in sustained and enhanced activation of p38 over a period of 120 minutes. Coculturing G1E-ER2 cells on stroma expressing S-SCF resulted in an equivalent activation of p38 at 10 minutes, but unlike cultivation with MA- or MR-SCF, this activation was transient, reaching baseline at 60 minutes.
To evaluate the functional significance of the p38 MAP kinase pathway
in SCF isoform-mediated proliferation of erythroid cells and to
determine if transient versus sustained activation of p38 via different
SCF isoforms results in differential effects on proliferation, G1E-ER2
cells were cocultured on stroma expressing various SCF isoforms in the
presence or absence of increasing concentrations of the p38-specific
inhibitor SB203580 (Figure 8A). After 48 hours of coculture, [3H]thymidine was added for 6 hours, and [3H] incorporation was determined. As shown in
Figure 8A, the extent of SB203580-induced inhibition in proliferation
of G1E-ER2 cells depended on the nature of the stimulating SCF isoform.
In contrast to reduced sensitivity to the inhibitor of ERK noted
previously (Figure 6A), soluble SCF-induced proliferation of G1E-ER2
cells was more sensitive to the p38 inhibitor SB203580 compared with MA
or MR forms of SCF, and this sensitivity was similar to that of rrSCF
(Figure 8A). No inhibition in proliferation of G1E-ER2 cells was
observed via MA- or MR-SCF stimulation in the presence of 12.5 µM
SB203580 (Figure 8A). In contrast, at this same concentration of the
inhibitor, soluble SCF- or rrSCF-induced stimulation of G1E-ER2 cells
resulted in a reduction of 10% in proliferation. As seen in Figure 8A,
approximately 40% inhibition in proliferation was observed when
GIE-ER2 cells were stimulated with S-SCF or rrSCF protein in the
presence of 25 µM SB203580. At this same concentration of SB203580,
stimulation of G1E-ER2 cells via MA- or MR-SCF was inhibited only 2%
and 5%, respectively (Figure 8A). Consistent with these observations,
treatment of G1E-ER2 cells with 12.5 µM and 25 µM SB203580 almost
completely inhibited p38 activation in response to soluble SCF
treatment (Figure 8B, lanes 3 and 4), but not in response to membrane
SCF stimulation (Figure 8B, lanes 7 and 8). In summary, these results
suggest that sustained activation of p38 in G1E-ER2 cells in response
to the membrane form of SCF is associated with more resistance to the
inhibitory effects of SB203580 compared with the soluble form.
One of the most overt hematopoietic phenotypes observed in mutants of Sl lacking the expression of MA-SCF (Sld) or possessing a defect in its presentation (Sl17H) is lifelong macrocytic anemia.1-4,9-10 Therefore, erythroid cells provide a unique model to study the biologic and biochemical consequences of Kit stimulation by soluble and membrane forms of SCF in the appropriate cellular context. To more clearly delineate the role of soluble and membrane presentation of SCF in erythropoiesis, we have compared the effect of soluble and membrane presentation of SCF on erythroid cell proliferation and activation of downstream kinase-signaling pathways. The major conclusions of this study are that (1) MR-SCF is as effective as MA isoform in mediating survival, proliferation, and cell cycle progression of erythroid cells; (2) the soluble form of SCF results in significantly less proliferation of G1E-ER2 cells; (3) stimulation of erythroid cells via MA- or MR-SCF results in sustained and enhanced activation of ERK and p38 MAP kinase pathways; and (4) MR- or MA-SCF-induced proliferation of G1E-ER2 cells is significantly more sensitive to the inhibitory effects of ERK MAP kinase inhibitor compared with S-SCF. In contrast, S-SCF-induced proliferation is significantly more sensitive to the inhibitory effects of p38 MAP kinase inhibitor compared with MR- or MA-SCF. The finding that membrane presentation of SCF is critical for erythroid cell proliferation and survival is consistent with previously published reports.8-9,11 Further, consistent with the inability of soluble SCF to sustain the proliferation of hematopoietic stem and progenitor cells described previously in vivo,6 stimulation of G1E-ER2 erythroid cells via soluble SCF results in less proliferation compared with MR- or MA-SCF over a 2-day coculture period in vitro. We have previously shown that soluble and MA-SCF stimulate qualitatively different responses in vitro in MO7e cells.6 In combination with other growth factors, MA-SCF but not S-SCF is capable of sustaining the survival of long-term hematopoietic progenitors and erythroblastic leukemia cells.25-28 More recently, using transgenic mice, we have shown that the transgene expression of MR-SCF but not S-SCF results in correction of erythroid cell deficiency associated with the lack or altered expression of MA-SCF in Sld and Sl17H mice, respectively.8-9 In addition, Tajima et al, using an elegant knockin strategy, also demonstrated that the exclusive expression of MA-SCF in vivo is sufficient for normal erythroid cell development.11 We extend these observations by investigating the response of Kit to an obligate MR form of SCF. Our findings, using stromal cells expressing exclusively the MR form of SCF, provide the biochemical mechanism that is possibly responsible for these in vivo observations. Further, our findings demonstrate no significant differences in erythroid cell proliferation, cell cycle progression, survival, and activation of downstream signaling molecules as a result of stimulation by MR- or MA-SCF. We have exploited the G1E-ER2 cell line in order to better study the biochemical events associated with activation of Kit by different SCF presentation. Although the biochemical basis for the differences between S-SCF,
MA-SCF, and MR-SCF are not completely understood, some signaling differences previously have been reported. After ligand binding to
receptor tyrosine kinases such as Kit, signal transduction cascades are
initiated by receptor autophosphorylation on key tyrosine
residues.29 In the case of S-SCF, tyrosine phosphorylation of Kit is rapid (within minutes), followed by a decline in
phosphorylation. This decline in phosphorylation coincides with
receptor internalization,30 leading ultimately to receptor
degradation. In contrast, phosphorylation of Kit by MA-SCF persists
over much longer periods in both MO7e cells as well as in primary
erythroid progenitor cells.8,24 This persistence in
tyrosine phosphorylation has been attributed to enhanced stability of
the Kit receptor on the cell surface after MA-SCF stimulation, likely
because of the delayed receptor internalization by this form of ligand
presentation. Our results, demonstrating sustained and enhanced
phosphorylation of p38 and ERK after stimulation by MA- or MR-SCF,
correlate with the persistent phosphorylation of Kit noted previously
via these 2 isoforms.8,24 Interestingly, sustained and
enhanced activation of p38 by the MA or MR form of SCF did not result
in greater dependency of erythroid cells on the p38 pathway for
proliferation. In contrast, activation of ERK via these 2 isoforms
correlated with significantly greater dependency of erythroid cells on
this pathway, as measured by sensitivity to inhibition. The biochemical
bases for these differences are currently not known. Differences in the
use of other downstream signaling pathways after stimulation with
soluble and MA-SCF have been recently described.31 In
these studies, significant differences in the use of
phospholipase C (PLC)- In summary, the results described in the present study, along with the elegant knockin studies by Tajima et al11 demonstrating exclusive expression of MA-SCF in vivo, suggest that, at least under steady-state conditions, S-SCF may play only a minimal role in hematopoiesis in vivo.
Reuben Kapur is a recipient of an American Society of Hematology Junior Faculty Scholar Award.
Submitted July 30, 2001; accepted March 28, 2002.
This work was supported in part by National Institutes of Health grant 2R01 DK48605-06.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Reuben Kapur, Herman B Wells Center for Pediatric Research, Cancer Research Building, 1044 W Walnut St, Room 425, Indianapolis, IN 46202; e-mail: rkapur{at}iupui.edu.
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S. Chandra, R. Kapur, N. Chuzhanova, V. Summey, D. Prentice, J. Barker, D. N. Cooper, and D. A. Williams A rare complex DNA rearrangement in the murine Steel gene results in exon duplication and a lethal phenotype Blood, November 15, 2003; 102(10): 3548 - 3555. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and necrotic (annexin V+/PI+) cells using
fluorescence-activated cell-sorter (FACS) analysis. G1E-ER2
cells cocultured on untransfected parental Sl/Sl
signaling pathway downstream from
Kit were observed in the 32D leukemic cell line.