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INTRODUCTION |
Suppression of apoptosis by an activated
IGF-IR1 plays an important
role in the survival of many cell types and in response to diverse
death stimuli. Epithelial cells and fibroblasts are protected from
apoptosis induced by oncogene activation or growth factor withdrawal
(1), toxic stimuli such as chemotherapeutic drugs, and irradiation (2).
Cells of the nervous system can be protected by IGF-I from osmotic
stress or growth factor withdrawal (3). Hematopoietic cells are
protected by IGF-I from IL-3 withdrawal (4), and in T lymphocytes
stimulation through the CD28 receptor leads to increased IGF-IR
expression levels and enhanced IGF-I-mediated protection from
Fas-induced apoptosis (5).
In addition to anti-apoptotic activity the IGF-IR also has strong
mitogenic and transforming activity, and there is considerable evidence
to indicate that the presence of the IGF-IR is necessary for
tumorigenesis (reviewed in Refs. 6-9). Fibroblasts derived from IGF-IR
null mice cannot be transformed by a series of oncogenes (6).
Inhibition of IGF-IR expression or signaling capacity by antibodies
(7), triple helix formation (8), or antisense strategies (9) results in
induction of apoptosis, failure to grow in anchorage-independent
conditions, as well as failure to form tumors in nude mice.
The signaling pathways activated by the IGF-IR have been extensively
studied, and it appears that there is considerable overlap in the
pathways used for all of the receptor functions. One major signaling
pathway activated by the IGF-IR is through its interaction with IRS-1
or IRS-2 leading to activation of PI 3-kinase and AKT (3, 10), which
promotes suppression of apoptosis via phosphorylation of several
proteins. These include the forkhead transcription factor FKHRL1 (11),
cAMP-responsive element-binding protein (12), caspase 9 (13), GSK3-
(14), and the Bcl-2 regulatory family member Bad (15). Other signaling
pathways leading to Bad phosphorylation can also be activated by the
IGF-IR including the Ras MAP kinase pathway via Rsk-1 in neuronal cells
(16) and via Shc activation or via translocation of RAF to the
mitochondria in 32D myeloid cells (17).
Interestingly, despite the prevalence of the AKT pathway in
anti-apoptotic signaling from the IGF-IR and the role of the AKT regulatory phosphatase PTEN in tumorigenesis (18, 19), there is
also compelling evidence for anti-apoptotic signaling from the IGF-IR
that is independent of AKT activation or Bad phosphorylation. Rat-1
fibroblasts exhibited IGF-IR-induced PI 3-kinase-dependent anti-apoptotic activity but also exhibited anti-apoptotic activity that
could not be blocked by PI 3-kinase inhibitors or by dominant-negative AKT constructs (20). In the IL-3-dependent cell line
FL5.12, which we have previously used as a model to study
IGF-IR-mediated suppression of apoptosis in response to IL-3
withdrawal, two mutants of the IGF-IR that could not suppress apoptosis
could still activate PI 3-kinase and AKT (21). These studies suggest
that the AKT pathway is not sufficient for suppression of apoptosis by
the IGF-IR in these cells.
The mitogen-activated protein (MAP) kinase family has been investigated
for its contribution to anti-apoptotic signaling. However, although
IGF-I induces ERK phosphorylation, this is not sufficient to mediate
the PI 3-kinase-independent anti-apoptotic activity of the IGF-IR (20,
22). Another candidate pathway that has not been investigated much in
IGF-IR anti-apoptotic signaling is that controlled by the MAP
kinase-related stress-activated kinases (SAPKs), consisting of c-Jun
N-terminal kinases (JNKs) and p38 kinase. Although the role of SAPKs in
either promoting or suppressing apoptosis appears to be quite complex
(reviewed in Ref. 14), there is considerable evidence to support a role for these proteins in transformation (23, 24), a function that overlaps
with the anti-apoptotic function of the IGF-IR. JNKs have also recently
been strongly associated with the differentiation of T
lymphocyte subsets (25).
The role of SAPKs in promoting apoptosis or cell survival appears to
depend on the stimulus a cell receives. Neurons activate JNKs in
response to growth factor withdrawal (26), and fibroblasts derived from
mice with two isoforms of JNK knocked out (JNK1 and JNK2) are more
resistant to UV-induced apoptosis than their wild type littermates
(27). However, the JNK kinase SEK can protect thymocytes from apoptosis
(28), and in a biphasic response to TNF-
there is a correlation with
prolonged activation of JNK and induction of apoptosis, whereas there
is a correlation with transient activation of JNK and suppression of
apoptosis (29). Transient activation of JNK by TNF-
in HeLa cells
could be inhibited by the quinone reductase inhibitor dicumarol, and
this lead to potentiation of apoptosis (30), suggesting that transient
JNK activation is associated with the survival of tumor cells.
Activation of JNK and p38 by cytokines such as erythropoietin has also
been associated more tightly with cell survival than with apoptosis (31). IGF-I has been shown to suppress JNK activity and activate p38 in
response to high glucose-induced stress in neuroblastoma cells (32) and
to suppress prolonged JNK activation by anisomycin in fibroblasts (33).
In contrast transient JNK activation has recently been shown to occur
in response to IGF-I stimulation of the breast carcinoma cell line
MCF-7 (34). However, the consequences of JNK activation by IGF-I for
regulation of apoptosis, cell growth, or transformation by the IGF-IR
are not known.
In this work we investigated SAPK activation by the IGF-IR and further
investigated the effects of dicumarol on the anti-apoptotic activity of
the IGF-IR using FL5.12 cells that overexpress the wild type (WT)
IGF-IR and that are protected by IGF-I from IL-3 withdrawal. We show
that JNK is transiently activated by IGF-I stimulation leading to
phosphorylation of c-Jun, whereas p38 is constitutively phosphorylated
in these cells. JNK activation by IGF-I does not require PI 3-kinase or
AKT activation. Dicumarol could block JNK activation and also abrogated
suppression of apoptosis by IGF-I but did not affect p38 activity or
AKT activation. Similar concentrations of dicumarol induced apoptosis
in the breast carcinoma cell line MCF-7. These data suggest that
IGF-I-mediated activation of JNK plays a role in suppression of
apoptosis by the IGF-IR and that the JNK inhibitor dicumarol can induce
apoptosis in tumor cells that are dependent on IGF-IR-mediated survival signals.
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EXPERIMENTAL PROCEDURES |
Materials--
Recombinant IGF-I was purchased from PeproTech
(Rocky Hill, NJ). The anti-ERK2, anti-phospho-ERK, anti-phospho-SAPK,
anti-SAPK, anti-phospho-p38, anti-p38, anti-AKT, anti-phospho-AKT
antibodies, and the non-radioactive SAPK assay kit were obtained from
New England Biolabs (Beverly, MA). Anti-phospho-Jun, anti-Shc
polyclonal, anti-Grb-2, anti-Gab-2 and anti-PI 3-kinase p85 subunit
antibodies were all obtained from Upstate Biotechnology, Inc. (Lake
Placid, NY). An anti-Shc monoclonal antibody was obtained from
Transduction Laboratories (Lexington, KY). The p38, ERK, and PI
3-kinase inhibitors PD98049, SB203580, and LY294002, respectively, were
purchased from Calbiochem, and dicumarol was from Sigma. The monoclonal anti-actin antibody and the FITC-conjugated anti-mouse IgG, antibody used for immunofluorescence, was purchased from Sigma.
Cells Lines--
FL5.12 and 32D cells were maintained in
Iscove's modified defined medium (IMDM) supplemented with 1 mM glutamine, 10% fetal bovine serum (FBS) (all from
BioWhittaker, Verviers, Belgium), and 10% (v/v) conditioned medium
(CM) from the IL-3-producing cell line WEHI-3B. FL5.12 cells that
overexpress the wild type (WT) and mutant IGF-IRs and the empty
pcDNA3 vector-expressing Neo cells were described previously (21).
MCF-7 cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 1 mM glutamine, 10% serum FBS.
Induction of Apoptosis by IL-3 Withdrawal and Cell Viability
Assays in the Presence of PI 3-Kinase and MAP Kinase
Inhibitors--
FL5.12/WT cells were cultured at 3 × 105 cells/ml in medium containing IL-3 for 24 h,
washed three times in serum-free medium, and then cultured at 5 × 105 cells/ml in IMDM containing 5% FBS (2 ml/well) in
24-well plates. IGF-I (100 ng/ml) or IL-3 (WEHI CM 10%) was added to
triplicate cultures. At the indicated times, 200-µl aliquots were
removed from each well, and viability was determined by counting live and dead cells after trypan blue staining. The percentage of viable cells was calculated from the total number of cells per well, and all
data are presented as the mean of triplicate cultures for each culture
condition. Survival assays were performed in medium supplemented with
5% FBS to reduce the amount of IGF-I and IGF-II that is available in
FBS that would mask the effects of exogenously added IGF-I. IGF-I- and
IL-3-mediated suppression of apoptosis was also assayed in the presence
of a number of inhibitors of signal transduction pathways. To inhibit
PI 3-kinase, ERKs, p38, or JNK, respectively, LY294002 (10 µM), PD98049 (50 µM), SB203580 (10 µM), or dicumarol (50, 100, or 150 µM) was
added to cultures that were exposed to IGF-I, IL-3, or 5% FBS. Cell viability was monitored by trypan blue exclusion over a 72-h period, and the data are presented as described above.
Western Blotting and Immunoprecipitations--
FL5.12 cells
overexpressing WT and mutant forms of the IGF-IR were seeded at 5 × 105 cells/ml in IMDM containing 10% FBS plus 10% WEHI
CM and cultured for 24 h, washed three times in serum-free medium,
and then starved for 3.5 h in serum-free medium. Following serum
starvation 7 × 106 cells were stimulated with IGF-I
(100 ng/ml) for up to 60 min, or not, and then washed in cold PBS.
Cells were then lysed in 40 µl of ice-cold SDS-lysis buffer (1%
Nonidet P-40, 0.1% SDS, 20 mM Tris, 50 mM
NaCl, 50 mM sodium fluoride, 1 µM pepstatin, 1 mM phenylmethylsulfonyl fluoride, 1 µM
aprotinin, and 1 mM sodium orthovanadate, pH 7.6). Debris
was removed by centrifugation at 15,000 × g at 4 °C
for 15 min, and samples were then denatured by boiling in 2× SDS-PAGE
sample buffer for 5 min. Proteins were resolved by 12 or 4-20%
gradient SDS-PAGE and blotted onto nitrocellulose membranes (Schleicher
& Schuell). Blots were blocked in TBS-T (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6) with 5% milk for 30 min
at room temperature. Primary phospho-specific antibodies were diluted
1/1000 in TBS-T, 5% goat serum, and all other antibodies were diluted
1/1000 in TBS-T, 5% milk and incubated for 1.5 h or overnight.
Horseradish peroxidase-conjugated secondary antibodies (Dako Glostrup,
Denmark) were used for detection using chemiluminescence with the ECL
reagent (Amersham Pharmacia Biotech) or with the Supersignal reagent (Pierce).
For immunoprecipitation cell lysates (107 cells in 500 µl
per sample) were pre-cleared with 20 µl of protein G-agarose beads for 1 h, followed by incubation with specific antibodies (5 µg) for 18 h. Protein G-agarose beads (20 µl) were then added for 3 h, and the beads were washed three times with lysis buffer
before boiling in gel sample buffer and separation of the released
proteins by SDS-PAGE. Proteins present in the immunoprecipitates were
identified by Western blotting as described above.
A non-radioactive kinase assay kit from PerkinElmer Life Sciences was
used to assay JNK activity in FL5.12 cells. This kit utilized
glutathione S-transferase-Jun beads that pull-down JNK protein from cell lysates. Cell lysates from cells that had been stimulated with IGF-I under different conditions, or that were not
stimulated, were incubated with glutathione
S-transferase-Jun beads for 18 h, washed, and then
incubated at 30 °C for 30 min in kinase reaction buffer according to
the manufacturer's instruction. The beads were then boiled in SDS
sample buffer and pelleted at 3,000 × g for 3 min.
Proteins in the supernatants were resolved by 12% SDS-PAGE, and
phosphorylated Jun was detected by Western blotting with an
anti-phospho-Jun antibody, which specifically detects JNK
phosphorylation on serine 63 of c-Jun.
MCF-7 Cell Viability Assays--
MCF-7 cells were seeded at
1 × 104 cells/ml in 24-well plates and cultured for
24 h. Dicumarol was then added to the cells at various
concentrations. These and control cultures cells were incubated in
Dulbecco's modified Eagle's medium supplemented with 10% FBS for 24, 48, or 72 h. At the indicated times cultures were photographed and
then washed. The remaining cells were stained with Giemsa, and the
viable cells in each well were counted by microscopic analysis. Cell
architecture was also analyzed by immunofluorescence staining of actin
in the cells. Cells were fixed in 100% methanol at
20 °C for 5 min and then blocked in blocking solution consisting of 2.5% goat
serum in PHEM buffer (60 mM Pipes, 25 mM Hepes,
10 mM EGTA, 2 mM MgCl2, pH 6.9) for
1 h. An anti-actin monoclonal antibody was then added to cells for
1.5 h at room temperature. After washing, a FITC-conjugated
anti-mouse IgG antibody, diluted 1/100 in blocking solution, and
Hoechst dye (0.5 µg/ml) were incubated with cells for a further
1.5 h at room temperature. The cells were washed again and
photographed using a SPOT-RT digital camera mounted on a Nikon TE300
inverted microscope equipped for epifluorescence.
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RESULTS |
JNK and the ERKs Become Phosphorylated in FL5.12/IGF-IR Cells
following IGF-I Stimulation, whereas p38 Is Constitutively
Phosphorylated--
Activation of the MAP kinase family members in
response to IGF-I has been demonstrated in different cell types (35).
However, the activation of SAPK by the IGF-IR in cells where IGF-I
mediates suppression of apoptosis has not been studied. To
investigate this, we used FL5.12 cells overexpressing the wild type
IGF-IR (FL5.12/WT) that are efficiently protected from IL-3 withdrawal by IGF-I (21). FL5.12/WT cells were removed from IL-3 for 3.5 h
and then stimulated with IGF-I or left unstimulated, and Western blotting was performed using antibodies against the
active/phosphorylated forms of the MAP kinase and SAPK proteins. Fig.
1A demonstrates that ERKs 1 and 2 are activated upon IGF-I stimulation, and this is in agreement
with published studies (17). Levels of p38 phosphorylation are high in
unstimulated cells and remain at similar levels in response to IGF-I
stimulation. This finding is in contrast to that observed with
IGF-I-induced phosphorylation of p38 in neuroblastoma cells (32) and
suggests that p38 is constitutively active in FL5.12 cells or,
alternatively, that its phosphorylation is induced very rapidly by
brief starvation from IL-3.

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Fig. 1.
Activation of MAPK and SAPK pathways in
response to IGF-I stimulation of FL5.12/WT cells.
A, cells were washed in serum-free medium, starved
for 3.5 h, and then stimulated with IGF-I for 20 or 40 min or left
unstimulated (0). Cells were then lysed as described under
"Experimental Procedures," and Western blotting was performed using
antibodies against the phosphorylated or native forms of ERK, p38, and
JNK. B, comparison of FL5.12/Neo with FL5.12/WT cells for
JNK activation and phosphorylation of c-Jun in response to IGF-I
stimulation for 20 and 60 min. Western blots were probed with
antibodies against phospho-JNK or against phospho-Jun (serine 73).
Anti-JNK and -actin antibodies were used to re-probe blots to control
for protein loading. C, Jun phosphorylation (serine 73) in
starved un-stimulated FL5.12/WT cells was investigated using lysates
from cells treated the same as in A but without IGF-I
stimulation. This is compared with stimulation with IGF-I for 20 min.
D, a time course of c-Jun phosphorylation was performed
using cells that were treated as in A above and a SAPK kit
to detect serine phosphorylation of glutathione
S-transferase-Jun fusion peptides (serine 63) by Western
blotting. Levels of JNK in the cell lysates are shown in the
bottom panel. All of the Western blots are representative of
several experiments with similar results.
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JNK phosphorylation is very low or not detectable in FL5.12/WT cells
but is greatly increased in response to IGF-I stimulation of cells at
20 and 40 min (Fig. 1A). Interestingly, although the levels
of JNK2 (56 kDa) protein seem to be higher than JNK1 (46 kDa) in these
cells, the increase in phosphorylation is predominantly in JNK1 rather
than in JNK2 (Fig. 1A). JNK activation is not detectable in
mock-transfected FL5.12/Neo cells compared with FL5.12/WT cells (Fig.
2B). An increase in
phosphorylation of c-Jun is also detectable between 20 and 60 min in
FL5.12/WT cells, and this is also not detectable in FL5.12/Neo cells
(Fig. 2B). JNK and c-Jun are also robustly phosphorylated in
response to stimulation with IL-3 for 15 min (data not shown).

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Fig. 2.
The JNK inhibitor dicumarol abrogates
IGF-I-mediated suppression of apoptosis in FL5.12/WT cells.
A, FL5.12/WT cells were cultured at 5 × 105/ml in medium containing IL-3 for 24 h, washed
extensively, and then cultured in medium containing 5% FBS (control),
FBS plus IGF-I, or FBS plus IL-3 either in the presence or absence of
100 µM dicumarol. Live and dead cells were counted as
triplicate cultures by trypan blue exclusion at 24, 48, and 72 h.
Data are presented as percent viability of total cells in the cultures.
Each point represents the mean and standard deviation of triplicate
cultures. B, a dose-response curve was determined using
FL5.12/WT cells cultured in FBS plus IGF-I in the presence of 0, 25, 75, or 100 µM dicumarol. Data are presented as in
A above. C, the effect of dicumarol on Jun
phosphorylation in FL5.12/WT cells was assessed by Western blotting
with anti-phospho-Jun antibodies. Cells were starved for 3.5 h and
pretreated with dicumarol for 5 min before stimulation with IGF-I for
up to 60 min. The 1st two lanes show Jun phosphorylation in
unstimulated and IGF-I-stimulated cells, and the next 4 lanes show levels of Jun phosphorylation in cells treated with
dicumarol. Levels of actin were assessed to control for loading.
D, Western blotting with anti-phospho-AKT antibody was used
to measure AKT phosphorylation in response to IGF-I in untreated FL5.12
cells cells (left 3 lanes) or in dicumarol-treated cells
(right 3 lanes), and actin was used as protein loading
control. E, the status of p38 activity was assessed in
FL5.12 cells in response to IGF-I (left 3 lanes) or in cells
treated with dicumarol (right 3 lanes) by Western blotting
with anti-phospho-p38 and against p38 as a control. F,
phosphorylation of I B in unstimulated cells, IL-3, stimulated cells,
or IGF-I-stimulated cells was assessed by Western blotting with
anti-phospho-I B antibodies. This was compared with phosphorylation
of c-Jun, and blots were re-probed with anti-I B antibody to control
for loading.
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To rule out the possibility that the increase in JNK activation and Jun
phosphorylation in FL5.12/WT cells was due to a stress response caused
by withdrawal of IL-3, cells were monitored for JNK activation at
different times up to 60 min in the absence of any stimulation. Results
shown in Fig. 1C compare levels of c-Jun phosphorylation in
starved cells with levels of c-Jun phosphorylation following IGF-I
stimulation for 20 min. This demonstrates that although low levels of
JNK activity are detected in FL5.12 cells upon IL-3 withdrawal, these
levels do not increase over time, whereas stimulation with IGF-I
induces a robust activation of JNK.
By having established that IGF-I induces JNK activation in FL5.12
cells, we were next interested in examining the time course of JNK
activation and c-Jun phosphorylation. This was investigated using a
non-radioactive assay kit that is based on an antibody that
specifically detects phosphorylation on serine 63 of Jun, which is
phosphorylated by JNK. Fig. 1D shows that JNK
phosphorylation activity peaks between 20 and 40 min of IGF-I
stimulation and begins to decrease by 60 min. This is indicative of a
transient activation of JNK in response to IGF-I and is different from
prolonged activation of JNK induced by anisomycin and stress signals
(36). Thus, overall these data demonstrate that IGF-I induces transient phosphorylation of both ERKs and JNK in FL5.12/WT cells and that p38 is
constitutively active in these cells.
Inhibition of JNK or p38 Abrogates Suppression of Apoptosis by
IGF-I in FL5.12/WT Cells--
Transient activation of JNKs has been
associated with suppression of apoptosis in response to TNF-
in T
lymphocytes (29), whereas dicumarol blocks JNK and NF-
B activation
leading to potentiation of apoptosis in HeLa cells (30). In order to
determine whether the activation of JNK by IGF-I in FL5.12/WT cells
shown above is associated with suppression of apoptosis, we sought to
block JNK activation using dicumarol and also to determine the effects of dicumarol on IGF-I-mediated suppression of apoptosis. Previously, we
have shown that IGF-I protects FL5.12/WT cells from IL-3 withdrawal, without promoting a proliferative response (21). To determine if
dicumarol had an affect on IGF-I-mediated suppression of apoptosis FL5.12/WT cells were cultured in the presence of IL-3, IGF-I, or 5%
FBS (control) with or without dicumarol at different concentrations ranging from 25 to 150 µM. The results shown in Fig.
2A demonstrate that IGF-I-mediated suppression of apoptosis
is drastically inhibited by 100 µM dicumarol, as is
suppression of apoptosis afforded by 5% FBS in the control cells (Fig.
2A). The viability of cells cultured in the presence of IL-3
is not altered significantly by incubation with 100 µM
dicumarol (Fig. 2A), although we did notice that the
proliferation of cells cultured in the presence of IL-3 was suppressed
and that higher concentrations of dicumarol (150 and 200 µM) lead to loss of viability in IL-3 cultures (not shown). A dose-response curve showing the effects of dicumarol on
IGF-I-mediated suppression of apoptosis is shown in Fig. 2B. This demonstrates that in the presence of 25 µM dicumarol
there is no effect on IGF-I-mediated anti-apoptotic activity, but at 50 µM there is some abrogation of IGF-I activity, with a
progressive abrogation at 75 µM to complete inhibition at
100 µM dicumarol.
We next tested the effects of dicumarol on JNK activation by IGF-I as
well as on the other signaling pathways that are activated by IGF-I or
constitutively active in FL5.12/WT cells. The results shown in Fig.
2C demonstrate that IGF-I-mediated phosphorylation of c-Jun
is completely inhibited up to 60 min in FL5.12/WT cells by
preincubation of the cells with 100 µM dicumarol for 5 min. IL-3-mediated phosphorylation of c-Jun is also inhibited under these conditions. In the same cells induction of AKT phosphorylation by
IGF-I is not affected in the presence of dicumarol (Fig.
2C). We also investigated whether dicumarol had any
influence on p38 or NF-
B activity in these cells. p38 is
constitutively phosphorylated in FL5.12/WT cells as shown above in Fig.
1A. The status of NF-
B activity in FL5.12/WT cells was
determined by measuring I
B phosphorylation. As shown in Fig.
2D I
B has very low basal levels of phosphorylation in
unstimulated cells, and this does not significantly increase with
either IGF-I or IL-3 stimulation. Pretreatment of FL5.12/WT cells with
dicumarol did not alter the levels of p38 and very slightly increased
the levels of I
B phosphorylation (Fig. 2D). Taken
together these data indicate that inhibition of JNK activation with
dicumarol abrogates IGF-I-mediated suppression of apoptosis despite the
fact that AKT and presumably the PI 3-kinase pathway as well as p38 and
NF-
B all remain active. This result suggests that activation of the
JNK pathway in these cells occurs independently of PI 3-kinase activation.
We next compared the effects of dicumarol with the effects of other
MAPK inhibitors on IGF-I-mediated anti-apoptotic activity in FL5.12
cells. IGF-I-mediated protection from IL-3 withdrawal was measured in
the absence or presence of the ERK inhibitor, PD98059, or the p38
inhibitor, SB203580. The ERK inhibitor, PD98059, does not have any
affect on the viability of cells cultured in the presence of IGF-I or
IL-3 (Fig. 3A), although it
does inhibit ERK1 and -2 activation in response to IGF-I stimulation
(Fig. 3B). We were also interested to determine whether
inhibition of p38 activity could affect IGF-I-mediated suppression of
apoptosis because this protein is apparently constitutively active in
FL5.12/WT cells. Results shown in Fig. 3C indicate that the
viability of the cells cultured in the presence of IGF-I is diminished
in the presence of the p38 inhibitor to below the levels of cells
cultured in FBS alone at 48 h. The viability of cells cultured in
the presence of 5% FBS only was also significantly decreased, but the
viability or proliferation of cells cultured in IL-3 is not affected by SB203580. These data suggest that although IGF-I does not induce phosphorylation of p38 in FL5.12 cells, p38 activity contributes to
IGF-I-mediated anti-apoptotic function. To confirm that the p38
inhibitor is acting on p38, we measured p38 phosphorylation in cells
stimulated with IGF-I in the absence and presence of SB203580. p38
phosphorylation levels remain constant in the presence of IGF-I up to
60 min and slightly decrease at 90 min of stimulation (Fig.
3D). This phosphorylation is clearly inhibited by 10 µM SB203580 in the absence of IGF-I and also up to 60 min
of IGF-I stimulation (Fig. 3D). However, SB203580 does not
inhibit JNK activation in response to IGF-I (Fig. 3D),
suggesting that inhibition of JNK is not responsible for the loss of
IGF-I-mediated anti-apoptotic activity.

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Fig. 3.
Viability of FL5.12/WT cells in the presence
and absence of the ERK inhibitor PD98049 or the p38 inhibitor
SB203580. A, FL5.12 cells were cultured at 5 × 105/ml in medium containing 5% FBS (control), FBS plus
IGF-I (50 ng/ml), or FBS plus IL-3 (WEHI CM, 10%) in the presence or
absence of the MAPK inhibitor PD98049 (50 µM), and cell
viability was monitored at the indicated times using trypan blue
exclusion. Data are presented as the mean and standard deviation from
triplicate cultures. B, FL5.12/WT cells were seeded at
5 × 105 cells/ml and grown overnight. Cells were then
starved for 3 h and either left untreated (left 3 lanes) or incubated in the presence of 50 µM PD98049
for 30 min (right 3 lanes) before stimulation with IGF-I for
the indicated times. Western blotting was performed using
anti-phospho-ERK and anti-ERK2 antibodies. C, viability
curve for FL5.12/WT cells treated with the p38 inhibitor SB303580.
Cells were cultured as described in A but in the presence or
absence of 10 µM of the p38 inhibitor SB203580. Data are
presented as mean and standard deviation from triplicate cultures.
D, FL5.12/WT cells were seeded at 5 × 105
cells/ml and grown overnight. Cells were then starved for 3 h and
treated with 10 µM SB203580 or not for 30 min before 100 ng of IGF-I was added for up to 90 min or not (0). Cells
were then washed and lysed as described under "Experimental
Procedures," and Western blots were probed with antibodies against
phospho-p38, p38, and phospho-Jun.
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Taken together, these data demonstrate that dicumarol can block JNK
activation by IGF-I and block IGF-I-mediated suppression of apoptosis,
without affecting AKT, p38, or NF-
B activation. The activity of
dicumarol is more potent than that of a p38 inhibitor and an ERK
inhibitor that has no effect. This suggests that dicumarol inhibits a
pathway involving JNK activation that is required for IGF-I-mediated
suppression of apoptosis in these cells.
Transient Activation of the JNK Pathway by IGF-I Does Not Require
PI 3 Kinase Activity--
Recruitment and activation of PI 3-kinase
leading to activation of AKT is required for suppression of apoptosis
by the IGF-IR in many cell types (10, 15) but is not sufficient in
fibroblasts and FL5.12 cells (21, 24). Since inhibition of JNK
activation by dicumarol did not affect AKT activation by IGF-I, but
still blocked suppression of apoptosis by IGF-I, we were interested to
determine whether activation of JNK by IGF-I requires PI 3-kinase activity. To investigate this we measured JNK activation and
suppression of apoptosis in response to IGF-I stimulation of FL5.12/WT
cells in the absence or presence of the PI 3-kinase inhibitor LY294002. As shown in Fig. 4A, LY294002
completely suppresses IGF-I-mediated protection from IL-3 withdrawal in
FL5.12/WT cells, whereas IL-3-mediated cell viability is not affected.
However, in these cells although AKT activation is suppressed by
LY294002, JNK activation still occurs in response to IGF-I as shown by
phosphorylation of c-Jun in the presence of LY294002 (Fig.
4B).

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Fig. 4.
Effects of PI 3-kinase inhibitor LY294002 on
FL5.12/WT cell viability and IGF-I-mediated JNK activation.
A, FL5.12/WT cells were cultured at 5 × 105/ml in medium containing 5% FBS (control), FBS plus
IGF-I (50 ng/ml), or FBS plus IL-3 (WEHI CM, 10%) in the presence or
absence of 10 µM LY294002. Cell viability was determined
by counting triplicate samples at the indicated times using trypan blue
exclusion. B, FL5.12/WT cells were seeded at 5 × 105 cells/ml and grown overnight. Cells were then starved
for 3 h and treated with 10 µM LY294002 or nothing
for 30 min either without or with IGF-I stimulation for the indicated
times. Cell lysates were prepared for Western blotting with antibodies
against phospho-Jun or phospho-AKT. Blots were also probed with
anti-AKT antibody to control for AKT levels in the cells and for
protein loading. C, FL5.12 cells were washed in serum-free
medium, starved for 3.5 h, and then stimulated with IGF-I for 5 or
10 min or left unstimulated. Lysate were prepared and split for
immunoprecipitation (I.P.) (107 cells/sample)
with Shc or Gab-2. In a separate experiment cells were stimulated with
IGF-I for 2 or 5 min, and p85 was immunoprecipitated from lysates
prepared in the same way as described above. Immunoprecipitated
proteins were separated by PAGE, and Western blotting performed with
the antibodies indicated below each panel.
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To determine how PI 3-kinase is activated in response to IGF-I in
FL5.12 cells, we investigated recruitment of p85 into IRS-1/2 and to
Shc complexes. We could not detect p85 IRS-2 complexes or IRS-1 in
FL5.12 cells, but as shown in Fig. 4C we found that IGF-I
induced phosphorylation of Shc and the formation of Shc-Grb-2 complexes. A Shc-Grb-2 pathway to PI 3-kinase has been shown to occur
via Gab-2 recruitment of p85 in BaF3 cells (37). Therefore, we
investigated a role for Gab-2 in FL5.12 cells. We found that IGF-I
induces the formation of phosphorylated Shc-Gab-2 complexes and
p85-Gab-2 complexes. Grb-2 is in all Gab-2 complexes in agreement with
Gu et al. (37). By immunoprecipitation of p85 we also
saw IGF-I-induced formation of p85-Grb-2 complexes. Altogether this indicates that IGF-I stimulation can lead to p85 recruitment and PI
3-kinase activation through recruitment of Shc-Grb-2 and Gab-2 in
FL5.12 cells.
In summary these results demonstrate that JNK activation by IGF-I is
not dependent on PI 3-kinase activity in FL5.12 cells. In addition, the
results suggest that PI 3-kinase and JNK control signaling pathways are
differentially activated by the IGF-IR. The effects of LY294002 and
dicumarol indicate that each of these pathways contributes to, but
neither is sufficient for, suppression of apoptosis by the IGF-IR.
IGF-I-activated Increase in JNK Is Not Dependent on Residues in the
IGF-IR C Terminus or on IRS-1 and IRS-2, but Requires Tyrosine 950 for
Maximal Activation--
By having established that activation of JNK
by IGF-I is through a signaling pathway that is distinct from the PI
3-kinase pathway, we were next interested to determine if any of the
domains of the IGF-IR previously associated with suppression of
apoptosis were necessary for JNK activation. To do this IGF-I-mediated
stimulation of JNK activity was measured in FL5.12 cells transfected
with a series of IGF-IR mutants (Table I)
and also in 32D cells, which lack IRS-1 and IRS-2. We were particularly
interested in mutants of the IGF-IR C terminus. These mutants included
receptors that have tyrosine 1251 mutated to phenylalanine (Y1251F) or
histidine 1293 and lysine 1294 mutated to phenylalanine and arginine
(H1293F/K1294R). Cells expressing either of these mutant receptors do
not exhibit IGF-I-mediated anti-apoptotic activity (21) but can
activate PI 3-kinase and AKT. However, as shown in Fig.
5A FL5.12 cells expressing
these receptors could activate Jun phosphorylation in response to IGF-I
stimulation. FL5.12 cells expressing a receptor truncated at amino acid
1245 (1245d) could also activate JNK (Table I), suggesting that
the last 92 amino acids of the IGF-IR C terminus are not required for
JNK activation. We then investigated whether an IGF-IR mutated at
tyrosine 950 in the juxta-membrane region and also deleted at residue
1245 in the C terminus could activate JNK, and we found that FL5.12
cells expressing this mutant had greatly diminished JNK phosphorylation
compared with FL5.12/WT cells (Fig. 5B). This result
suggests that proteins that interact with tyrosine 950 are required for
JNK activation.
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Table I
JNK activation in FL5.12 cells overexpressing C-terminal mutants of the
IGF-IR
Cells expressing mutant IGF-IRs were washed in serum-free medium,
starved for 3.5 h, and stimulated with IGF-I for 20 or 40 min or
left unstimulated. Cells were then lysed as described under
"Experimental Procedures," and the lysates were separated on
4-20% gradient SDS-PAGE. Western blots were performed using
anti-phospho-JNK or anti-phospho-Jun antibodies to detect
IGF-I-stimulated phosphorylation and activation of JNK. Blots were
re-probed with anti-JNK antibody as a loading control.
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Fig. 5.
IGF-I-induced activation of JNK in FL5.12
cells overexpressing mutant IGF-IRs and in 32D/IGF-IR cells.
A, the PerkinElmer Life Sciences SAPK assay kit was used to
determine whether IGF-I activates JNK in FL5.12 cells that express
mutant IGF-IRs (Y1251F and H1293F/K1294R). Cells were starved and
stimulated as for FL5.12/WT cells described above. B, JNK
activation in FL5.12 cells expressing the Y950F/1245d mutant
compared with FL5.12/WT by Western blotting with an anti-phospho-JNK
antibody. Cells were starved and stimulated as in Fig. 1B.
32D cells that overexpress the WT IGF-IR were assayed for their ability
to phosphorylate c-Jun in response to IGF-I stimulation for the
indicated times. Cells were treated in the same way as FL5.12 cells,
and Western blotting was performed with anti-phospho-Jun antibody.
Blots were re-probed with an anti-p38 antibody to control for protein
levels.
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To determine whether JNK activation requires signals from IRS-1 or
IRS-2 that could bind to Tyr-950, we investigated IGF-I-mediated activation of JNK in the myeloid cell line 32D that stably expresses a
WT IGF-IR. Stimulation of these cells with IGF-I in the absence of IL-3
resulted in activation of JNK with similar kinetics to those seen in
FL5.12 cells (Fig. 5C). This suggests that IRS-1 and IRS-2
are not required for IGF-IR-mediated activation of JNK. Therefore,
taken together with the results from FL5.12 cells expressing the
different mutant receptors, we conclude that Tyr-950 is required for
maximal JNK activation and that a major part of the IGF-IR C terminus
as well as IRS-1 and IRS-2 are dispensable for JNK activation in
response to IGF-I.
IGF-I and Anisomycin Can Induce Additive SAPK Activity in FL5.12/WT
Cells--
Anisomycin has been used to study SAPK signaling and has
been shown to be an enhancer of both persistent SAPK activity and apoptosis (38). Anisomycin desensitizes cells to further JNK activation
in fibroblasts (44), and pretreatment of human embryonic kidney cells
with IGF-I resulted in inhibition of anisomycin-induced SAPK activation
(33). To further characterize JNK activation by IGF-I in FL5.12/WT
cells and determine its association with suppression of apoptosis, we
were therefore interested to determine whether IGF-I pretreatment could
affect anisomycin-induced JNK activation. Cells were deprived of IL-3
and stimulated with IGF-I or nothing for 5 min, after which anisomycin
was added to the cultures for a further 20 min. As shown in Fig.
6 anisomycin induced JNK phosphorylation,
and this was enhanced in cells that were pre-stimulated with IGF-I.
Therefore, in FL5.12/WT cells IGF-I does not block anisomycin-induced
JNK activation. This result suggests that the IGF-IR activates a
signaling pathway upstream of JNK in FL5.12 cells that is distinct from
that activated by anisomycin.

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Fig. 6.
Anisomycin has an additive effect on
IGF-I-induced JNK/Jun phosphorylation. FL5.12/WT cells were
cultured in the presence of IL-3 for 24 h, starved for 3.5 h,
pretreated with 100 ng of IGF-I for 5 min (+) or not ( ), and then
treated with anisomycin (+) or not ( ) at a final concentration of 10 µg/ml. Western blots were probed with anti-phospho-JNK and
anti-phospho-Jun antibodies, and blots were probed with anti-actin
antibody to control for protein levels.
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Dicumarol Is Cytotoxic to the Breast Carcinoma Cell Line
MCF-7--
There is considerable evidence to support the idea that
suppression of apoptosis by the IGF-IR contributes to tumorigenesis and
to the survival and chemo-resistance of several kinds of tumor cells
including those derived from breast, lung, and prostate (39). The
results shown above suggest that JNK kinase activation by IGF-I is
associated with IGF-I-mediated suppression of apoptosis and that
dicumarol, a JNK inhibitor, is an effective inhibitor of IGF-I-mediated
suppression of apoptosis. Therefore, we were interested to determine if
dicumarol could induce apoptosis in tumor cells. To test this we used
the breast carcinoma cell line MCF-7, which expresses high levels of
IGF-IR, is very IGF-I responsive, and is quite resistant to
chemotherapeutic drugs. As shown in Fig.
7A exposure of cultures of
MCF-7 cells to dicumarol for 24 or 48 h results in a
dose-responsive decrease in cell viability with more than 75% of the
cells dead in the presence of 100 µM dicumarol by 48 h. The morphology of remaining cells indicates shrinkage and loss of
actin organization as shown by immunofluorescence staining with
anti-actin antibodies (Fig. 7B). These results demonstrate that dicumarol potently induces apoptosis in MCF-7 cells and further suggest that targeting this pathway could be a specific way in which to
inhibit IGF-IR anti-apoptotic signaling in tumor cells.

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Fig. 7.
Dicumarol inhibits MCF-7 cell survival and
disrupts cytoskeletal organization. MCF-7 cells were cultured at
1 × 105 cells/ml in triplicate wells of 24-well
plates for 24 h. Dicumarol was then added to the cultures at the
indicated concentrations, and duplicate plates were further incubated
for 24 or 48 h. A dose curve was determined by counting cell
viability using trypan blue exclusion 48 h after the addition of
dicumarol (A). Cells were also photographed live under phase
contrast at 24 h at × 10 (B, top panels). Cells
cultured for 48 h were fixed in methanol for immunofluorescence
staining with anti-actin antibody and FITC-labeled secondary antibody.
Hoechst dye was used to counter-stain the nuclei, and cells were
photographed at × 40.
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DISCUSSION |
The role of the JNK signaling pathway in regulating responses to
cellular stress and its association with induction of apoptosis in
neuronal cells has been well documented. There has also been some
evidence to indicate a role for the JNK pathway in promoting cell
survival and tumorigenesis (40). Our findings that JNK activation
occurs transiently in response to IGF-I stimulation of FL5.12 cells and
that inhibition of JNK by dicumarol in these cells where the IGF-IR
mediates anti-apoptotic signaling, but not proliferation, support a
role for the stress pathways and for JNK activation in suppression of
apoptosis by the IGF-IR. Our findings also suggest that activation of
the JNK pathway by IGF-I is distinct from but might operate in addition
to the PI 3-kinase/AKT pathway.
Activation of JNK in response to IGF-I stimulation of FL5.12 cells is
reminiscent of the activity of p38 and JNK in TNF signaling described
by Roulston et al. (29). This study showed that activated JNK and p38 were protective against apoptosis, during the early part of
the signaling response to TNF, an effect that could be inhibited by
actinomycin D. Similarly transforming growth factor-
, which has been
shown to prevent cell death upon serum withdrawal, induced a sustained
phosphorylation of c-Jun with diminution of JNK activation later in the
death response (41). Dicumarol could inhibit JNK activation by IGF-I in
FL5.12/WT cells at concentrations where it did not inhibit AKT
activation or constitutive p38 and NF-
B activity. Dicumarol also
inhibited IGF-I-mediated suppression of apoptosis at concentrations
that did not inhibit the anti-apoptotic activity of IL-3. All these
data suggest that transient activation of JNK is involved in mediating
anti-apoptotic signaling from the IGF-IR.
The potential molecular mechanisms of anti-apoptotic signals from
transient activation of JNK in FL5.12 cells remain unclear. Two
possible mechanisms are likely to be enhanced transcription of
anti-apoptotic genes in the nucleus or signaling events in the
cytoplasm that lead to activation of anti-apoptotic proteins. JNK
activation induces the phosphorylation of several transcription factors
including c-Jun, Elk-1, and ATF-2, which regulate immediate early gene
expression (42, 43). A role for c-Jun activity in survival signaling
has been supported by the activity of non-phosphorylatable dominant-negative forms of c-Jun (44, 45). These rendered fibroblasts
incapable of survival following UV irradiation (46) and could also
attenuate the anti-apoptotic effects of TGF-
upon serum withdrawal
in A549 cells (41).
The effects of JNK may not be entirely due to transcriptional
responses, and JNK has been shown to regulate signaling events near the
plasma membrane and in the cytoplasm. For example JNK has recently been
shown to phosphorylate IRS-1 on a regulatory serine in its
phosphotyrosine binding domain resulting in inhibition of IRS-1
activity in Chinese hamster ovary cells (47). Other apoptosis-regulating proteins have also been shown to interact with JNK
such as Bcl-XL and NFATc1 (48, 49). Thus, the mechanisms by
which JNK might regulate cell survival could include both
transcriptional output in the nucleus and anti-apoptotic signaling
events in the cytoplasm.
We did not detect increased p38 activity in response to IGF-I
stimulation of FL5.12 cells, which is in contrast to observations in
neuroblastoma cells where IGF-I suppressed JNK activity but enhanced
p38 activity under conditions of stress (32). However, in FL5.12 cells
p38 activity also appears to be a mediator of survival signals from the
IGF-IR because inhibition of p38 activity resulted in diminution of
IGF-I-mediated anti-apoptotic activity. This is in contrast to the
observation that inhibition of ERK activity had no effect on cell
viability either in the presence of IGF-I, IL-3, or 5% FBS. p38 has
also been shown to have a role in survival signaling in neuronal PC12
cells where a requirement for p38 activity was demonstrated in the
IGF-I-mediated transcriptional activation of the cAMP-responsive
element-binding protein, which can lead to increased Bcl-2 expression
and increased cell survival (51). We have not been able to detect
enhanced expression of Bcl-2 in FL5.12 cells in response to IGF-I
stimulation, but we have detected slight increases in
Bcl-XL levels (not shown). It also remains possible that
other Bcl-2 family members or other survival genes could be targets of
p38 or JNK in these cells.
JNK activation following IGF-I stimulation of FL5.12/IGF-IR cells is PI
3-kinase-independent, and this result defines the JNK signaling pathway
as a candidate to be regulated by residues in the IGF-IR C terminus
that abrogate the anti-apoptotic activity of the IGF-IR (21). However,
an IGF-IR deleted at amino acid 1245 could still induce JNK activation
in response to IGF-I (Table I and Fig. 5). Activation of JNK following
IGF-I stimulation of the IRS-1 and -2 deficient 32D/IGF-IR cells
indicates that IRS-1 and IRS-2 are not required for JNK activation by
the activated IGF-IR. However, mutation of tyrosine 950 almost
completely inhibited JNK activation, suggesting that residues that
interact with Tyr-950 such as Shc or Crk (52) are required for JNK
activation. There are many candidate pathways leading to JNK including
the Ste20-like kinase (53), Src, Cas, reactive oxygen species (54), and
Gi (55). We could not inhibit the IGF-I-mediated
activation of JNK using Gi inhibitors, but we could detect
an increase in the autophosphorylation of the hematopoietic progenitor
kinase (HPK), 3 min after IGF-I stimulation (data not shown). HPK has
been shown to activate JNK via its interaction with Crk in a Rac and
Cdc42-independent manner (56). Thus, a possible scenario is that an
activated IGF-IR directly recruits Crk, or recruits SHC, which then
forms a complex with Grb-2 and Crk that can associate with HPK.
Activated HPK has been shown to activate JNK via MEKK/Tak 1 and MKK4
(57). All of this makes SHC, Crk, and HPK likely to be the molecules that mediate the signaling pathway between the IGF-IR and JNK.
The drastic effect that dicumarol had on MCF-7 cell survival suggests
that this could be a useful therapeutic approach for killing tumor
cells that overexpress the IGF-IR such as prostate, lung, and breast
carcinomas. The IGF-IR and c-Jun have both been shown to be associated
with cellular transformation, and both c-Jun and AP-1 signaling have
been postulated to influence breast cancer phenotype (50). The
tightening of the actin bundles that was observed following dicumarol
treatment of MCF-7 cells (Fig. 7B) is in contrast to the
loss of actin architecture seen in prostate carcinoma cells following
the overexpression of PSK, a Ste20-like kinase that activates JNK
(53).
In summary, we have demonstrated that transient activation of JNK
occurs in response to IGF-I stimulation in FL5.12 cells and that
dicumarol can block both IGF-I-mediated JNK activation and
IGF-I-mediated suppression of apoptosis. The JNK pathway in these cells
is distinct from the PI 3-kinase/AKT activated by the IGF-IR and may be
important in maintaining the survival and transformation of tumor cells.