|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 94 No. 5 (September 1), 1999:
pp. 1568-1577
By
From the Department of Internal Medicine II,
Hokkaido University School of Medicine, Sapporo; and the
Division of Hematology, Department of Internal
Medicine, and the Blood Center, Keio University,
Tokyo, Japan.
Little is known about the physiologic role of phosphatidylinositol
3-kinase (PI-3K) in the development of erythrocytes. Previous studies
have shown that the effects of the PI-3K inhibitor wortmannin on
erythropoietin (EPO)-dependent cell lines differed depending on the
cell type used. Wortmannin inhibited EPO-induced differentiation of
some cell lines without affecting their proliferation; however, the
EPO-induced proliferation of other cell lines was inhibited by
wortmannin. In neither case were signs of apoptosis observed. We have
previously reported that signaling in highly purified human colony
forming units-erythroid (CFU-E), generated in vitro from
CD34+ cells, differed from that in EPO-dependent cell
lines. In the current study, we examined the effects of a more specific
PI-3K inhibitor (LY294002) on human CFU-E. We found that LY294002
dose-dependently inhibits the proliferation of erythroid progenitor
cells with a half-maximal effect at 10 µmol/L LY294002. LY294002 at
similar concentrations also induces apoptosis of these cells, as
evidenced by the appearance of annexin V-binding cells and DNA
fragmentation. The steady-state phosphorylation of AKT at Ser-473 that
occurs as a result of PI-3K activation was also inhibited by LY294002 at similar concentrations, suggesting that the effects of LY294002 are
specific. Interestingly, the acceleration of apoptosis by LY294002 was
observed in the presence or absence of EPO. Further, deprivation of EPO
resulted in accelerated apoptosis irrespective of the presence of
LY294002. Our study confirms and extends the finding that signaling in
human primary cultured erythroid cells is significantly different from
that in EPO-dependent cell lines. These data suggest that PI-3K has an
antiapoptotic role in erythroid progenitor cells. In addition, 2 different pathways for the protection of primary erythroid cells from
apoptosis likely exist: 1 independent of EPO that is LY294002-sensitive
and one that is EPO-dependent and at least partly insensitive to LY294002.
ERYTHROPOIETIN (EPO) is a glycoprotein
hormone essential for normal erythropoiesis.1-4 Signaling
through the EPO receptor (EPO-R) regulates the proliferation,
differentiation, and survival of erythroid progenitor cells.
Homodimerization of the receptor in response to EPO binding transiently
activates the receptor-associated protein tyrosine kinase
Jak2.5-8 It has been reported that activation of Jak2 is
accompanied by tyrosine phosphorylation of numerous proteins, including
Jak2 itself, STAT proteins, SHP-1, Shc, Vav, the EPO-R, and the classic
phosphatidylinositol 3-kinase (PI-3K), although tyrosine kinases other
than Jak2 may also phosphorylate these proteins.4,6-22 The
classic PI-3K is a heterodimeric enzyme composed of a regulatory p85
subunit and a catalytic p110 subunit that phosphorylates
phosphoinositides at the D3 position of the inositol
ring.23 Published data have implicated PI-3K and its downstream target, the serine/threonine kinase AKT, in a pathway that
conveys survival signals within various systems.24-26
Activation of PI-3K results in the generation of 2 lipid products
(PI-3,4-P2 and PI-3,4,5-P3), which serve as
second-messenger molecules and activate AKT. Activated AKT may
phosphorylate the proapoptotic factor BAD on a serine residue,
resulting in its dissociation from BCL-XL and its
association with 14-3-3.24 Released BCL-XL may
then suppress cell-death pathways that involve the activity of APO-1,
cytochrome c, and the caspase protease cascade.26 Such a
mechanism may function in human erythroid cells, as it has been
reported that these cells express BCL-XL and its expression is positively regulated during the final stage of
erythropoiesis.27,28 Another possible target of activated
AKT may be glycogen synthase kinase-3 (GSK-3), and it was reported that
phosphorylation of GSK-3 by activated AKT may promote the survival of
Rat-1 and PC12 cells.29
Little is known about the physiologic significance of PI-3K in the
survival, proliferation, and differentiation of erythroid progenitors.
Wortmannin, an inhibitor of PI-3K, was reported to inhibit the
proliferation of HCD-57 and DA-3 cells in response to
EPO.18,23 More recently, Sui et al30 reported
that wortmannin inhibited the proliferation of human erythroid
precursors expanded in vitro. The mechanism involved in the
antiproliferative effects was unknown. Moreover, in certain cells,
wortmannin had no antiproliferative effects. However, in some of the
studies cited, high concentrations of wortmannin were used that
probably had nonspecific effects. Lysophosphatidic acid (LPA) has been
shown to be a major survival factor for murine macrophages. While high
concentrations of wortmannin were necessary to inhibit the survival
effects of LPA,31 LY294002, a more specific inhibitor of
PI-3K,32 opposed the action of LPA at appropriate
concentrations. We postulate that the use of wortmannin and different
cell lines could explain the controversy regarding the role of PI-3K in erythropoiesis.
We previously used primary cultured human erythroid progenitor
cells33 to gain insight into the physiologic role of PI-3K activation to support proliferation and differentiation of erythroid progenitors. Using these cells, we found that EPO induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. In the present study, we
show that LY294002 induces apoptosis in human erythroid progenitors in
a time- and dose-dependent manner, and that it blocks phosphorylation of AKT at concentrations similar to those required for the induction of
apoptosis. Our results suggest that PI-3K may be essential for the
survival of human erythroid precursor cells by preventing apoptosis.
Reagents.
HEPES, sodium dodecyl sulfate (SDS), 2-mercaptoethanol (2-ME), sodium
orthovanadate, bovine serum albumin (BSA), chicken egg albumin,
Iscove's modified Dulbecco's medium (IMDM), propidium iodide (PI),
protein A-Sepharose, Triton X-100, and Tris were purchased from Sigma
(St Louis, MO). Polyvinylidene difluoride (PVDF) membranes (pore size,
0.45 µm) were from Millipore Corp (Bedford, MA). SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) molecular standards and enhanced
chemiluminescence reagents including secondary antibodies were
purchased from Amersham (Arlington Heights, IL). Insulin (porcine
sodium; activity, 26.3 USP U/mg) was purchased from Calbiochem and
Behring Diagnostics (La Jolla, CA). Antibodies against AKT and
phosphorylated AKT were from Upstate Biotechnology Inc (Lake Placid,
NY) and New England Biolabs (Beverly, MA). Nitroblue tetrazolium
chloride and 5-bromo-4 chloro-3-indolyl phosphate p-toluidine
salt were from GIBCO-BRL (Gaithersburg, MD). Horse tendon type I
collagen was from Nycomed (Munich, Germany).
Ex vivo generation of erythroid progenitor cells.
Human erythroid progenitor cells were generated ex vivo as previously
described.33 In brief, recombinant human granulocyte colony-stimulating factor ([G-CSF] Chugai Pharmaceutical Co and Kyowa
Hakko Pharmaceutical Co, Tokyo, Japan) was administered to healthy
subjects who previously signed consent forms approved by the Hokkaido
University School of Medicine and the Hokkaido Red Cross Blood Center
Committee for the Protection of Human Subjects, as described
previously.34 The mobilized peripheral blood (PB) CD34+ cells were isolated using immunomagnetic
beads.35,36 The cells were then cryopreserved and stored
until use in liquid nitrogen. The frozen PB CD34+ cells
were thawed, suspended in IMDM containing 30% FCS and 100 U/mL DNase,
and then centrifuged at 400g for 5 minutes at 4°C. The cells
were washed twice with IMDM containing 20% FCS and then resuspended in
IMDM containing 0.3% deionized BSA.37 The cells were next
cultured in liquid phase as described elsewhere.33,38 In
brief, cells at 0.5 × 104 to 2.0 × 104
cells/mL were suspended in a mixture containing 20% FCS, 10% heat-inactivated pooled human AB serum, 1% BSA, 10 µg/mL insulin, 10 µg/mL vitamin B12, 15 µg/mL folic acid, 100 U/mL IL-3,
100 ng/mL SCF, and 4 U/mL EPO in the presence of 5 × 10 Semisolid culture of progenitors.
Day 8 cells were incubated in triplicate at a concentration of 500 cells/mL in flat-bottom 48-well tissue culture plates (Linbro; Flow
Laboratories) in 0.25 mL serum-containing or serum-free fibrin clots
with EPO at 2 U/mL.38-41 After 7 days of incubation at
37°C in a 5% CO2/5% O2 incubator, the clots
were fixed and stained with benzidine-hematoxylin.37 The
aggregates consisting of 8 to 49 hemoglobinized cells were defined as
colony-forming units-erythroid (CFU-E), while aggregates consisting of
2 to 7 hemoglobinized cells were defined as small erythroid. Erythroid
colony-forming cells (ECFCs) were defined as cells that yield colonies
of 2 to 49 hemoglobinized cells after 7 days of culture of day 8 cells.40
Liquid suspension culture of progenitors.
Day 8 cells were incubated at concentrations of 1 × 105
to 5 × 105 cells/mL in flat-bottom 24- to 96-well tissue
culture plates (Linbro; Flow Laboratories) in serum-free medium with
EPO at 2 U/mL.39 After incubation at 37°C in a 5%
CO2/5% O2 incubator for the indicated periods,
the cells were collected and washed twice with IMDM containing 0.3%
BSA before the subsequent colony assay of these cells.
Immunoprecipitation, gel electrophoresis, and Western blotting.
After starvation, day 8 cells were stimulated with EPO. Cells were
lysed by adding an equal amount of lysis buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L PMSF, 10 mmol/L EGTA, 1 mmol/L sodium
orthovanadate, 0.8 µg/mL leupeptin, and 2% Triton X-100 vol/wt, pH
7.4). After 20 minutes on ice, the lysates were centrifuged at
10,000× g (at 4°C) for 20 minutes. The supernatant was
removed and precleared with preimmune serum and protein A-Sepharose
(40 µL of 50% slurry) for 1 hour. Anti-AKT polyclonal antibody was then added and the preparation was incubated for 2 to 3 hours on ice.
Protein A-Sepharose (40 µL of 50% slurry) was added and followed by
1 hour of incubation. The immune complexes were washed 3 times with 1 mL cold washing buffer (15 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L
PMSF, 10 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 0.8 µg/mL
leupeptin, and 1% Triton X-100 vol/wt, pH 7.4) and resuspended in
Laemmli sample buffer (10% glycerol, 1% SDS, 5% 2-ME, 50 mmol/L Tris
HCl (pH 6.8), and 0.002% bromophenol blue) with 10 mmol/L EGTA and 1 mmol/L sodium orthovanadate. After boiling at 95°C for 5 minutes,
1-dimensional electrophoresis was performed on SDS 10% or 7.5% to
15% polyacrylamide gels.42 Separated proteins were
electrophoretically transferred from the gel onto PVDF membranes or
nitrocellulose in a buffer containing Tris (25 mmol/L), glycine (192 mmol/L), and 20% methanol at 0.2 amps for 12 hours at room temperature. To block residual protein binding sites, membranes were
incubated in TBST (Tris-buffered saline [TBS], 10 mmol/L Tris, and
150 mmol/L NaCl, pH 7.6, with 0.1% Tween 20) with 10% chicken egg
albumin. The blots were washed with TBST and incubated overnight with
primary antibodies at a final concentration of 1.0 µg/mL in TBST. The
primary antibody was removed, and the blots were washed 4 times in TBST
and incubated with horseradish peroxidase-conjugated secondary
antibodies diluted 1:3,000 in TBST. The blots were then washed 4 times
in TBST. Antibody reactions were quantified by chemiluminescence
according to the manufacturer's instructions.
Flow cytometry.
Day 8 cells were cultured at a concentration of 5 × 105/mL in 1.0 mL serum-free medium supplemented with 2 U/mL
EPO with or without 50 µmol/L LY294002. After incubation for 24 hours, the cells were collected, washed twice with staining medium
(phosphate-buffered saline [PBS] containing 3% FCS and 0.005%
NaN3), counterstained with PI and annexin V conjugated with
FITC (Apoptosis Detection Kit; R&D Systems Inc, Minneapolis, MN), and
then analyzed by FACS Vantage (Becton Dickinson, Franklin Lakes, NJ).
DNA fragmentation.
DNA fragmentation was measured by quantitation of cytosolic
oligonucleosome-bound DNA using an enzyme-linked immunosorbent assay
(ELISA) kit (Cell Death Detection ELISA; Boehringer, Mannheim, Germany) according to the manufacturer's instructions.
Briefly, day 8 cells were incubated in 0.5 mL serum-free medium in the presence of 2 U/mL EPO with or without 50 mmol/L LY294002 at a concentration of 1 × 105 cells/mL. In the other set of
experiments, cells were incubated for 24 hours with various
concentrations of LY294002 (0, 1, 10, 20, 50, and 100 µmol/L). After
incubation for the indicated periods, the cells were collected and
washed twice with PBS and the cytosolic fraction (13,000g
supernatant) was extracted. The diluted cytosolic fraction equivalent
to 200 cultured cells was used as the Ag source in a sandwich ELISA
with a primary anti-histone Ab coated to the microtiter plate and a
secondary anti-DNA Ab coupled to peroxidase. From the absorbance
values, the percentage of fragmentation, in comparison to controls, was
calculated according to the following formula:
Platelet preparation.
Washed platelets were prepared from citrated whole blood as previously
described.43
Characteristics of expanded cells.
PB CD34+ cells cultured for 8 days in serum-containing
medium with SCF, IL-3, and EPO generated cells that predominantly
consist of erythroid cells (day 8 cells; Fig
1). The ECFCs in day 8 cells predominantly
consist of mature erythroid progenitor cells for which the maturation
level is equivalent to CFU-E as described elsewhere.33
Inhibition of erythroid colony growth by LY294002.
To gain insight into the role of PI-3K signaling in human erythroid
colony growth, day 8 cells were incubated in serum-free fibrin clots
with 2 U/mL EPO in the presence of various concentrations of LY294002,
a specific PI-3K inhibitor. The inhibition of erythroid colony growth
by LY294002 occurred in a dose-dependent manner, and half-maximal
inhibition occurred at 10 µmol/L LY294002 (P < .001).
Maximal inhibition occurred at about 50 µmol/L (Table 1).
Inhibition of proliferation and survival of erythroid progenitor
cells by LY294002.
To investigate the effect of LY294002 on the viability and
proliferation of erythroid progenitor cells, day 8 cells were incubated in liquid phase in serum-free medium with 2 U/mL EPO in the presence of
various concentrations of LY294002 (Table 2). After 7 days of culture,
the cells incubated with neither LY294002 nor 0.1% DMSO increased in
number by 27.8 ± 2.8-fold with 88.0% ± 5.3% viability, resulting
in a 24.5 ± 3.5-fold increase in the number of viable cells. The
presence of 0.1% DMSO, a vehicle of LY294002, did not affect the
proliferation or viability of erythroid progenitor cells. The
proliferation of day 8 cells was markedly inhibited by LY294002 in a
dose-dependent manner and was accompanied by a decrease in viability.
Inhibition of the proliferation and survival of day 8 cells was evident
at concentrations of 10 µmol/L (P < .001 for total cells
and P < .01 for viable cells) and 20 µmol/L LY294002
(P < .01), respectively, the maximum being about 50 µmol/L, which suggests that a blockage of PI-3K signaling by LY294002 inhibits the proliferation and differentiation of erythroid progenitor cells with an accompanying decrease in cell viability. These findings suggest that the inhibition of erythroid progenitor cells by LY294002 could be an early event during the course of erythroid proliferation and differentiation.
Time-course study of the effect of LY294002 on erythroid progenitor
cells.
To elucidate the early events initiated by LY294002, the time course of
the inhibitory effect of LY294002 on the proliferation and
differentiation of erythroid progenitor cells was investigated. Day 8 cells were suspended in liquid phase in serum-free medium with 2 U/mL
EPO in the presence of 50 µmol/L LY294002 and cultured for 0, 4, 8, 24, and 48 hours, and the number and viability of the cells were
assessed. To determine if the inhibitory effect of LY294002 is
reversible, the cells were collected and washed twice with IMDM
containing 0.3% BSA and then cultured in serum-containing fibrin clots
with 2 U/mL EPO for 7 days, the objective being to assess the
colony-forming ability after exposure to LY294002.
Titration of the effect of LY294002 on erythroid progenitor cells.
To titrate the inhibitory effect of LY294002 on erythroid progenitor
cells, day 8 cells were suspended in liquid phase in serum-free medium
with 2 U/mL EPO in the presence of various concentrations of LY294002
(0, 1, 10, 20, 50, and 100 µmol/L) and cultured for 4 and 24 hours,
and the number and viability were assessed. To investigate whether the
irreversibility of the inhibitory effect of LY294002 depends on the
dose of LY294002, the cells were collected and washed twice with IMDM
containing 0.3% BSA and then cultured in serum-containing fibrin clots
with 2 U/mL EPO for 7 days.
LY294002 induces apoptosis and DNA fragmentation in erythroid
progenitor cells.
A decrease in erythroid progenitor growth with intact viability after
exposure to LY294002 suggests that an early event in the induction of
an internal suicide program (apoptosis) could be responsible for the
mechanism. Because apoptosis is commonly associated biochemically with
the loss of phospholipid asymmetry on the cell surface and DNA
fragmentation, we examined these 2 issues. The loss of phospholipid
asymmetry on the cell surface was examined using FITC-conjugated
annexin V and FACS (Fig 4). When day 8 cells were incubated for 24 hours, 22.8% of cells incubated with LY294002 at 50 µmol/L were
detected as annexin V+/PI
LY294002 inhibits phosphorylation of the PI-3K-dependent kinase AKT.
To determine if PI-3K activity in situ was indeed inhibited by LY294002
in primary cultured human erythroid progenitor cells, we next examined
the effect of LY294002 on the steady-state phosphorylation of AKT at
Ser-473, a recognized downstream event of PI-3K activation. AKT was
modestly and constitutively phosphorylated at Ser-473 in human
erythroid progenitor cells, and phosphorylation of AKT was blocked by
treatment of the cells with LY294002 at concentrations similar to those
which suppressed erythroid growth or induced apoptosis (Fig
7). We acknowledge that the degree of AKT
phosphorylation was small. However, it should be noted that we have
tried to measure steady-state levels of AKT phosphorylation instead of
induced levels. In preliminary experiments, we could not find any
steady-state phosphorylation over the background levels by simple
Western blotting on 3 different occasions. However, we confirmed that
the reagents work well, because of the results of the following
experiments. Human washed platelets were prepared and stimulated by
type 1 horse tendon collagen (50 µg/L) as previously
described.43 Whole-cell lysates (107
cells/lane) were subjected to SDS-PAGE followed by Western blotting to
detect AKT phosphorylation using the same system. As expected from the
previous report,44 collagen induced a clear increase in AKT
phosphorylation and the increase was abolished by LY294002 (50 µmol/L), suggesting that the detection system and LY294002 are
appropriate (Fig 8). Similar amounts of AKT were detected on each lane
(lower panel). In both panels, the bands appear broad, probably due to
the presence of different isoforms of
AKT.26
We examined the role of PI-3K in the proliferation and viability of
human erythroid progenitors expanded in vitro. Because these cells,
unlike genetically engineered murine cell lines expressing the EPO-R or
murine and human leukemic cell lines, regularly undergo full terminal
differentiation and become reticulocytes, these studies are more likely
to reflect actual physiologic situations. However, we acknowledge the
possible presence of artifacts arising from in vitro
culture.30,32
We thank Dr C.I. Civin for the generous gift of the monoclonal
antibodies, without which this study could not have been performed. We
also thank Chugai Pharmaceutical Co, Kirin Brewery Co, Sankyo Pharmaceutical Co, and Takeda Pharmaceutical Co for the generous gift
of recombinant human growth factors and reagents. Finally, we thank S. Okazaki for manuscript preparation, M. Ohara for helpful comments, and
M. Kitayama and I. Sato for technical assistance.
Submitted November 6, 1998; accepted May 3, 1999.
Supported in part by grants-in-aid from the Ministry of Education,
Science, Sports and Culture of Japan (K.S., A.O., and Y.I.), a research
grant from the Idiopathic Disorders of Hematopoietic Organs Research
Committee of the Ministry of Health and Welfare of Japan (K.S.), and
the Ryoichi Naito Foundation for Medical Research (A.O.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Ken-ichi Sawada, MD, Department of Medicine
II, Hokkaido University School of Medicine, N-15, W-7, Sapporo,
Hokkaido 060-8638, Japan.
1.
Krantz SB:
Erythropoietin.
Blood
77:419, 1991
2.
Koury MJ, Bondurant MC:
The molecular mechanism of erythropoietin action.
Eur J Biochem
210:649, 1992[Medline]
[Order article via Infotrieve]
3.
Youssoufian H, Longmore G, Neuman D, Yoshimura A, Lodish HF:
Structure, function, and activation of the erythropoietin receptor.
Blood
81:2223, 1993
4.
Sayer ST, Penta K:
Erythropoietin cell biology.
Hematol Oncol Clin North Am
8:895, 1994[Medline]
[Order article via Infotrieve]
5.
Witthuhn BA, Quelle FW, Silvennoinen O, Yi T, Tang B, Miura O, Ihle JN:
Jak2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin.
Cell
74:227, 1993[Medline]
[Order article via Infotrieve]
6.
Miura O, Nakamura N, Quelle FW, Witthuman BA, Ihle JN, Aoki N:
Erythropoietin induces association of the Jak2 protein tyrosine kinase with the erythropoietin receptor in vivo.
Blood
84:1501, 1994
7.
Wakao H, Harada N, Kitamura T, Mui AL, Miyajima A:
Interleukin 2 and erythropoietin activate STAT5/MGF via distinct pathways.
EMBO J
14:2527, 1995[Medline]
[Order article via Infotrieve]
8.
Ohashi H, Maruyama K, Liu YC, Yoshimura A:
Ligand-induced activation of chimeric receptors between the erythropoietin receptor and receptor tyrosine kinases.
Proc Natl Acad Sci USA
1:158, 1994
9.
Sawyer ST, Penta K:
Association of Jak2 and STAT5 with erythropoietin receptors.
J Biol Chem
271:32430, 1996
10.
Quell FW, Wang D, Nosaka T, Thierfelder WE, Stravopodis D, Weinstein Y, Ihle JN:
Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for mitogenic response.
Mol Cell Biol
16:1622, 1996[Abstract]
11.
Ohashi T, Masuda M, Ruscetti SK:
Induction of sequence-specific DNA-binding factors by erythropoietin and the spleen focus-forming virus.
Blood
85:1454, 1995
12.
Klingmuller U, Bergelson S, Hsio JG, Lodish HF:
Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5.
Proc Natl Acad Sci USA
93:8324, 1996
13.
Penta K, Sawyer ST:
Erythropoietin induces the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1 and STAT5 in erythroid cells.
J Biol Chem
270:31282, 1995
14.
Mellitzer G, Wesseley O, Decker T, Meinke A, Hayman MJ, Beug H:
Activation of STAT5b in erythroid progenitors correlates with the ability of ErbB to induce sustained cell proliferation.
Proc Natl Acad Sci USA
93:9600, 1996
15.
Miura O, Nakamura N, Ihle JN, Aoki N:
Erythropoietin-dependent association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated erythropoietin receptor.
J Biol Chem
269:614, 1994
16.
Kubota Y, Tanaka T, Yamaoka G, Yamaguchi M, Ohnishi H, Kawanishi K, Takahara J, Irino S:
Wortmannin: A specific inhibitor of phosphatidylinositol-3-kinase, inhibits erythropoietin-induced erythroid differentiation of K562 cells.
Leukemia
10:720, 1996[Medline]
[Order article via Infotrieve]
17.
He TC, Zhuang H, Jiang N, Waterfield MD, Wojchowski DM:
Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.
Blood
82:3530, 1993
18.
Jaster R, Bittorf T, Brock J:
Involvement of phosphatidylinositol 3-kinase in the mediation of erythropoietin-induced activation of p70S6k.
Cell Signal
9:175, 1997[Medline]
[Order article via Infotrieve]
19.
Joneja B, Wojchowski DM:
Mitogenic signaling and inhibition of apoptosis via the erythropoietin receptor Box-1 domain.
J Biol Chem
272:11176, 1997
20.
Verdier F, Chretien S, Billat C, Gisselbrecht S, Lacombe C, Mayeux P:
Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.
J Biol Chem
272:26173, 1997
21.
Shigematsu H, Iwasaki H, Otsuka T, Ohno Y, Arima F, Niho Y:
Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity.
J Biol Chem
272:14334, 1997
22.
Gobert S, Porteu F, Pallu S, Muller O, Sabbah M, Dusanter-Fourt I, Courtois G, Lacombe C, Gisselbrecht S, Mayeux P:
Tyrosine phosphorylation of the erythropoietin receptor: Role for differentiation and mitogenic signal transduction.
Blood
86:598, 1995
23.
Damen JE, Cutler RL, Jiao H, Yi T, Krystal G:
Phosphorylation of tyrosine 503 in the erythropoietin receptor (EpR) is essential for binding the P85 subunit of phosphatidylinositol (PI) 3-kinase and for EpR-associated PI 3-kinase activity.
J Biol Chem
270:23402, 1995
24.
Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M:
3-Phosphoinositide-dependent protein kinase-1 (PDK1): Structural and functional homology with the Drosophila DSTPK61 kinase.
Curr Biol
7:776, 1997[Medline]
[Order article via Infotrieve]
25.
Blume-Jensen P, Janknecht R, Hunter T:
The kit receptor promotes cell survival via activation of PI 3-kinase and subsequent Akt-mediated phosphorylation of Bad on Ser136.
Curr Biol
18:779, 1998
26.
Downward J:
Mechanisms and consequences of activation of protein kinase B/Akt.
Curr Opin Cell Biol
10:262, 1998[Medline]
[Order article via Infotrieve]
27.
Chao DT, Korsmeyer SJ:
BCL-2 family: Regulators of cell death.
Annu Rev Immunol
16:395, 1998[Medline]
[Order article via Infotrieve] |
TOP
ABSTRACT
INTRODUCTION
5 mol/mL 
-ME, 50 U/mL penicillin, 50 U/mL
streptomycin, and IMDM in a 50-mL polystyrene flask (Corning Coster
Corp, Cambridge, MA). After incubation for 8 days at 37°C in a 5%
CO2/95% O2 atmosphere, the cells were
collected, washed twice with IMDM containing 0.3% BSA (day 8 cells).
) or without
(
) 50 µmol/L LY294002. After incubation and at the indicated
periods, the cells were collected, washed twice in IMDM containing
0.3% BSA, and then plated into serum-containing fibrin clots with 2 U/mL EPO. After 7 days of incubation, the clots were fixed and stained
with benzidine-hematoxylin. (A) Viability; (B) absolute number of
viable cells; (C) percent expression for the number of erythroid
colonies in relation to the value of 0 hours as 100%. The mean ± SD
of triplicates is shown for C. +++P < .001, decrease v control (
