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HEMATOPOIESIS
From the Institute of Biomedical Sciences, and
Institute of Biological Chemistry, Academia Sinica, and Graduate
Institute of Life Sciences, National Defense Medical Center, Taipei,
Taiwan.
Stem cell factor (SCF) has been suggested as essential for optimal
production of various hematopoietic lineages mainly because of its
apoptosis prevention function when it costimulates with other
cytokines. However, the underlying mechanism of this synergism of
apoptosis prevention is largely unknown. The present study examined the
expression of some Bcl-2 family members, including Bcl-2,
Bcl-XL, Mcl-1, and Bax, in response to cytokine
stimulation in TF-1 and JYTF-1 cells in which SCF costimulation is
differentially required for optimal proliferation. The results revealed
that only the expression of Mcl-1 highly correlated with the
antiapoptotic activity of interleukin-5 (IL-5) and the synergistic
effect of SCF. In TF-1 cells, the defect of IL-5 in apoptosis
suppression and Mcl-1 induction was associated with the incapability to
highly phosphorylate Janus kinases (JAK1, JAK2), signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase
(MAPK), and Akt/PKB, whereas SCF costimulation restored the potent
phosphorylation of MAPK and Akt/PKB, but not STAT5. The importance of
MAPK and Akt/PKB signaling pathways in regulating the expression of
Mcl-1 and cell survival was further supported by the observation that
inhibition of MEK by PD98059 or phosphatidylinositol-3 kinase (PI-3K)
by LY294002 independently resulted in the reduction of Mcl-1 expression
and loss of cell viability. Therefore, the data suggest that
Mcl-1 is a common antiapoptotic target of both early-stage
cytokine SCF and late-stage cytokine IL-5. Both MEK/MAPK and PI-3K/Akt
signaling pathways are essential in the regulation of Mcl-1 expression
and apoptosis prevention.
(Blood. 2000;96:1764-1771) The development of different cell lineages in
hematopoiesis depends on direct cell-cell contact and the release of
soluble factors. Among these external soluble factors, cytokines play important roles. Stem cell factor (SCF) is an early acting cytokine produced by marrow stromal cells. Although SCF alone does not stimulate
myelopoiesis, when combined with other cytokines, SCF increases the
cloning efficacy of hematopoietic progenitor cells from all
lineages.1 The maintenance of high levels of myelopoiesis and erythropoiesis in vitro by SCF was shown to be due to the suppression of apoptosis.2,3 By contrast, interleukin
(IL)-5 belongs to a group of late-acting cytokines with a restricted set of targets in hematopoiesis, because the major role of IL-5 is
limited to the eosinophilic/basophilic lineage in humans1 and to B-cell development additionally in mice.4-6
Recently, IL-5 was shown to be able to promote the eosinophilic
outgrowth from bone marrow stem and progenitor cells. However,
according to an in vitro assay,7 the maximal production of
eosinophils from stem cell population requires the costimulation
of SCF.
Binding of IL-5 to its surface high-affinity receptor complexes, which
are composed of an The involvement of several signaling pathways in the regulation of
apoptosis has been extensively investigated, and gene products including Ras, Raf, MAPK,29 PI-3 kinase, and
PKB/Akt30 have been suggested as having an important role
in transducing the antiapoptotic function. However, the target of these
signaling molecules is largely unclear. Recently, the Bcl-2
gene family, which is composed of 14 members, was shown to compose the
central players of apoptosis regulation (for review, see reference 31). Some members of this family, such as Bcl-2, Bcl-XL, A1, and
Mcl-1, inhibited apoptosis whereas other members, such as Bax, Bad, and Bak, accelerated apoptosis. Among Bcl-2 family members, Bcl-2, Bcl-XL, and Mcl-1 all have been shown to be
up-regulated by IL-3 in various IL-3-dependent cell
lines.30,32
TF-1 is a human hematopoietic progenitor cell line derived from the
bone marrow of an erythroleukemic patient.34 TF-1 cells, like normal progenitor cells, express high levels of
granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor Our previous study indicated that Mcl-1 plays a role in the viability
response of GM-CSF.33 When GM-CSF was withdrawn from the
culture medium of human GM-CSF-dependent cell line TF-1, the level of
endogenous Mcl-1 decreased rapidly down to the background level in 6 hours. On restimulation of these starved cells with GM-CSF,
mcl-1 is one of the immediate early genes induced and its
messenger RNA (mRNA) peaked at 30 minutes and its protein product was
accordingly resynthesized.33 Ectopic overexpression of
Mcl-1 in TF-1 cells significantly delayed cell death triggered by
GM-CSF deprivation and reduced expression of endogenous Mcl-1 by an
antisense construct decreased the viability of cells cultured in the
presence of cytokine.33 In the present study, we attempted to further identify the candidate Bcl-2 gene family members
through which the antiapoptotic signals of IL-5 and SCF merge and
to delineate the signaling pathways that lead to activation
of Bcl-2 family proteins from both IL-5 and SCF receptors. We
demonstrate that the Mcl-1, but not Bcl-2, Bcl-XL or
Bax, is one of the common targets of SCF and IL-5 for
their antiapoptosis function and that both MEK/MAPK and PI-3K/Akt
pathways are essential for the survival functions of these 2 cytokine receptors.
Cell lines, cytokines, and chemical reagents
Cell proliferation assay
Nucleosome-releasing assay and DNA fragmentation analysis Quantitative measurement of the apoptosis activity in cultured cells was performed by nucleosome-releasing assay (Boehringer Mannheim, Mannheim, Germany; catalogue no. 1774425) and qualitative demonstration of apoptosis was performed by DNA fragmentation assay.35Immunoprecipitation and Western blot analysis Cells were washed and starved in 0.5% fetal bovine serum (FBS) medium for 20 to 24 hours, then stimulated with 10 ng/mL GM-CSF or IL-5, or 50 ng/mL SCF (R&D Systems). For protein tyrosine kinase activation analyses, cells were stimulated with various cytokines for 5 minutes. For measurement of the expression of Bcl-2 family proteins, cells were incubated for 1 hour. After washing with phosphate-buffered saline (PBS), cells were lysed at 4°C in 0.5 mL of lysis buffer (1% NP-40, 50 mmol/L Tris-HCl, pH 7.4, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L PMSF, 1 µg/mL leupeptin, 1 mmol/L Na3VO4, 1 mmol/L NaF) and the unsolubilized material was removed by centrifugation for 10 minutes at 14 000 rpm. The lysates were precleared with protein A-Sepharose beads and then reacted with the indicated primary antibody overnight at 4°C. The antigen-antibody complexes were then precipitated with protein A-Sepharose beads for 2 hours at 4°C, washed 3 times with lysis buffer, and then boiled for 5 minutes with 2 × sodium dodecyl sulfate (SDS) sample buffer. The boiled supernatants were electrophoresed on a SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed with Western blotting. After binding with horseradish peroxidase-conjugated secondary antibodies, blots were visualized with an enhanced chemiluminescence (ECL) detection system (Amersham, Little Chalfont, Buckinghamshire, UK). The antibodies against Bcl-2 (N-19), Mcl-1 (S-19), Bax (N-20), Bcl-XL (M-125), human c chain
(S-16), JAK1 (HR-785), JAK2 (HR-758), and STAT5 (C-17) were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Antiphosphotyrosine
monoclonal antibody 4G10 was purchased from UBI (Lake Placid, NY).
Antibodies against phospho-MAPK(Thr202/Tyr204), MAPK,
phospho-Akt(Ser473), and Akt/PKB were all purchased from New England
BioLabs (Beverly, MA).
In vitro kinase assay The MAPK in vitro kinase assay was performed using the p44/42 MAP kinase assay kit (New England BioLabs). Briefly, cells were lysed in MAPK lysis buffer (20 mmol/L Tris-HCl, pH7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L-glycerolphosphate, 1 mmol/L
Na3VO4, 1 µg/mL leupeptin, 1 mmol/L PMSF),
and MAP kinases were immunoprecipitated with the immobilized
phospho-p44/42 MAPK monoclonal antibody. After washing twice with lysis
buffer and twice with kinase buffer (25 mmol/L Tris-HCl, pH7.5, 5 mmol/L -glycerolphosphate, 2 mmol/L DTT, 0.1 mmol/L
Na3VO4, 10 mmol/L MgCl2), the
immunoprecipitates were assayed for MAP kinase activity in kinase
buffer with 200 µmol/L adenosine triphosphate (ATP) and 2 µg Elk-1
fusion protein per reaction. The reaction was stopped with SDS sample
buffer and analyzed by Western blotting with specific
antiphospho-Elk-1 antibody.
The Akt in vitro kinase assays were performed as described by Franke
and colleagues.36 Cells were lysed in Akt lysis buffer (20 mmol/L Tris-HCl, pH7.5, 150 mmol/L NaCl, 10% glycerol, 1% NP-40, 10 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L
Na4P2O7, 2 µmol/L leupeptin, 2 µmol/L aprotinin, 1 mmol/L PMSF) and the Akt protein was
immunoprecipitated with anti-Akt antibody (New England BioLabs). The
immune complexes were collected with protein A-Sepharose and washed 3 times with lysis buffer, once with cold water, and once with kinase
buffer (20 mmol/L HEPES, pH7.4, 1 mmol/L DTT, 10 mmol/L MnCl2, 10 mmol/L MgCl2). The Akt kinase
activity was then assayed in kinase buffer with 5 µmol/L ATP, 3 µg
histone 2B, and 20 µCi of [
Mcl-1 expression is closely associated with apoptosis prevention ability of cytokines In our previous study,34 we demonstrated that either costimulation with SCF or increased expression of IL-5R allowed
hematopoietic progenitor cells to grow optimally in medium containing
IL-5. In the present study, we explored whether these 2 treatments lead to apoptosis prevention by a similar mechanism. When healthy TF-1 cells
were transferred from medium containing GM-CSF (Figure
1A, lane 2) into medium containing IL-5,
cell death was detectable by DNA ladder analysis after 12 hours of
incubation and became very obvious within 24 hours (Figure 1A, lanes
5-7). However, IL-5 was capable of suppressing apoptosis in JYTF-1
cells in which the expression of IL-5R is increased and the DNA
ladder was not detectable throughout the experimental period (Figure
1A, lanes 11-14). We previously reported that overexpression of Bcl-2
or Mcl-1 protein in TF-1 cells strongly suppresses cytokine
withdrawal-induced apoptosis and that Mcl-1 is one component of both
GM-CSF and IL-3 survival responses in cytokine-dependent cell
lines.33,34 We were curious whether Mcl-1 also plays an
important role in the survival effect of IL-5 and SCF in the
hematopoietic progenitor cell line. To address this issue, cell lysates
were prepared from cells grown in various culture conditions and then
subjected to Western blot analysis for the protein expression of
several Bcl-2 family members, including Bcl-2, Bcl-XL,
Mcl-1, and Bax. TF-1 and JYTF-1 cells cultured in GM-CSF were shifted
to medium containing IL-5 for various time periods. As shown in Figure
1B, the expression level of Mcl-1 in both cell lines was 3- to 4-fold
higher in medium containing GM-CSF than in cytokine-free medium (Figure
1B, lanes 1, 7, 8, and 14). IL-5 was not able to sustain the expression of Mcl-1 after transferring TF-1 cells from GM-CSF-containing into
IL-5-containing medium, and Mcl-1 expression reduced to basal level
within 18 to 24 hours (Figure 1B, lanes 5-7). The decrease in Mcl-1
expression correlated well with the appearance of the DNA ladder
(Figure 1A, lanes 6 and 7). On the other hand, IL-5 was capable of
maintaining the expression of Mcl-1 in JYTF-1 cells at a level similar
to the effect of GM-CSF within 24 hours (Figure 1B, lanes 10-13). Under
the same conditions, the expression levels of 3 other Bcl-2 family
members (Bcl-2, Bcl-XL, and Bax) remained the same
throughout the experimental period (Figure 1B). Next, we examined
whether cytokines (GM-CSF, IL-5, and SCF) capable of sustaining the
survival of JYTF-1 cells stimulate Mcl-1 expression. The result shown
in Figure 1C indicates that this is indeed the case, whereas the
expression levels of Bcl-XL, a Bcl-2 family member known to
be involved in the survival response of IL-3 in certain cell
systems,31 are not stimulated by any of these 3 cytokines.
The correlation between the induction of the Mcl-1 protein and the
apoptosis prevention ability of cytokine was further strengthened by
studying Mcl-1 induction by SCF costimulation. Although the optimal
proliferation of TF-1 cells required IL-5 and SCF costimulation (Figure
2A, left panel), as previously
demonstrated,34 IL-5 was able to promote the optimal
growth of JYTF-1 cells in an SCF-independent manner (Figure 2A, right
panel). These data were consistent with the responses previously
observed in an IL-5R
Differential activation of signaling components in TF-1 and JYTF-1 cells by IL-5 In response to IL-5, extensive tyrosine phosphorylation of intracellular proteins and activation of various kinases have been reported.17,37,38 However, the specific role of each individual signaling pathway in IL-5-dependent antiapoptotic activity and in Mcl-1 induction remains unclear. We next investigated how signaling molecules known to be activated by IL-5 are differentially affected in TF-1 cells when the cell's antiapoptotic function is lacking. As shown in Figure 3,c,
JAK1, and JAK2 were tyrosine phosphorylated after stimulation by GM-CSF
in both TF-1 and JYTF-1 cell lines (Figure 3, lanes 2 and 5). However,
in TF-1 cells, the phosphorylation of JAK1 was not detectable (Figure
3, lane 3) and c and JAK2 were only weakly phosphorylated by IL-5
(Figure 3, lane 3). The tyrosine phosphorylation of JAK2 in TF-1 cells,
although weak, was highly reproducible. However, we were not able to
detect the signal of JAK1 phosphorylation even when 5 mg of protein
lysate was used in an immunoprecipitation reaction and the blots were exposed for up to 1 hour after ECL reaction (data not shown). This
defect in JAK1 phosphorylation by IL-5 was restored and the phosphorylation of c and JAK2 was also greatly improved in JYTF-1 cells (Figure 3, lane 6) as well as in 2 of the cell lines
overexpressing IL-5R, TF1, and TF8 (data not shown).
Phosphorylation of JAK3 and Tyk2 was not detectable by GM-CSF and IL-5
in TF-1 and JYTF-1 cell lines (data not shown). Furthermore, GM-CSF and
IL-5 both were able to stimulate the phosphorylation of STAT5 in TF-1
and JYTF-1 cell lines (Figure 3, lanes 2, 3, 5, and 6), although the level of phosphorylation was much weaker in TF-1 cells stimulated by
IL-5 (lane 3). Among other STAT family members, STAT3 was slightly tyrosine phosphorylated on stimulation by GM-CSF, but not IL-5, in both
cell lines (data not shown).
As demonstrated by Kinoshita and colleagues,20 a mutant
Both MAPK and Akt/PKB pathways are activated by SCF To further explore the commitment of each signaling pathway in apoptosis prevention, we costimulated SCF with IL-5 and checked the activation of STAT5, MAPK, and Akt/PKB by Western blotting as described in Figure 3. As shown in Figure 4, SCF activated MAPK and Akt/PKB, but did not activate STAT5 in either TF-1 or JYTF-1 cell lines (Figure 4, lanes 4 and 8). When costimulated with IL-5, SCF complemented the defect in MAPK and Akt/PKB activation of IL-5, but did not complement the defect in STAT5 activation (Figure 4, lane 3). Therefore, activation of MAPK and Akt/PKB pathways seemed to be more closely correlated with antiapoptosis ability. When the proliferation of JYTF-1 cell line was optimally activated by IL-5, the levels of phosphorylation of MAPK at Thr202/Tyr204 and Akt/PKB at Ser473 were significantly less than when stimulated by the optimizing concentration of SCF (Figure 4, lanes 6 versus 8). The signal intensity of phospho-MAPK and phospho-Akt in SCF-treated samples was about 2- to 3-fold higher than that in IL-5-treated JYTF-1 samples. However, the proliferation response of JYTF-1 cells to IL-5 was constantly greater than the effect of SCF (Figure 2A), suggesting that the STAT5 pathway or other unidentified pathways may also play important roles in supporting the proliferation of hematopoietic cells.
Both MEK/MAPK and PI-3K/Akt activities are essential for apoptosis prevention and Mcl-1 induction To further confirm the roles of the MAPK and Akt/PKB pathways in apoptosis prevention, we applied specific inhibitors to abrogate each pathway and explored the possible interference of the growth property. PD98059, a specific MEK inhibitor, and LY294002, an inhibitor of PI-3 kinase, were used to treat JYTF-1 cells, and a time course study on the viable cell numbers was performed. As shown in Figure 5A, both PD98059 (100 µmol/L) or LY294002 (40 µmol/L) effectively suppressed growth of JYTF-1 cells in both IL-5- and SCF-containing medium. Both PD98059 and LY294002 completely inhibited the mitogenic activity of SCF and IL-5 (data not shown), whereas they showed differential inhibition of the antiapoptotic effect of cytokines (Figure 5B). As revealed by a nucleosome releasing assay, LY294002 abrogated the antiapoptotic effect of IL-5 slightly better than did PD98059 (Figure 5B, IL5+LY versus IL-5+PD). In contrast, both inhibitors completely inhibited the survival effect of SCF (Figure 5B, SCF+PD and SCF+LY).
To confirm the specificity of these kinase inhibitors, the activities
of MAPK and Akt/PKB kinases after treating with inhibitors were
measured. Results from Western blot analyses with phospho-MAPK (P-MAPK)
and phospho-Akt (P-Akt) specific antibodies revealed that the
phosphorylation of MAPK and Akt by their upstream kinases were
dramatically inhibited by PD98059 and LY294002, respectively (Figure
6A). Furthermore, the kinase activities
for both MAPK and Akt were also measured by in vitro kinase assay
(Figure 6B). A known MAPK substrate, Elk-1, was included in the MAPK
kinase reaction and its phosphorylation was detected with phospho-Elk specific antibody. In contrast, histone H2B was used as a
pseudosubstrate of Akt and the phosphorylation of H2B was detected by
autoradiography after labeling with 32P-ATP by kinase. The
results of the in vitro kinase reaction suggested that both PD98059 and
LY294002 have a very potent inhibition effect to their target kinases
(Figure 6B). Intriguingly, LY294002 also showed a partial inhibitory
effect on the activity of MAPK in both the phosphorylation assay
(Figure 6A) and in the in vitro kinase assay (Figure 6B). Additionally,
LY294002 inhibited the phosphorylation of MEK, an upstream kinase of
MAPK, by 50% (data not shown).
After demonstrating the inhibition of the MEK/MAPK signaling pathway by
PD98059 and PI-3K/Akt signaling pathway by LY294002, we next
investigated effects of these inhibitors on the induction of Mcl-1
protein. LY294002 completely suppressed the expression of Mcl-1 induced
by either IL-5 or SCF (Figure 7, lanes 8 and 9). However, PD98059 only reduced Mcl-1 expression 36% by IL-5 induction (Figure 7, lane 5 versus lane 2) and 77% by SCF induction (Figure 7, lane 6 versus lane 3). Meanwhile, the expression of Bcl-2
and Bax proteins was not affected at all when cells were treated with
either kinase inhibitor (Figure 7). It was previously demonstrated that
the phosphorylation of STAT5 protein by erythropoietin was not
inhibited by PI-3K inhibitor and PD98059 in primary erythroid colony-forming cells.39 We, therefore, investigated the
tyrosine phosphorylation of the STAT5 protein. As shown in Figure
8, the induction of tyrosine
phosphorylation of the STAT5 protein was not affected by either kinase
inhibitor in medium containing IL-5 (Figure 8, lanes 2, 5, and 8), and
SCF did not activate STAT5 tyrosine phosphorylation, the same result as
shown in Figure 4.
In this study, we used 3 systems to demonstrate the importance of
Mcl-1, a prosurvival member of the Bcl-2 gene family, in the
antiapoptotic effects of SCF and IL-5. First, the ability of IL-5 to
induce Mcl-1 expression is correlated with the ability of IL-5 to
suppress apoptosis in TF-1 and JYTF-1 cell lines. IL-5 was unable to
induce Mcl-1 expression in the IL-5R mcl-1 was originally identified as an early gene induced by
phorbol ester during differentiation of ML-1 myeloid leukemia cells.40 It was suggested that Mcl-1 may have a function
in development and differentiation of hematopoiesis. Indeed, in
terminally differentiated human neutrophils, the expression level of
Mcl-1 was shown to be tightly correlated with cell viability promoted by certain agents including GM-CSF, IL-1 Our data also suggest that both the MEK/MAPK and PI-3K/Akt signaling pathways are essential in regulating Mcl-1 expression and the antiapoptotic activities of IL-5 and SCF. However, several lines of evidence suggest that these 2 signaling pathways do not cross-talk with each other. A Ras mutation (G12V/T35S), which effectively activates Raf/MAPK activity, does not stimulate PI-3K function.29 Inversely, the G12V/V45E mutation of Ras causes the phosphorylation of S6 kinase via PI-3K without affecting the function of MAPK.29 In the present study, MEK kinase-specific inhibitor efficiently blocked the activation of MAPK by cytokines, but did not interfere with the function of Akt (Figure 6A,B). Overexpression of the constitutively active mutant of Akt (ie, Myr-Akt) also independently confers a survival ability on hematopoietic cells30 without activating MAPK (our unpublished data). Overexpression of the activated Ras mutants,29 which causes activation of either the Raf/MAPK or PI-3K/Akt pathway, independently prevents cytokine withdrawal-induced apoptosis suggesting that each signal is sufficient for suppressing apoptosis. These observations are consistent with the results from our Myr-Akt overexpression experiment, but not with the results from our kinase inhibitor experiments. For example, although SCF still fully activated the function of the PI-3K/Akt pathway in the presence of MEK inhibitor, SCF almost completely lost its antiapoptotic function (Figure 5B). In the presence of PI-3K inhibitor, SCF also completely lost its antiapoptotic activity (Figure 5B). These later observations suggested an essential, but not sufficient, role of each signaling pathway in achieving a survival effect from cytokine. We cannot currently account for these discrepancies among results reached by overexpression of constitutive active mutants29,30 and by chemical inhibitor treatments or from the expression of receptor binding site mutants.50 One possibility may be that the strength of the enzymatic activity of Akt in myr-Akt-expressing cells is much stronger than that of the endogenous Akt activated by cytokine receptors under the physiologic condition. Hypothetically, in this case the quantitative difference in the strength of the 2 identical Akt signals would result in the qualitative difference in physiologic responses; that is, the strong Myr-Akt signal protects cells from death, whereas the weak endogenous Akt signal does not. Intriguingly, although most of our results suggested that similar, if
not identical, extents of activation in the MEK/MAPK and PI-3K/Akt
pathways were achieved by IL-5 and SCF (Figure 3), JYTF-1 cells
cultured in IL-5 were always slightly more viable than SCF-treated
cells (Figure 2A). In addition, although the enzymatic activity of MAPK
and Akt were suppressed to similar levels by kinase inhibitors (Figure
6A,B), cells treated with IL-5 still possessed a better survival rate
than cells cultured in SCF (Figure 5A,B). We demonstrated that STAT5 is
activated only by IL-5, but not SCF, and that STAT5 was not affected by either PD98059 or LY294002 in our experiments (Figure 8). These differential responses of JYTF-1 cells toward IL-5 versus SCF may be
due to the existence of an additional STAT5 signal of IL-5. Recently,
expression of the dominant negative mutant of STAT5 was shown to be
deleterious to the proliferation of factor-dependent cells.51 Furthermore, STAT5 was shown to be an essential
antiapoptotic component of IL-3, IL-5, and GM-CSF in in vitro colony
formation assays with STAT5a
The authors thank Mr Yung-Luen Yu for preparation of the manuscript and Dr Hsin-Fang Yang-Yen for her careful reading of this manuscript and helpful suggestions.
Submitted October 25, 1999; accepted May 14, 2000.
Supported by grants from Academia Sinica and the National Science Council of Taiwan, NSC 86-2314-B-001-026 to J.J.-Y.Y.
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: Jeffrey Jong-Young Yen, Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan; e-mail: bmjyen{at}novell.ibms.sinica.edu.tw.
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D. Miao, X.-K. Tong, G. K. Chan, D. Panda, P. S. McPherson, and D. Goltzman Parathyroid Hormone-related Peptide Stimulates Osteogenic Cell Proliferation through Protein Kinase C Activation of the Ras/Mitogen-activated Protein Kinase Signaling Pathway J. Biol. Chem., August 17, 2001; 276(34): 32204 - 32213. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-32P]ATP per reaction.
Akt kinase reaction was stopped with SDS sample buffer and the products
were separated by SDS-PAGE for autoradiography.
), SCF (
), IL-5 plus SCF (
), or
no cytokine (
) and the viable cell numbers were measured every 24 hours by trypan blue dye exclusion assay. Left panel is for TF-1 cells
and right panel is for JYTF-1 cells. Each number is the average of 3 independent measurements and SDs are shown as error bars. (B) SCF
costimulation induced Mcl-1 expression in TF-1 cells. TF-1 (lanes 1-4)
and JYTF-1 (lanes 5-8) cells were starved in cytokine-free medium for
24 hours (lanes 1 and 5) and then treated with IL-5 (lanes 2 and 6),
SCF (lanes 4 and 8), or IL-5 plus SCF (lanes 3 and 7) for 1 hour before
harvesting for lysate preparation. Western blot analysis was performed
for the expression of Mcl-1, Bcl-2, Bax, and 
), SCF plus 100PD (
), SCF plus 40LY (
). (B) Differential
effect on antiapoptotic activity in IL-5- and SCF-cultured cells.
JYTF-1 cells were treated as described in panel A and the extent of
nucleosome released from apoptotic cells was measured after 24 hours of
incubation. Each value is the average of 3 independent quadruplet
experiments, and the SEs are shown as error bars.
/