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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3757-3773
By
From the Department of Pharmacology and Toxicology, Medical College
of Virginia campus of Virginia Commonwealth University, Richmond, VA;
the Department of Medicine, Stanford University, San Francisco, CA; and
Hematopoietic Stem Cell Laboratory, University of Illinois, Chicago,
IL.
We found that erythropoietin (EPO) and stem cell factor (SCF)
activated protein kinase B (PKB/Akt) in EPO-dependent HCD57 erythroid
cells. To better understand signals controlling proliferation and
viability, erythroid cells that resist apoptosis in the absence of EPO
were subcloned and characterized (HCD57-SREI cells). Constitutive activations of PKB/Akt, STAT5a, and STAT5b were noted in these EPO-independent cells. PI3-kinase activity was an upstream activator of
PKB/Akt because the PI3-kinase inhibitor LY294002 blocked
both constitutive PKB/Akt and factor-dependent PKB/Akt activity. The LY294002 study showed that proliferation and viability of both HCD57-SREI and HCD57 cells correlated with the activity of PKB/Akt; however, PKB/Akt activity alone did not protect these cells from apoptosis. Treatment of HCD57 cells with SCF also activated PKB/Akt, but did not protect from apoptosis. This result suggested that PKB/PI3-kinase activity is necessary but not sufficient to promote viability and/or proliferation. Constitutive STAT5 activity, activated through an unknown pathway not including JAK2 or EPOR, may act in
concert with the constitutive PI3-kinase/PKB/Akt pathway to protect the
EPO-independent HCD57-SREI cells from apoptosis and promote limited proliferation.
ERYTHROPOIETIN (EPO) is the primary
hormone which regulates the maturation of immature erythroid
cells1-3 into erythrocytes. EPO is necessary for the
proliferation, erythroid differentiation, and prevention of apoptosis
of erythroid cells; it is not yet clear whether these three processes
are regulated by distinct signaling pathways.1-3 Some
studies suggest that EPO might act primarily to maintain viability by
protecting mature erythroid cells from programmed cell death or
apoptosis in the relatively narrow window of EPO-dependent
differentiation.1 The "knock out" of EPO or EPO
receptor (EPOR) results in EPO EPO acts through cell-surface receptors on immature erythroid
cells.1-3,5 The EPOR does not have a kinase domain and
transmits an intracellular signal by interacting with cytoplasmic
nonreceptor tyrosine protein kinases. Two of these kinases have been
reported to become phosphorylated after EPO binding to cells: the Janus kinase, JAK2, and the c-fes kinase.6-8
JAK2 can physically associate with the EPOR.6,9,10 This
interaction is constitutive in HCD57 erythroid cells, but JAK2 appears
to be selective in interacting with a subset of highly modified EPOR
molecules.10 Known substrates of tyrosine protein kinases
activated by EPO binding to its receptor include phosphatidylinositol-3
kinase (PI3-kinase), SHC proteins, c-raf, STAT1, STAT3, STAT5A,
STAT5B, and other molecules.4,9-23
Recent studies have suggested that c-Kit, the receptor for
stem cell factor, interacts with the EPOR and transduces an
intracellular signal through the EPOR.24 Our laboratory has
shown that stem cell factor (SCF) can stimulate proliferation of HCD57
cells but cannot protect them from apoptosis.5 EPO
activates JAK2/STAT signaling and other signaling which
protects the cells from apoptosis and stimulates proliferation. Because
SCF does not activate JAK2/STAT5 signaling in this system, we have
speculated that this pathway may protect erythroid cells from apoptosis.
Constitutive STAT activation has been observed in transformed cell
lines and cancerous cells.25-35 In most cases,
constitutively activated Janus kinases seem to be responsible for
activating the STAT proteins; however, in other cells and cancers,
other kinases may phosphorylate STAT molecules. Oncogenes such as
v-abl,30 bcr/abl,35 and
v-src30 may directly phosphorylate and activate STAT proteins. These studies suggest a possible role of STAT proteins, independent of Janus kinases, in signaling the proliferation and protecting these transformed cells from apoptosis. However, the role of
STAT5 signaling in erythroid cells is controversial. STAT5 activation
may be linked to enhanced proliferation19,36; in contrast,
a mutant EPOR incapable of activating STAT5 still promotes proliferation.37 Wakao et al38 and Iwatsuki et
al39 have separately demonstrated that STAT5 is required
for erythroid differentiation; in contrast, deletion of STAT5A and/or
STAT5B results in STAT5 null mice that have no obvious reduction or
defects in erythroid cells.40-42
The involvement of PI3-kinase in cell transformation has been
implicated in several cell lines.43,44 PI3-kinase plays an important role in regulation of cell proliferation and apoptosis; in
addition, constitutive PI3-kinase activity has been observed in
cancerous cells and, therefore, may contribute to the malignant transformation of cells. The insulin receptor substrate proteins (IRS)
are substrates of many tyrosine kinases, including the JAK2 kinase
bound to the EPOR.45,46 Activated IRS-1, -2, -3 are known
to be docking sites for PI3-kinase and are implicated in the activation
of PI3-kinase either in complexes with receptors or independently of receptors.
An important downstream effector activated by the PI3- kinase signaling
pathway is protein kinase B (PKB/Akt), also known as c-Akt and RAC-PK.
PI3-kinase activity leads to accumulation of polyphosphatidylinositols
lipids in the plasma membrane. PKB/Akt is apparently targeted to
translocate to the polyphosphatidyinositol rich membrane via a
pleckstrin homology domain and is activated at the membrane by
phosphorylation at Thr308 and Ser474 from two
distinct phosphatidylinositol dependent kinases.47 PKB/Akt
activity is linked to the control of many diverse cellular functions
that included insulin-dependent glucose transport, glycolysis, glycogen
synthesis and protein translation, and viability of
cells.47 Recent work has shown that PKB/Akt is able to
phosphorylate the pro-apoptotic protein BAD. Phosphorylation of BAD
correlates with the cell survival and it is suspected that
phosphorylated BAD is ineffective in disrupting the protective action
of Bcl anti-apoptotic proteins.
In the current study, we found that EPO and SCF activated PKB/Akt in
the EPO-dependent erythroid cells but the PI3-kinase/PKB/Akt pathway
was constitutively active in the apoptosis-resistant, EPO-independent
HCD57-SREI cells. The finding of constitutive activity of STAT5 in this
apoptosis-resistant subclone of HCD57 cells suggests that STAT5
activity which arises through a signal independent of Janus kinase
activity and the EPOR may also act in concert with PKB/Akt activity to
prevent cells from undergoing apoptosis induced by growth factor withdrawal.
Cell culture.
HCD57 cells, obtained from Sandra Ruscetti (National Cancer Institute,
Frederick, MD),6,31 were cultured in the presence of 1.0 U
EPO/mL in Iscove's modified Dulbecco's medium and 25% fetal calf
serum. In experiments to test the action of signaling by EPO, the cells
were further cultured overnight in the same medium devoid of EPO. As
previously reported,6 this deprivation of EPO increased
cell-surface EPOR but was an insufficient time without EPO for
apoptosis to begin. HCD57-SREI cells were subcloned by transferring
HCD57 cells from medium containing EPO to medium free of EPO but
containing 100 ng SCF/mL. A subclone of cells that survived and
proliferated was then cultured in the same medium indicated above
except that both EPO and SCF were omitted.
Apoptosis studies.
HCD57 and HCD57-SREI cells were cultured either in the absence of
cytokines or in the presence of the indicated factors for 72 hours. The
cells were then harvested and genomic DNA was isolated.5 Ten micrograms of genomic DNA was resolved on a 2.25% agarose/1X TAE/300 ng EtBr/mL gel. DNA laddering indicative of apoptosis was
visualized using UV light.
Western blot analysis.
Tyr(P)-containing proteins and STAT5 tyrosine phosphorylation and
nuclear translocation were detected by either Western blotting alone or
the immunoprecipitation and Western blotting method described previously.5,10 Polyclonal anti-Tyr(P) antibodies from
Zymed (San Francisco, CA) were used to immunoprecipitate
and concentrate the Tyr(P)-containing proteins before they were
analyzed by Western blot. The blots were stripped of bound antibody and
reprobed with anti-JAK2 (Upstate Biotechnology, Inc [UBI], Lake
Placid, NY) antisera. The blot was stripped as described by the
Amersham literature with the ECL kit (Amersham, Arlington Heights,
IL). Briefly, the bound antibody was released in the
presence of 2% sodium dodeyl sulfate (SDS) and reducing agents at
50°C for 30 minutes. STAT5 tyrosine phosphorylation and nuclear
translocation were detected by Western blot analysis of 10 µg of
nuclear extracts probed with the anti-Tyr(P) antisera described above
or anti-STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA) antisera. For
the Bcl-Xl and Bcl-2 Western blots, 10 µg of total cellular lysate
were probed with anti-Bcl-Xl and anti-Bcl-2 antisera (Santa Cruz
Biotechnology). For PKB/Akt assay, 100,000 cells were
lysed in SDS sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, 10%
glycerol, 50 mmol/L dithiothreitol [DTT], and 0.1% bromphenol blue).
The blots were probed with anti-phospho Akt (Ser473) antibody (New
England Biolabs, Beverly, MA) followed by detection using the
Phototope-HRP Western detection system (New England
Biolabs). After stripping of phosphospecific antibody, the blot was
reprobed with a general anti-PKB/Akt antibody (New
England Biolabs) that recognizes both phosphorylated and unphosphorylated PKB/Akt.
Gel mobility shift assay of STAT5 binding.
Nuclear STAT5 binding to radiolabeled DNA (prolactin inducible element)
(PIE) was done as described previously.11 For the supershift assays to verify the presence or absence of STAT protein in
the DNA binding complex, 2 µg of anti-STAT1, anti-STAT3, anti-STAT5A or anti-STAT5B antibodies (Zymed) were preincubated with 20 µg of
nuclear extract for 15 minutes on ice before addition of the radiolabeled DNA.
Detection of cell-surface c-Kit expression.
Surface c-Kit expression was detected using flow cytometry analysis.
For each sample, 1 × 105 HCD57 or HCD57-SREI cells were
washed in FACS buffer (Tris-buffered saline/4% fetal calf serum).
After preincubation of the cells in a monoclonal antibody (MoAb) that
blocks nonspecific binding of fluorescein isothiocyanate
(FITC)-conjugated antibodies to IgG (rat MoAb 2.4G2; a
generous gift from Dr William Paul, National Institutes of Health,
Bethesda, MD), the cells were incubated with FITC anti-cKit or FITC
anti-CD4 (Becton Dickinson, San Diego, CA) (to control for background
antibody staining) at 10 mg/mL for 30 minutes at 4°C. The cells were
then washed in fluorescence-activated cell sorter (FACS) buffer before
analysis using a FACScan flow cytometer (Becton Dickinson).
Sequencing of the EPOR in HCD57 and HCD57-SREI cells.
cDNA encoding the EPOR was cloned using reverse
transcriptase-polymerase chain reaction (RT-PCR) techniques. Briefly,
mRNA was isolated from HCD57 and HCD57-SREI cells using the Quick Prep Micro mRNA kit (Pharmacia, Piscataway, NJ). cDNA was generated from
mRNA using the First Strand cDNA Synthesis kit (Pharmacia). The cDNA
was subjected to PCR using Taq polymerase (GIBCO-BRL) and
oligonucleotides corresponding to the 5' and 3' end of the murine EPOR
gene. PCR fragments were isolated using the Geneclean procedure (BIO
101, Vista, CA) and cloned into the pCrII vector (Invitrogen, Carlsbad,
CA). Multiple clones of EPOR cDNA from both HCD57 and HCD57-SREI cells
were sequenced using the dideoxy method described by Sanger et
al48 using the Sequenase version 2.0 kit (Amersham,
Piscataway, NJ).
Immunoprecipitation, ERK-1, PI3-kinase, and PKB/Akt assay.
Immunoprecipitation of ERK-1 and quantitation of activity as determined
by incorporation of 32P from [ Proliferation and apoptosis of EPO-independent HCD57-SREI cells.
Earlier studies in this laboratory5 showed that the
treatment of HCD57 cells with SCF in the absence of EPO increased
proliferation for 3 days but the effect was limited because the cells
underwent apoptosis. While SCF clearly acted as a mitogen, EPO was also required to prevent apoptosis. The proliferation of HCD57 cells in
either EPO or SCF is also shown in Fig 1A.
In contrast, other workers have shown that introduction of the EPOR
into nonerythroid cells will allow SCF to promote proliferation for
infinite generations, possibly through an interaction of c-kit with the
EPOR.24 We subcloned the HCD57 cells, hoping to select for
an exclusively SCF-dependent cell line which had enhanced signaling of
SCF through the EPOR. To accomplish this, the EPO-dependent HCD57 cells
were cultured in the absence of EPO but in the presence of 100 ng
SCF/mL.
HCD57-SREI do not produce EPO.
Some erythroleukemic cell lines may produce autocrine EPO. To
investigate whether the HCD57-SREI cells were secreting EPO into the
medium, we tested whether the parental HCD57 cells could survive in
medium conditioned by the HCD57-SREI cells. HCD57 cells did not
proliferate and underwent apoptosis in this conditioned medium even
when the HCD57-SREI medium was concentrated (data not shown). In
addition, the proliferation of HCD57-SREI cells was not dependent on a
high concentration of cells in culture and was not affected by the
addition of an anti-EPO antibody that blocked the proliferation of
HCD57 cells in EPO-containing medium (data not shown). To investigate
whether HCD57-SREI cells might produce EPO that is not secreted, we
probed a Northern blot of RNA prepared from HCD57-SREI cells with a
probe to EPO. No EPO mRNA was detected (data not shown). Thus, it
appeared that the EPO-independent phenotype of the HCD57-SREI cells did
not result from either autocrine EPO production or secretion of another
factor that maintains the viability of erythroid cells.
EPOR in HCD57-SREI is not activated through an auto-activating
mutation.
We tested whether the EPO-independent phenotype resulted from a
mutation of the EPOR gene. The arginine 129 to cysteine
mutation in the EPOR is an autoactivating mutation50 that
allows sulfhydryl bonding to form covalent dimers of EPOR in the
absence of EPO. Therefore, we isolated 10 cDNA clones of the EPOR from
these HCD57-SREI cells for sequencing. None of the 10 clones were found
to have the arginine 129 mutation. Full-length sequencing of two
HCD57-SREI cDNA clones showed four identical mutations compared with a
previously published sequence51 (amino acid 5, A to G
mutation in the second base of codon; amino acid 273, T to G mutation
of the first base of the codon; amino acid 334, mutation of A to G in
the third base of the codon; amino acid 350 mutation of T to G in the
third base of the codon). However, these mutations are conservative such that the amino acid sequence of the protein is not altered. Thus,
the EPOR peptide in HCD57-SREI cells is wild type and has not undergone
an autoactivating mutation.
Processing and expression of EPOR and c-Kit in HCD57-SREI cells.
In the parental HCD57 cells and other primary erythroid cells, most of
the cell-surface EPOR molecules are posttranslationally processed by
extensive N-linked glycosylation and phosphorylation of nontyrosine
residues into a 78-kD form.6,10 We examined the posttranslational processing and metabolism of the EPOR in HCD57-SREI cells to test whether overexpression of EPOR peptide, alternative posttranslational processing, or failure to downregulate in
the presence of EPO might contribute to EPO independence (or apparent
independence as a result of hypersensitivity to trace levels of EPO).
Figure 2A shows that the EPOR in HCD57-SREI
cells appears to be identically processed from the 62-kD peptide into the 78-kD EPOR as the parental HCD57 cells; however, the level of
expression of the EPOR was reduced in the HCD57-SREI cells. This
reduced level of EPOR expression correlated with the determination that
HCD57 cells (deprived of EPO overnight to upregulate receptors) had
3,800 binding sites for 125I-EPO whereas the HCD57-SREI
cells only expressed 500 binding sites for 125I-EPO. The
binding affinity for 125I-EPO was similar in both cell
lines (kd = 810 nmol/L and 780 nmol/L). In the presence
of EPO, the high-molecular-weight 68- to 78-kD forms of EPOR in both
HCD57 and HCD57-SREI cells were similarly downregulated within 40 minutes (Fig 2A, lanes D and H). Thus, there appeared to be no
alteration of the EPOR protein in HCD57-SREI cells that would explain
the EPO-independent phenotype.
Expression of Bcl-XL and Bcl-2 in HCD57 and HCD57-SREI cells.
The above experiments indicated a nonreceptor explanation for
EPO-independence in HCD57-SREI cells. Overexpression of the Bcl family
of apoptosis modulating proteins can result in resistance to apoptosis.
Previous work had implicated Bcl-Xl but not Bcl-2 as a critical control
point in protecting from apoptosis in HCD57 cells.52
Therefore, we examined the expression of Bcl-Xl and Bcl-2 in the
HCD57-SREI cells. There was a similar level of expression of these two
apoptosis suppressing proteins in the HCD57-SREI cells (Fig
3, lanes D and H) as the parental HCD57
cells (Fig 3, lanes B and F). Thus, the protection from apoptosis in
the HCD57-SREI cells did not result from overexpression of the Bcl gene
products. However, we found that whereas the withdrawal of EPO
suppressed the expression of both Bcl-Xl and Bcl-2 proteins in HCD57
cells, Bcl-Xl levels were maintained in the presence or absence of EPO
in HCD57-SREI cells (Fig 3, lanes C and G). The EPO-independent
phenotype of HCD57-SREI cells, therefore, may be caused by an
alteration in signaling upstream of the regulation of Bcl-Xl gene that
maintains the expression of the Bcl-Xl protein even in the absence of
EPO.
Tyrosine protein phosphorylation in HCD57 and HCD57-SREI cells.
Alterations in cellular signaling in HCD57-SREI cells might explain why
Bcl-Xl levels were maintained in the absence of EPO. Therefore, we
examined the phosphotyrosine-containing proteins in either untreated
cells or cells treated with SCF or EPO in HCD57-SREI and parental HCD57
cells. If an autoactivating mutation in the EPOR or a mutation
resulting in expression of EPO were present, we would expect to see
constitutive phosphorylation of the EPOR and JAK2 kinase as previously
reported.53 In addition, the constitutive secretion of SCF
would be expected to lead to constitutive tyrosine protein
phosphorylation of c-Kit. However, as shown in Fig
4A (top
panel), there was no apparent constitutive phosphorylation of bands
corresponding to EPOR, JAK2, c-Kit, or other major phosphotyrosine
[Tyr(P)] containing protein in HCD57-SREI cells, virtually ruling out
the possibility of an autoactivating mutation in either EPOR or JAK2 or
autocrine production of EPO or SCF.
STAT5A/B is constitutively phosphorylated, translocated to the
nucleus, and bound to DNA in the HCD57-SREI cells.
Although not evident in the above experiments, a band of about 100 kD constitutively tyrosine phosphorylated in the SREI
subclone but not the HCD57 cells, was seen in some experiments. To test whether this approximately 100-kD band might be an activated STAT protein, we immunoprecipitated STAT5A/B from HCD57 cells and HCD57-SREI cells and analyzed the tyrosine phosphorylation state of STAT5 in the
presence and absence of EPO (Fig 4D). This experiment confirmed that
STAT5 was constitutively phosphorylated on tyrosine residues in the
HCD57-SREI cells (Fig 4D, lane G). Additional experiments with STAT5A-
and STAT5B-specific antisera show that both forms of STAT5 were
phosphorylated, but we did not find other STATs to be constitutively
active. Interestingly, the major tyrosine phosphorylated band at 95 kD
seen in EPO-treated HCD57 and HCD57-SREI cells was not the STAT5
protein as many have assumed. STAT5 was a minor component of this 95-kD
band (STAT5 migrates at the very top of this broad band). Although
STAT1 is also activated by EPO in HCD57 cells, the level of
phosphorylation of STAT1 is an order of magnitude less than STAT5 and,
therefore, does not contribute significantly to this band, leaving the
identity of the band a mystery.
Does a phosphatase defect explain the HCD57-SREI resistance to
apoptosis?
The observation showing that sodium vanadate
(Na3VO4), an inhibitor of tyrosine protein
phosphatase activity, can activate STAT protein independently of Janus
kinases suggested that constitutive activation of STAT5 might be due to
the loss of phosphatase activity in HCD57-SREI cells.55,56
The SHP-1 tyrosine protein phosphatase has been strongly implicated in
the modulation of both JAK2/EPOR and STAT
signaling.22,57-60 We and others have previously reported that HCD57 cells are hypersensitive to EPO.10,61 Therefore, we tested the hypothesis that the hypersensitivity of HCD57 cells to
EPO might be caused by a reduction of the SHP-1, and that the loss of
EPO-dependence in the HCD57-SREI cells could be the result of the
further reduction or complete loss of this phosphatase. We compared the
levels of SHP-1 in these two cell lines with a population of CFU-E and
proerythroblasts purified from normal human blood. Figure
6 shows that identical levels of SHP-1 were present in normal immature human erythroid cells, HCD57 cells, and the
apoptosis-resistant HCD57-SREI cells.
Constitutive activation of PI3-kinase in HCD57 and HCD57-SREI cells.
PI3 kinase has been reported to be activated as part of the EPO
signaling pathway. Because PI3-kinase has been implicated in several
cell types as critical to the regulation of cell proliferation, differentiation, and survival,62-64 we measured PI3-kinase
activity and tested for interaction of PI3-kinase with proteins known
to associate with PI3-kinase. This study compared these parameters in
the EPO-dependent HCD57 cells and the EPO-independent HCD57-SREI cells.
Figure 7A shows that the PI3-kinase
activity measured by incorporation of 32P from
gamma-labeled ATP into phosphatidylinositol by the immunoprecipitated PI3-kinase (anti-p85 subunit of PI3-kinase) appeared constitutive in
both HCD57 and HCD57-SREI cells and was not additionally increased by
treatment of EPO. The bottom panel of Fig 7A shows the combined results
from five experiments that confirm the lack of EPO effect. To verify
that this unexpected constitutive activity which phosphorylated phosphatidylinositol was truly PI3-kinase activity, we added the PI3-kinase inhibitor, LY294002, to the reaction mixture and showed that
the activity was inhibited in a dose-responsive fashion at concentrations of inhibitor known to specifically block PI3-kinase activity (shown in Fig 7B). For a further control, we examined the
PI3-kinase activity in immunoprecipitated STAT5, JAK2, Vav, as well as
PI3-kinase. PI3-kinase activity was only detected in the
immunoprecipitated PI3-kinase (anti-p85 immunoprecipitate), as shown in
Fig 7C.
Factor-dependent and constitutive activation of PKB/Akt.
The observation of both EPO-dependent and constitutive PI3-kinase
activity led us to test if the downstream effectors of PI3-kinase correlated with observed PI3-kinase activity. Activity of PKB/Akt was
assessed two ways, by activity (phosphorylation)-specific antibodies
and the ability of immunoprecipitated PKB/Akt to phosphorylate a
peptide substrate. In sharp contrast to the finding of constitutive activity of PI3-kinase in the anti-p85 PI3-kinase immunoprecipitate in
both EPO-dependent HCD57 cells and EPO-independent HCD57-SREI cells (in
Fig 7), there was constitutive activity of PKB/Akt activity only in the
EPO-independent HCD57-SREI. There was no detectable phosphorylation of
PKB/Akt in the HCD57 cells in the absence of added factors (Fig
10). The constitutive activation of
PKB/Akt in HCD57-SREI cells and EPO-dependent activation in HCD57 cells was confirmed by the in vitro assay of PKB/Akt. The in vitro assay of
PKB/Akt gave the following result when PKB/AKt activity was determined
(assayed as described in Materials and Methods and expressed as the
average of three assays to determine the cpm of 32P from
ATP incorporated into a substrate peptide ± standard deviation): HCD57 |
TOP
ABSTRACT
INTRODUCTION
/
-32P]
adenosine triphosphate (ATP) into myelin basic protein was performed as
described previously.
-glycerophosphate). Lysates were centrifuged at 14,000g for
10 minutes at 4°C, and the supernatants were equalized for protein
concentration (1 mg/mL) by addition of buffer A. PKB/Akt was
immunoprecipitated from the supernatants containing 1 mg of protein
with an anti-PKB/Akt antibody (New England Biolabs Inc)
and incubated at 4°C for 1.5 hour. Protein A-agarose
beads (UBI) were added and incubated for another 1 hour at 4°C. The
beads were washed three times with buffer A containing 0.5 mol/L NaCl, twice with buffer B (50 mmol/L Tris-HCl, pH 7.5, 0.03% Brij-35, 0.1 mmol/L EGTA, and 0.1% 2-mercaptoethanol), and twice with assay dilution buffer (20 mmol/L MOPS, pH 7.2, 25 mmol/L b-glycerophosphate, 5 mmol/L EGTA, 1 mmol/L
Na3VO4, and 1 mmol/L DTT). The beads were
suspended in 30 µL of assay dilution buffer containing 10 mmol/L PKA
inhibitor peptide (Sigma, St Louis, MO). PKB/Akt-specific substrate
peptide (UBI) was added to reaction mixture at a final concentration of
100 mmol/L. The reaction was initiated by addition of 10 mCi
[
; HCD57-SREI, 
) or
absence of EPO (HCD57-SREI, x) were treated with LY294002 at the
indicated concentrations for 72 hours, and the viable cells were
counted in the presence of trypan blue. Values are expressed as a
percentage of the corresponding control (treated with vehicle
dimethylsulfoxide [DMSO]). Data are means ± SE of at least
triplicate measurements. (B) Cells were cultured in the presence of 1 U
EPO/mL (HCD57) or absence of EPO (HCD57-SREI). HCD-57 (lanes 2 through
6) and HCD57-SREI (lanes 7 through 11) cells were treated with vehicle
DMSO (lanes 3 and 8), LY294002 at indicated concentrations (lanes 4 through 6 and 9 through 11), or untreated (lanes 2 and 7) for 24 hours.
The genomic DNA was isolated and analyzed for fragmentation of
DNA characteristic of apoptosis as described in Materials and
Methods.