From the Immuno-apoptose, U503 INSERM CERVI, 21 avenue Tony Garnier, 69007 Lyon, France and the ¶ Laboratoire de
physiopathologie métabolique et rénale, INSERM U499,
Faculté de médecine Laennec, 12 rue Guillaume Paradin,
69372 Lyon, cedex 08, France
Received for publication, August 7, 2000, and in revised form, January 8, 2001
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ABSTRACT |
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Baf-3 cells are dependent on interleukin-3 (IL-3)
for their survival and proliferation in culture. To identify
anti-apoptotic pathways, we performed a retroviral-insertion
mutagenesis on Baf-3 cells and selected mutants that have acquired a
long term survival capacity. The phenotype of one mutant, which does
not overexpress bcl-x and proliferates in the absence of
IL-3, is described. We show that, in this mutant, Akt is constitutively
activated leading to FKHRL1 phosphorylation and constitutive glycolytic
activity. This pathway is necessary for the mutant to survive following IL-3 starvation but is not sufficient or necessary to protect cells
from DNA damage-induced cell death. Indeed, inhibition of the
phosphatidylinositol 3-kinase (PI3K)/Akt pathway in Baf-3 cells does
not prevent the ability of IL-3 to protect cells against Growth factors are necessary to inhibit the intrinsic apoptotic
machinery, which is constitutively expressed in all cells. Baf-3 cells
are dependent on IL-31 for
their proliferation and survival (1). A number of signaling pathways
that are activated by IL-3 play a role in the inhibition of apoptosis
(2-4). One major signaling pathway involved in the control of cell
death by growth factors is the PI3K/Akt pathway. Activation of
PI3K leads to the generation of 3'-phosphorylated phosphatidylinositides that act by multiple mechanisms to activate Akt
(5). Akt will in turn phosphorylate proteins that play a key role in
the control of apoptosis. These proteins include Bad, a pro-apoptotic
bcl-2 family member, which when phosphorylated by Akt,
releases Bcl-x allowing it to perform its anti-apoptotic function (6,
7). Caspase 9, another effector protein of the intrinsic cell-death
machinery can also be inactivated following phosphorylation by Akt (8).
Finally, the transcription factor FKHRL1 that regulates the expression
of genes encoding pro-apoptotic proteins such as Fas-ligand
is located in the cytoplasm following its phosphorylation by Akt on
serine 253 and threonine 32 (9, 10).
Growth factors can also delay DNA damage-induced death leading in some
systems to an increased clonogenic survival (11, 12). It has previously
been shown that IL-3 protects Baf-3 cells from DNA damage-induced
apoptosis (13, 14). Indeed, in the absence of IL-3, the kinetics of
cell death is accelerated following DNA damage. In contrast, in the
presence of IL-3, cells are resistant to high doses of DNA
damage-inducing agents. The increased rate of death observed when cells
are irradiated in the absence of IL-3 is dependent on functional p53,
indicating that IL-3 acts by inhibiting a p53-dependent
apoptotic pathway (15). There are multiple pathways downstream of p53
that are potentially involved in the induction of apoptosis (16-18).
p53 regulates the transcription of pro-apoptotic genes such as
Bax, Fas, or PERP (19-21), which could play a role in this process, although some of them such as
Bax could have a redundant function because p53-induced
death is not affected by its absence (22). Recently it was shown that, in irradiation or myc-induced
p53-dependent death, APAF-1 and Caspase
9 were essential down-stream targets of p53 (23).
The signaling pathways involved in the inhibition of
p53-dependent apoptosis by growth factors have been studied
in a number of systems. In erythropoietin-dependent myeloid
cell lines, Jak2 kinase activation by erythropoietin receptor mutants
was shown to be necessary and sufficient to inhibit
p53-dependent apoptosis induced by We have performed a retroviral insertion mutagenesis to obtain mutants
that are resistant to apoptosis following growth factors starvation. We
describe the characterization of one mutant that proliferates in the
absence of growth factors and that, in contrast to previously described
mutants, does not overexpress bcl-x (27). This mutant (the
S4 mutant) is able to survive for prolonged periods of time in the
absence of IL-3 but shows no resistance to Cell Culture and Reagents--
The bone marrow-derived
IL-3-dependent Baf-3 cells were maintained in DMEM
containing 6% fetal calf serum (Roche Molecular Biochemicals), 2 mM L-glutamine (Life Technologies, Inc.), and 5% WEHI 3B cell-conditioned medium as a source of IL-3. Cells were
grown at a density of 5 × 104 to 8 × 105 per ml. To remove IL-3, cells were washed twice with
warm DMEM/fetal calf serum 6%/glutamine 2 mM and cultured
in this medium. Baf-3 cells overexpressing bcl-x (Baf-bcl-x)
and bcl-2 (Baf-bcl-2) have been described elsewhere (27,
29). Recombinant murine IL-3 was obtained from PeproTech. Ly294002 (20 mM, Calbiochem) was resuspended in Me2SO (Sigma
Chemical Co.). 2-Deoxy-glucose was obtained from Sigma. Retroviral Infection of Baf-3 Cells--
Baf-3 cells (1)
(6-7 × 107) were infected by co-culture with
M3Pneo-sup-producing cells (PAPM3 cells) (30). Baf-3 cells were
co-cultivated for 48 h with semi-confluent PAPM3 cells.
Polybrene at the concentration of 8 µg/ml (Sigma) and 5%
newborn calf serum were added to the Baf-3 medium. Cells were
washed and cultured for another 2 days to allow retrovirus-induced
expression of neighboring cellular genes. Thereafter, cells resistant
to IL-3 starvation-induced apoptosis were selected.
Individual clones were obtained by limiting dilutions. After a first
IL-3 starvation resistance screen, mutants were further characterized
for their survival capacity to other apoptotic pathways.
Measurement of Apoptosis and Proliferation--
Apoptotic and
dead cells were detected by propidium iodide staining. 5 × 103 to 104 cells were incubated a few minutes
with propidium iodide (Sigma) at a concentration of 5 µg/ml. For each
sample, 5000 cells were counted on a FACScan (Becton-Dickinson) and
analyzed on a FSC/FL2 dot plot using the CellQuest software. This
staining protocol allows the distinction between dead (FL-2 bright, FSC
low), apoptotic (FL-2 dull, FSC intermediate), and live cells
(FL-2-negative, FSC high). The percentage of viable cells corresponds
to the percentage of FL-2-negative cells.
Annexin V staining was realized according to the manufacturer's
instructions (PharMingen). For proliferation assay, life cells were
plated at 104 cells per well and pulsed for 16 h with
0.5 µCi of [3H]thymidine/well (2.0 Ci/mmol, Amersham
Pharmacia Biotech).
Glucose Metabolism Evaluation--
1.5 × 107
cells were incubated in 4 ml of Krebs-Henseleit buffer (31), pH 7.4, containing 0.1 unit/ml recombinant IL-3, 12.5 mM
[14C]glucose uniformly labeled (3.14 Bcq/ml) in 25-ml
stoppered conical flasks with an isolated central well and filled with
a 95%O2/5%CO2 mixture. Incubations were
performed for 2 h in a shaking water bath at 37 °C. Reactions
were stopped by adding perchloric acid (2% final concentration) in
each flask. 14CO2 collection was performed by
adding NaOH 5 N in the central well of the flask and
incubating for 2 h at room temperature. 14CO2 release was measured by liquid
scintillation counting as described previously (32). The Krebs medium
from each flask was centrifuged to remove denatured proteins, and the
supernatant was neutralized using a 20% KOH/1% phosphoric acid
solution before the measurement of metabolite concentration. Glucose
consumption and pyruvate and lactate accumulation were evaluated as
previously described (32, 33). Substrate utilization or metabolites
production were calculated as the difference between metabolites
content in the flask before incubation and after 2-h incubation.
Lactate Production Measurement--
Cells were washed twice in
prewarmed DMEM containing 1% fetal calf serum and 2 mM
glutamine and resuspended at 2 × 105 cells per ml in
the same medium with or without addition of IL-3. For lactate
measurement, a 1-ml cell suspension was centrifuged and the
concentration of lactate in the supernatant was measured using a
lactate measurement kit (Sigma Diagnostics) according to the
manufacturer's instructions.
2-Deoxy-D-[3H]Glucose Uptake
Measurement--
Cells were cultured in the presence or absence of
IL-3 during 8 h and then washed once in DMEM medium containing no
glucose (Life Technologies, Inc.). 8 × 105 cells were
maintained 30 min in the presence or absence of IL-3 in glucose-free
DMEM containing 160 mM
2-deoxy-D-[3H]glucose (0.5µCi for 8 × 105 cells) (Amersham Pharmacia Biotech). The reaction was
stopped by addition of one volume of an ice-cold phloretin solution
(0.3 µM ICN in PBS). Cells were then washed once with
ice-cold PBS and the 2-deoxy-D-[3H]glucose
incorporated by cells was measured using liquid scintillant (Wallac)
and a liquid scintillation counter (Wallac).
Western Blot--
Cells were washed in cold PBS and pellets were
lysed for 10 min at 4 °C in lysis buffer (20 mM Tris, pH
7.5, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40,
20 µg/ml aprotinin, 10 µg/ml pepstatine, 20 µg/ml leupeptine, 200 µg/ml PMSF, and 1 mM orthovanadate). Cells were then
centrifuged 15 min at 4 °C to eliminate cellular debris. Protein
concentration was determined by the Bradford procedure using the
Bio-Rad protein assay. Proteins were separated on 12.5%, 10%, or
7.5% polyacrylamide SDS-polyacrylamide gel electrophoresis gels. Gels
were electroblotted onto polyvinylidene difluoride membranes
(Millipore) using a liquid transfer apparatus (Bio-Rad).
Murine phosphospecific Akt and total Akt antibodies were purchased from
New England BioLabs and p53 antibody was purchased from Novo Castra
(CM5). Phosphospecific FKHRL1 antibodies were obtained from A. Brunet,
and Bcl-x antibody was obtained from J. Ham.
Bound antibodies were detected with horseradish peroxidase-coupled
anti-rabbit antibodies (DAKO) and the chemiluminescence detection
system from PerkinElmer Life Sciences.
RNase Protection Assay--
Total cellular RNA was isolated
using the RNA Now method (Ozyme) according to the
manufacturer's instructions. The bcl-x mRNA level was
measured by RNase protection assays using the Riboquant kit
(Becton-Dickinson) following the instructions of the supplier. Briefly,
5 µg of RNA was hybridized overnight to the 32P-labeled
RNA probe, which had previously been synthesized from the supplied
template (m-apo2). Single-stranded RNA and free probe were digested by
RNase A and T1. Subsequently, protected RNA was purified and analyzed
on a 6% denaturing polyacrylamide gel. The quantity of protected RNAs
was determined using a PhosphorImager and ImageQuaNT (both from
Molecular Dynamics, Sunnyvale, CA).
RNA Preparation, Northern Blot Analysis, and DNA
Probes--
Total cellular RNA was isolated by the RNA Now method
(Biogentex). 10 µg of total RNA were separated on 1% agarose
formaldehyde gel, transferred to Hybond N nylon membrane (Amersham
Pharmacia Biotech), hybridized overnight at 42 °C with
32P-labeled probes and washed using a standard protocol.
The murine bcl-x probe is a 0.8-kbp
SacI/XhoI fragment and the murine
GAPDH is a 1-kpb PstI fragment.
The S4 Mutant Survives and Proliferates in the Absence of
IL-3--
The IL-3 starvation-resistant Baf-3 mutant S4 was obtained
by retroviral insertion mutagenesis as described under "Experimental Procedures." The survival capacity of this mutant was compared with
Baf-3 cells overexpressing bcl-2 or bcl-x.
Results in Fig. 1A show that
the S4 mutant survives to IL-3 starvation for prolonged periods of
time, more than 70% of the cells being viable after 5 days in culture
in the absence of IL-3. In contrast, nearly all parental Baf-3 control
cells were dead after 1-day culture in the absence of IL-3.
Overexpression of bcl-2 or bcl-x leads to an
increased survival in the absence of growth factor but was unable to
confer long term survival, because most cells were dead by 4 days of
culture. The proliferative capacity of this mutant in the absence of
IL-3 was also assessed. As previously described, the Baf-3 cell lines
overexpressing bcl-2 or bcl-x did not proliferate in the absence of IL-3 (Fig. 1B) (27, 29). In contrast,
48 h following IL-3 removal, the S4 mutant did incorporate
thymidine, indicating that it is able to cycle independently of IL-3
(add Fig. 1). The phenotype of this mutant is different from
bcl-2- or bcl-x-overexpressing cells, suggesting
that the gene that has been activated by the retroviral insertion is
not a bcl-2 family member. This was confirmed by Northern
analysis, which shows that the S4 mutant and parental Baf-3 cells
express similar levels of RNA coding for bcl-x in the
presence of IL-3 and a similar down-regulation of this gene in the
absence of growth factor (Fig. 1C). Finally, we have
excluded the possibility that this mutant is able to survive through
the production of its own growth factor. Indeed we have been unable to
induce the survival of parental Baf-3 cells with the supernatant of the
S4 mutant or by co-culture experiments (data not shown).
The S4 Mutant Is Not Resistant to DNA Damage-induced Cell
Death--
To further characterize the survival capacity of the S4
mutant, we have measured its resistance to DNA damage-induced
apoptosis. Cells overexpressing bcl-2 or bcl-x
and the S4 mutant were irradiated with The Glycolytic Activity Is IL-3-independent in the S4
Mutant--
To investigate the signaling pathways that leads only to
an increased survival of the mutant in the absence of IL-3, we decided to identify genes that are overexpressed in the S4 mutant compared with
parental Baf-3 cells using a cDNA differential display approach (CLONTECH PCR-select cDNA subtraction kit).
Among a number of differentially expressed genes, the cDNA coding
for the phosphoglycerate kinase and the lactate dehydrogenase (LDH)
enzymes were found. Both enzymes are involved in glycolysis, LDH being
more specifically involved in anaerobic glycolysis. The glycolytic
pathway is regulated by IL-3 and is the main ATP-generating source in
IL-3-dependent cell lines (35, 36). To characterize the
glucose metabolism in Baf-3 cells and in the S4 mutant, we have
measured the glucose metabolites produced by glycolysis (lactate and
pyruvate) and citric acid cycle (CO2). For this purpose,
Baf-3 and S4 cells were incubated in Krebs medium containing
[14C]glucose uniformly labeled in the presence of IL-3.
The consumption of glucose as well as the quantity of lactate,
pyruvate, and 14CO2 produced were measured
after 2-h incubation as described under "Experimental Procedures."
Almost no 14CO2 (less than 5% equivalent
glucose) was produced by either cell line indicating that
[14C]glucose-derived pyruvate was not metabolized in the
citric acid cycle or the pentose pathway. Indeed, the glucose taken up
by cells was mainly transformed into lactate as described in Table I. As a whole, these results
indicate that Baf-3 and S4 cells mainly perform anaerobic glycolysis,
confirming results obtained with other IL-3-dependent bone
marrow-derived cell lines (35, 36).
It has been previously shown that IL-3 deprivation leads to a decline
in glycolysis, which can be monitored by a drop in lactate production.
This occurs before apoptotic cells can be detected in the culture (36).
To determine whether the overexpression of phosphoglycerate kinase and
LDH correlates with a deregulation of the glycolytic pathway in the S4
mutant in the absence of IL-3, we have measured the lactate produced by
these cells in the presence or absence of IL-3, because we showed that
most of the glucose is degraded into lactate in these cells. As shown
in Fig. 3A, in the presence of
IL-3, Baf-3, Baf-bcl-x, and S4 cells produced lactate, confirming that
under these culture conditions they are undergoing anaerobic
glycolysis. In the absence of IL-3, Baf-3 and Baf-bcl-x cells showed a
strongly reduced lactate production that was not due to cell death
because more than 85% of the cells are alive after 9 h of culture
in these conditions. This confirms that, in the absence of IL-3, there
is a decline in glycolysis. In contrast, in the S4 mutant a significant
lactate production was found after 9 h in the absence of IL-3. The
production of lactate was not transient, because the lactate
concentration produced by 105 S4 cells over more than 3 days was similar in the presence or absence of IL-3 (Fig.
3B). These results indicate that glycolysis is activated
independently of IL-3 in the S4 mutant.
We next wanted to determine whether constitutive glycolysis correlated
with a constitutive glucose uptake in S4 cells in the absence of IL-3.
For this purpose, we measured 2-deoxy-[3H]glucose
uptake in Baf-3, Baf-bcl-x, and S4 cell lines in the presence or
absence of IL-3. In the presence of IL-3, Baf-3, Baf-bcl-x, and S4
cells incorporate glucose, reflecting the fact that glucose transport
is active under these conditions (Fig. 3C). Following IL-3
withdrawal, glucose uptake is reduced by 50% in Baf-3 and Baf-bcl-x
cells, confirming that glucose transport is regulated by IL-3. In
contrast, no decline in glucose uptake is observed in S4 cells
following growth factor withdrawal. These results indicate that glucose
transport and glycolysis, as measured by glucose uptake and lactate
production, have become independent of the presence of IL-3 in the S4 mutant.
Because IL-3 regulates the level of glycolytic activity in Baf-3 cells,
we evaluate the importance of the glycolytic pathway for Baf-3 cell
survival in the presence of IL-3. For this purpose, Baf-3 cells were
cultured in the presence of 2-deoxy-glucose, a glucose analogue, which
is a potent inhibitor of glycolysis acting as a competitive inhibitor
of glucose transport. Cell viability was then measured at different
time points. After 16 h in these conditions, more than 30% of
Baf-3 cells are apoptotic as measured by propidium iodide and Annexin V
staining (Fig. 3D). Similarly, 48% of control Baf-3 cells
are apoptotic 16 h following IL-3 starvation. These results show
that regulation of glucose metabolism by IL-3 in Baf-3 cells is
essential for their survival.
Akt and FKHRL1 Are Constitutively Phosphorylated in the Absence of
IL-3 in the S4 Mutant--
Insulin regulates glycolysis through
activation of the Phosphatidylinositol 3-kinase/PKB pathway.
Phosphorylation by Akt/PKB leads to the activation of
phosphofructo-2-kinase and the inhibition of glycogen synthase
kinase-3, which are key regulators of the glycolytic pathway (37, 38).
Akt has also been involved in glucose transporters translocation,
mainly GLUT4 and GLUT1, leading to an increase in glucose uptake
(39-41). To test if glycolysis, as mirrored by lactate production, is
regulated by IL-3 through a PI3K-dependent pathway, we have
measured the lactate produced by Baf-3 and S4 cells in the presence or
absence of the PI3K inhibitor Ly294002. As shown in Fig.
4A, the lactate concentration
produced by Baf-3 and S4 cells in the presence of IL-3 was reduced by
more than 70% in the presence of the PI3K inhibitor Ly294002. This inhibition was independent of cell death, because cells were more than
80% viable at that time point. These results indicate that PI3K
activation by IL-3 regulates anaerobic glycolysis in Baf-3 cells and in
the S4 mutant. Similarly, the lactate production, which is observed in
S4 cells in the absence of IL-3, was inhibited by the addition of the
PI3K inhibitor Ly294002 (Fig. 4B). This indicates that a
PI3K-dependent pathway is activated in the absence of IL-3
in the S4 mutant.
To confirm these results, the activation status of the serine/threonine
kinase Akt/PKB was measured. PI3K activation following growth factor
stimulation leads to the activation and phosphorylation of Akt on
serine 473 and threonine 308 (42). The phosphorylation status of Akt in
the S4 mutant was thus monitored using a phospho-Akt (Ser-473)-specific
antibody. In IL-3-starved Baf-3 and S4 cells, Akt phosphorylation was
strongly induced by addition of IL-3 (Fig. 5A), confirming Akt regulation
by this growth factor. In Baf-3 cells, no phosphorylation of Akt could
be detected following 8 h culture in the absence of IL-3. In
contrast, in S4 cells deprived of IL-3 for 16 h we detected a
level of Akt phosphorylation similar to the physiological level, which
is observed in Baf-3 or S4 cells continuously maintained in the
presence of IL-3. This phosphorylation could be inhibited by the
addition of the PI3K inhibitor Ly294002 (Fig. 5B),
indicating that in the S4 mutant constitutive Akt activation was
dependent on the generation of 3'-phosphorylated
phosphatidylinositides.
To test if the constitutive phosphorylation of Akt on Ser-473 observed
in the S4 mutant in the absence of IL-3 leads to an in vivo
Akt activation, we have monitored the level of phosphorylation of
different known Akt substrates. In Baf-3 cells we have been unable to
detect the phosphorylation of the Bad protein in response to IL-3. In
contrast, an IL-3-dependent phosphorylation of FKHRL1 on
threonine 32 was found in Baf-3 and S4 cells (Fig. 5C).
FKHRL1 phosphorylation on serine 253 was also found in both cell lines but was independent of IL-3. In the S4 mutant an IL-3-independent, 3'-phosphorylated phosphatidylinositides-dependent FKHRL1
phosphorylation was found (Fig. 5D), confirming that the
constitutive Akt phosphorylation, which is observed in the S4 mutant in
absence of IL-3, leads to an in vivo Akt activation.
Akt Activation Is Required for the Survival of the S4 Mutant in the
Absence of IL-3--
To determine whether activation of Akt was
necessary for the survival of the S4 mutant in the absence of IL-3, we
have measured the survival of these cells when grown in the presence of
the PI3K inhibitor Ly294002. The survival of the S4 mutant is strongly decreased by using 10 µM Ly294002 (Fig.
6A). This concentration inhibits the constitutive Akt activation (Fig. 5B) and the
glycolytic activity (Fig. 4B) observed in the S4 mutant in
the absence of IL-3. In contrast, using similar doses of the PI3K
inhibitor Ly294002, IL-3-induced survival was maintained confirming
that a PI3K-independent survival pathway is activated by IL-3 in
Baf-3-derived cells (43). These results confirm that the constitutive
Akt activation observed in the S4 mutant is necessary for the survival
of these cells in the absence of IL-3. The constitutive Akt activation
did not allow the maintenance of bcl-x mRNA or protein
levels (Fig. 6, B and C) normally found in the
presence of IL-3, indicating that inhibition of the intrinsic death
pathway can take place in the absence of high Bcl-x levels.
Inhibition of DNA Damage-induced Apoptosis by IL-3 Is Independent
of Akt--
Although Akt activation in S4 cells is necessary and
sufficient for survival in the absence of growth factor, it is not able to delay DNA damage-induced apoptosis. The acceleration of death that
is observed in the absence of growth factor following DNA damage is
dependent on functional p53 (15). Hence our results would indicate that
the Akt pathway is not involved in the inhibition of
p53-dependent apoptosis by growth factors. To confirm this hypothesis we tested the ability of IL-3 to inhibit DNA damage-induced death following PI3K inhibition by Ly294002 in Baf-3 cells. Results in
Fig. 7A show that following
irradiation the death rate of Baf-3 cells was increased in the absence
of IL-3 but not in the presence of growth factors. In both conditions
1.5-Gy irradiation was associated with a p53 up-regulation within
1 h (Fig. 7B), which was maintained up to 10 h
(Fig. 7C), at a time where, in the absence of IL-3, more
than 50% of irradiated Baf-3 cells had already undergone apoptosis.
The relative increase in p53 protein level induced by irradiation was
not affected by the concentration of Ly294002 used, indicating that the
p53-dependent apoptotic pathway was also activated in these
conditions. Inhibition of PI3K by Ly294002 did inhibit the Akt
phosphorylation observed in response to IL-3 (Fig. 7C) but
did not abrogate the ability of IL-3 to protect against DNA
damage-induced death (Fig. 7A). We reproducibly found that
the level of total Akt protein was reduced in Baf-3 cells irradiated
and cultured in the absence of IL-3. This could reflect a selective
degradation of that protein in apoptotic cells that represents more
than 50% of the pellet. These results confirm that Akt activation by
IL-3 is not necessary for the protection against DNA damage-induced
apoptosis. In contrast, there was a strong correlation between
expression of high levels of Bcl-x protein and resistance to DNA
damage-induced cell death (Fig. 7, A and C).
In this study we report that, in a Baf-3 mutant cell line,
the S4 mutant, which was obtained after retroviral-insertion
mutagenesis, shows an IL-3 independent Akt phosphorylation on serine
473, which leads to FKHRL1 phosphorylation on threonine 32 and
constitutive glycolysis. Akt activation was necessary for the
inhibition of the intrinsic death pathway and long term survival of
these cells in the absence of growth factor. The gene modified upstream
of Akt responsible for the constitutive activation of Akt has not yet
been identified. Several pathways downstream of Akt could be
responsible for the survival of the S4 mutant in the absence of growth
factor. In neurones, inactivation of the FKHRL1 transcription factor
following phosphorylation by Akt is potentially involved in the
inhibition of apoptosis by growth factors (5, 9). Indeed, FKHRL1
mutants where the three residues phosphorylated by Akt have been
converted to alanine are strong transactivators and trigger apoptosis
when overexpressed in a number of cell types. Conversely, replacement
of these residues by aspartic acid, which mimics the presence of a
phosphate group, disrupts the transactivation function of FKHRL1 (44).
FKHRL1-mediated death could result from the expression of death genes,
such as Fas-ligand, which expression is regulated by FKHRL1
and which are strongly up-regulated following growth factor withdrawal
in neurones (9). In Baf-3 cells we have found that FKHRL1 threonine 32 phosphorylation is controlled by IL-3. In contrast, FKHRL1 serine 253 remains phosphorylated in Baf-3 cells grown in the absence of IL-3 up
to a stage when cells are starting to enter apoptosis. Because FKHRL1
phosphorylation on serine 253 should maintain FKHRL1 in the cytoplasm
(9), these results suggest that IL-3 starvation-induced apoptosis is not mediated by FKHRL1-dependent gene transactivation. This
is in agreement with data showing that IL-3 starvation-induced
apoptosis proceeds with similar kinetics in the presence of protein or
RNA synthesis inhibitors (45). In the Baf-3 cells or the S4 mutant we
have been unable to detect a phosphorylation of the Bad protein. Even
if we cannot completely exclude a role for Bad in the inhibition of
death, we have shown that in the S4 mutant the Bcl-x mRNA and protein are down-regulated following IL-3 starvation, suggesting that
the Bad-Bcl-x pathway is not involved in the survival of these cells in
the absence of IL-3. In contrast, there is a strong correlation between
expression of Bcl-x and protection against DNA damage-induced death.
Hence, pathways such as Jak/Stat5 or MAPK, which are involved in the
up-regulation of Bcl-x by IL-3 could play a role in the inhibition of
DNA damage-induced death (45-47). The PI3K/Akt pathway has also been
involved in the regulation of bcl-x expression (43, 48).
Indeed, PI3K inhibition delays bcl-x mRNA up-regulation
induced by IL-3 restimulation of growth factor-starved cells. However,
the addition of the PI3K inhibitor does not affect the steady-state
level of Bcl-x protein in the presence of IL-3 (Fig. 7C),
and constitutive activation of the PI3K/Akt pathway does not induce
bcl-x overexpression (Fig. 6, B and
C), suggesting that the activation of the PI3K/Akt pathway is not sufficient for bcl-x expression.
The maintenance of a glycolytic activity in the absence of IL-3 could
contribute to the survival of the S4 mutant in these conditions.
Indeed, glucose deprivation or inhibition of glycolysis by
2-deoxyglucose can lead to growth arrest or to apoptosis, suggesting that some as yet undefined checkpoint able to induce apoptosis in these
conditions exists (49). In IC.DP IL-3-dependent cells, survival in the absence of IL-3 following v-abl transfection
has been shown to be dependent on glucose transport activation. Indeed v-ABL activation in these cells induced an increased survival in the
absence of growth factor which correlated with a stimulation of glucose
uptake. Hence the control of glycolytic activity by growth factors
could be an essential step in the inhibition of the intrinsic apoptotic
pathway (50). Acquisition of a growth factor-independent glycolytic
activity could delay the onset of apoptosis and contribute to the
process of transformation. Indeed, tumor cells frequently exhibit a
high rate of anaerobic glycolysis even under aerobic conditions (51).
In Baf-3 and S4 cells, we confirmed that anaerobic glycolysis is the
main glucose-derived ATP-generating source, because these cells do not
undergo aerobic glycolysis. The S4 mutant shows constitutive glucose
transport and glycolytic metabolism in the absence of IL-3 as measured
by glucose uptake and lactate production. In the presence or absence of
IL-3, glycolytic activity was dependent on the PI3K/Akt pathway. In the
absence of IL-3, inhibition of the glycolytic pathway using PI3K
inhibitors, glucose deprivation, or 2-deoxy-glucose all resulted in
rapid apoptotic death of the S4 mutant but not of Bcl-x-overexpressing cells (data not shown). These results indicate that the maintenance of
the glycolytic activity of these cells was essential for their survival
in the absence of growth factor.
We also show that the inhibition of p53-dependent DNA
damage-induced death is not inhibited by Akt activation. Indeed, the activation of Akt in the S4 mutant in the absence of growth factor is
unable to protect these cells against DNA damage-induced death. Similarly, in parental Baf-3 cells inhibition of the PI3K/Akt pathway
does not abrogate the protection conferred by IL-3. These results are
in contradiction with data showing that overexpression of PI3K or
activated Akt can protect against apoptosis following p53 transfection
(25). This discrepancy could be due to difference in the level of Akt
activation obtained by overexpressing an activated form of Akt compared
with the level of Akt activation obtained in the S4 mutant. Indeed, the
level of Akt phosphorylation in these cell in the absence of growth
factors is lower than the level obtained when growth factor-starved
cells are synchronously restimulated with IL-3. However, it is similar
to the physiological level observed in Baf-3 or S4 cells continuously
maintained in the presence of IL-3 and might mimic more accurately the
functions fulfilled by Akt in IL-3-dependent cell lines.
Alternatively, one could imagine that the apoptotic pathway induced by
p53 following DNA damage-induced death is different from the pathway
induced by p53 overexpression. p53 has multiple functions that are
regulated by post-translational modifications such as protein
phosphorylation or protein/protein interaction. A number of these
functions are involved in the induction of apoptosis by p53 (16, 18).
Therefore increasing the level of p53 protein by overexpression or by
DNA damage-induced phosphorylation and stabilization might not result in the activation of the same function and apoptotic pathway. A better
understanding of the apoptotic pathways activated by p53 in these
different experimental conditions might help to resolve this issue.
-irradiation-induced DNA damage. This protective effect of IL-3 rather correlates with the expression of the anti-apoptotic Bcl-x protein. Taken together, these data demonstrate that the PI3K/Akt pathway is sufficient to protect cells from growth factor
starvation-induced apoptosis but is not required for IL-3 inhibition of
DNA damage-induced cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation (24). In
the same system, activation of other signaling pathways, including
PI3K, STATs, and Ras was not required to inhibit death. In contrast, it
has recently been shown that death induced by p53 expression could be
inhibited by activating the PI3K/Akt pathway (25). Finally, it has been suggested that IL-3 could delay p53-dependent apoptosis
induced by
-irradiation by regulating the levels of p21 and Rb, two
proteins involved in the regulation of the G1/S
transition (14, 26). Although in one report, the expression of v-Src or
activated c-Raf could mimic the effect of IL-3 on p21 levels, the
signaling pathway activated by IL-3, involved in the control of
G1 arrest or apoptosis following DNA damage, has not been identified.
-irradiation-induced cell
death. This mutant shows a constitutive IL-3-independent, 3'-phosphorylated phosphatidylinositides-dependent, Akt kinase activation. In this mutant, we could demonstrate that Akt activation leads to FKHRL1 phosphorylation and constitutive glycolytic activity. In Baf-3 cells, glycolysis is regulated by IL-3 and is the main ATP-generating source (28). The activation of Akt was necessary for the
survival observed in the absence of IL-3. In contrast we show in the S4
mutant and in Baf-3 cells that Akt activation is neither sufficient nor
necessary to inhibit p53-dependent DNA damage-induced cell
death. These results indicate that IL-3 activates multiple signaling
pathways, which can inhibit growth factor starvation-induced apoptosis
(called intrinsic apoptosis here after); however, only some of these
can delay DNA damage-induced p53-dependent apoptosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Irradiation
was performed using an IBL 637 machine (CIS Bio International)
at a dose rate of 1.58 Gy/min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
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Fig. 1.
S4 mutant survival and proliferation in the
absence of IL-3. A, cells were grown in the absence of
IL-3 and the percentage of viable cells was measured by propidium
iodide exclusion at indicated time. One representative experiment out
of three is presented. Full circle, S4 cells; open
square, Baf-bcl-x cells; open circle, Baf-bcl-2 cells;
and full diamond, Baf-3 cells. B, cells were
deprived of IL-3 for 48 h. Proliferation in the absence of IL-3
was assessed on 1 × 104 viable cells by a 16-h
[3H]thymidine incorporation pulse. Data are presented as
mean values ± S.D. of three independent experiments.
C, Baf-3 and S4, Baf-bcl-x cells were cultured in the
presence or in the absence of IL-3 during 8 and 24 h,
respectively. Total RNA was purified as described under "Experimental
Procedures," and 10 µg was used to perform a Northern blot
analysis. Expression of bcl-x and GAPDH was
detected using the probes described under "Experimental
Procedures."
-rays. Their survival was
monitored after 24-h culture in the absence of IL-3 (Fig.
2A). As previously described,
bcl-2- or bcl-x-overexpressing cells survived to
irradiation doses up to 12.5 Gy, confirming that overexpression of
these proteins confers a resistance to growth factor starvation and DNA
damage-induced cell death (13, 34). In contrast, the S4 mutant, which
showed more than 70% viable cells after 24 h culture in the
absence of IL-3, was unable to survive irradiation doses as low as 1.5 Gy. Indeed, following irradiation with 1.5 Gy and culture in the
absence of IL-3, this mutant showed a death kinetic similar to parental Baf-3 cells (Fig. 2B). Results obtained with the S4 mutant
suggest that the signaling pathway activated by IL-3, which protects
against DNA damage-induced death, differs from the signaling pathway
leading to the inhibition of the intrinsic death program triggered in the absence of growth factors. Indeed the S4 mutant can be maintained in culture in the absence of IL-3 for more than 5 days, but shows the
same susceptibility as Baf-3 cells to apoptosis induced by irradiation
in the absence of growth factors. The absence of protection was not due
to an increased sensitivity of S4 mutant to DNA damage that could not
be inhibited by IL-3, because this mutant showed the same death kinetic
as parental Baf-3 cells when irradiated in the presence of IL-3 (data
not shown). This indicates that, in this mutant, the survival pathway
that protects cells from DNA damages has not been activated, whereas
one survival pathway leading to the inhibition of the intrinsic death
program is preserved.
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Fig. 2.
S4 mutant survival following
irradiation. A, cells were irradiated on ice at
indicated doses following IL-3 removal and maintained in culture in the
absence of IL-3 for a further 24 h. The percentage of viable cells
was then measured by propidium iodide exclusion. One representative
experiment out of three is presented. Full square, Baf-bcl-2
cells; open circle, Baf-bcl-x cells; and full
diamond, S4 cells. B, cells were irradiated on ice at
1.5 Gray following IL-3 removal and maintained in culture in the
absence of IL-3. Percentage of viable cells was determined at indicated
times by propidium iodide exclusion. One representative experiment out
of three is presented. Full square, Baf-bcl-2 cells;
open circle, Baf-bcl-x; open square, Baf-3 cells;
and full diamond, S4 cells.
Glycolytic metabolism
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Fig. 3.
Glucose transport and glycolytic activity are
independent of IL-3 in the S4 mutant. A, cells were
maintained in the absence or in the presence of IL-3 as indicated, at a
concentration of 1 × 105 cells per ml. Viability
(open bars) and lactate concentration (full bars)
for 1 × 105 viable cells were measured after 9 h. Data are presented as mean values ± S.D. of three independent
experiments. B, the S4 mutant was cultured in the presence
(full circle) or in the absence (full square) of
IL-3 at a concentration of 1 × 105 cells per ml, and
the lactate concentration in the supernatant was measured. Values
correspond to the lactate concentration per 1 × 105
viable cells at the indicated times. One representative experiment out
of three is presented. C, Baf-3, Baf-bcl-x, and S4 cells
were cultured in the presence or absence of IL-3 during 8 h.
2-Deoxy[3H]glucose uptake was measured as
indicated under "Experimental Procedures." Data are presented as
mean values ± S.D. of four experiments. D, Baf-3 cells
were cultured in the presence of IL-3 (+IL-3), in the
absence of IL-3 ( IL-3), or in the presence of IL-3 in a
low glucose Dulbecco's modified Eagle's medium containing 6 mM 2-deoxyglucose (+IL-3+2DG). Viability was
measured after 16 h by staining cells with Annexin V
(FL1) and propidium iodide (FL2) and analysis by
flow cytometry. Percentages indicated correspond to percent apoptotic
cells (propidium iodide-positive and Annexin V-positive) contained in
the R1 gate.
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Fig. 4.
Lactate production is dependent on PI3K
activity. A, 1 × 105 cells per ml
were cultured in the presence of IL-3 with 50 µM Ly294002
where indicated, and the lactate production was measured 8 h
latter (full bars). Viability was also measured at the same
time point by propidium iodide exclusion (open bars).
Results are presented as mean values ± S.D. of three independent
experiments. B, S4 cells were cultured at a concentration of
1 × 105 cells per ml in the absence of IL-3 with 10 µM Ly294002 where indicated. Survival (open
bars) and lactate production (full bars) was measured
12 h latter. Results are presented as mean values ± S.D. of
three independent experiments.
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Fig. 5.
Akt and FKHRL1 are constitutively
phosphorylated in the mutant S4 in the absence of IL-3.
A, Baf-3 and S4 cells were maintained 8 and 16 h,
respectively, in the presence (+) or absence of IL-3 ( ). Half of the
IL-3-starved cells were restimulated with recombinant IL-3 for 10 min
(Re 10 min). Cells were then lysed, and 100 µg of total
protein from each sample was loaded onto a 10% acrylamide gel.
Detection of phosphospecific Akt or total Akt was done as described
under "Experimental Procedures." One representative experiment out
of three is presented. B, S4 cells were maintained 16 h
in the absence of IL-3 (
), with (+) or without (
) 10 µM Ly294002. As control for steady-state Akt
phosphorylation levels, S4 cells cultured in the presence of IL-3 were
also added in the test (+). Cells were lysed, and 100 µg of total
protein from each sample was loaded onto a 10% acrylamide gel.
C, cells were prepared as in A, and 100 µg of
total protein was loaded onto a 7.5% acrylamide gel. Detection of
phosphospecific FKHRL1 was done as described under "Experimental
Procedures." One representative experiment out of three is presented.
D, cells were prepared as in B, and 100 µg of
total protein was loaded on a 7.5% acrylamide gel.
View larger version (20K):
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Fig. 6.
S4 survival in the absence of IL-3 is
dependent on the PI3K pathway. A, cells were cultured
in the absence or in the presence of IL-3 as indicated by (+) or ( ).
10 µM Ly294002 was added at the beginning of the culture
as indicated by (+) or (
). Cell viability was determined 48 h
later by propidium iodide exclusion. Data are presented as mean
values ± S.D. of three independent experiments. B,
Baf-3 cells and S4 cells were cultured in the absence of IL-3 for 8 or
24 h, respectively. Half of the cells were then stimulated for
2 h with IL-3. Total RNA was purified as described under
"Experimental Procedures." bcl-x mRNA content in 5 µg of total RNA was measured by a RNase protection assay as described
under "Experimental Procedures." C, Baf-3 and S4 cells
were cultured in the presence (+) of IL-3 or in the absence (
) of
IL-3 during 8 and 24 h, respectively. Cells were then lysed, and
80 µg of total proteins from each sample was loaded on a 12.5%
acrylamide gel. Detection of Bcl-x protein was done as described under
"Experimental Procedures."
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Fig. 7.
Akt activation is not required for
IL-3-mediated inhibition of DNA damage-induced death.
A, Baf-3 cells were cultured with or without IL-3 and
irradiated on ice at 1.5 Gy in the presence or absence of 10 µM Ly294002 as indicated by (+) or ( ). Viability was
assessed 10 h later. B, cells were cultured in the same
conditions as in A, and lysates were prepared as described
under "Experimental Procedures" 1 h after irradiation. 100 µg of total protein from each sample was loaded onto a 10%
acrylamide gel and analyzed for its p53 content. C, cells
were cultured in the same conditions as in A, and lysates
were prepared as described under "Experimental Procedures" 10 h after irradiation. 100 µg of total protein from each sample was
loaded onto a 10% acrylamide gel and analyzed for its Bcl-x, p53,
phosphospecific Akt, and total Akt protein content.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank A. Brunet and J. Ham for kindly providing us with the phospho-specific FKHRL1 and the Bcl-x antibodies, respectively. We also thank E. Goillot for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by institutional grants from CNRS and the Ministère de l'Enseignement Supérieur et de la Recherche and by additional support from the Association pour la Recherche sur le Cancer, the Région Rhône-Alpes and the Comité Départemental (Rhône and Saône et Loire) de la Ligue Nationale Française contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a fellowship from CNRS.
To whom correspondence should be addressed: Tel.:
334-37-28-23-50; Fax: 334-37-28-23-41; E-mail:
marvel@cervi-lyon.inserm.fr.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M007147200
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ABBREVIATIONS |
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The abbreviations used are: IL-3, interleukin-3; PI3K, phosphatidylinositol 3-kinase; STAT, signal transducers and activators of transcription; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffed saline; kbp, kilobase pair(s); LDH, lactate dehydrogenase; PKB, protein kinase B; MAPK, mitogen-activated protein kinase.
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