From the Unit of Molecular Carcinogenesis,
International Agency for Research on Cancer,
69372 Lyon Cedex 08, France and ¶ Ruttenberg Cancer Center,
Mount Sinai School of Medicine, New York, New York 10029
Received for publication, July 23, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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WR1065 is an aminothiol with selective
cytoprotective effects in normal cells compared with cancer cells. In a
previous study (North, S., El-Ghissassi, F., Pluquet, O.,
Verhaegh, G., and Hainaut, P. (2000) Oncogene 19, 1206-1214), we have shown that WR1065 activates wild-type p53 in
cultured cells. Here we show that WR1065 induces p53 to accumulate
through escape from proteasome-dependent degradation. This
accumulation is not prevented by inhibitors of phosphatidylinositol 3-kinases and is not accompanied by phosphorylation of Ser-15, -20, or
-37, which are common targets of the kinases activated in response to
DNA damage. Furthermore, WR1065 activates the JNK (c-Jun N-terminal
kinase), decreases complex formation between p53 and inactive JNK, and
phosphorylates p53 at Thr-81, a known site of phosphorylation by JNK. A
dominant negative form of JNK (JNK-APF) reduces by 50% the activation
of p53 by WR1065. Thus, WR1065 activates p53 through a
JNK-dependent signaling pathway. This pathway may prove
useful for pharmacological modulation of p53 activity through
non-genotoxic mechanisms.
The radioprotective aminothiol amifostine (WR2721;
S-2[3-aminopropylamino]-ethylphosphothioic acid,
Ethyol®) is a pro-drug that is converted to its active free thiol
form, WR1065, by dephosphorylation by alkaline phosphatase in tissues
(1). WR1065 penetrates into cells by both passive and active mechanisms
(2, 3) and protects cells against toxicity by radiation, alkylating
agents, and platinum compounds in both animals and humans (4-7).
WR1065 is currently used as a cytoprotector in patients treated with
cisplatin for ovarian cancer and, in some protocols, for colorectal or
lung cancers (8-10). The administration of WR1065 in vivo
protects normal cells without impairing the efficacy of radio- and
chemotherapy on tumor cells (11-13). WR1065 acts primarily as a free
radical scavenger with anti-clastogenic and anti-mutagenic effects
in vitro as well as in We have shown recently (20-22) that WR1065 induces the accumulation
and activation of the tumor suppressor protein p53 and of several of
its target genes, including p21waf-1 and Mdm2. In
MCF-7 cells (expressing wild-type p53), WR1065 induces a
p53-dependent increase in the level of p21waf-1,
correlated with an arrest in the G1 phase of the cell cycle (22). p53 acts as a transcription regulator to arrest proliferation or
to induce cell death in response to multiple types of stresses (23).
Broadly, these stresses can be classified in three groups, including
genotoxic stress (e.g. DNA strand breaks, base oxidation, and formation of bulky DNA adducts), oncogenic stress (constitutive activation of Ras/E2F or In non-stressed cells, p53 is in a latent form and is constitutively
repressed by the binding of two proteins, Mdm2 and the inactive form of
JNK,1 which mediate the
degradation of p53 by the proteasome (28-31). In response to stress,
p53 undergoes multiple post-translational modifications, both in the N-
and C-terminal regions. These modifications stabilize the protein and
turn it from a latent to an active form that can bind specific DNA
sequences with a high affinity. These post-translational modifications
(reviewed in Refs. 24, 26, and 32-34) include phosphorylations at
Ser-15, -20, -33, -37, and -392 (32, 33, 35-37), acetylation of lysine
residues in the C terminus by co-activators of transcription such as
p300/CBP or PCAF (38, 39), as well as sumoylation at Lys-386 (40, 41).
Phosphorylation at specific sites may play additional roles in the
control of p53 activities, such as phosphorylation of Ser-46, which
appears to correlate with p53-dependent apoptosis (36, 42).
Transduction of DNA-damage signals involves kinases of the PI 3-kinase
superfamily (ATM, ATR, and DNA-PK) (43, 44), the cell cycle kinases
Chk1 and Chk2 (45, 46), and kinases of the mitogen-activated protein
kinase/SAPK family including p38 and JNK (47, 48). Members of the PI
3-kinase family and of the Chk family phosphorylate p53 at several
sites in the N terminus, in particular Ser-15 and Ser-20 (37, 49).
These serines are located within the domain of interaction with Mdm2,
and their phosphorylations allow p53 to escape Mdm2-mediated
degradation (50). Escape from degradation can also occur through the
binding of Mdm2 by p14arf, the alternative product of the
CDKN2a locus (24, 25). In contrast, JNK acts through an
Mdm2-independent mechanism to regulate p53 stability. Binding of the
inactive kinase to residues 97-116 earmarks p53 for proteasome
degradation (29). After kinase activation, this complex dissociates,
and active JNK may further participate in p53 induction by
phosphorylation of Thr-81 (51). The same dual role of JNK in the
regulation of the stability and activity of transcription factors has
also been demonstrated with c-Jun, Elk-1, and ATF-2 (52, 53).
Activation of JNK is induced by many forms of genotoxic and well
non-genotoxic stress such as heat shock, osmotic shock, and
anti-oxidative agents (47).
There is evidence that p53 may play a role in the mechanism of
cytoprotection by WR1065. For example, in a recent study, Shen et
al. (54) have shown that WR1065 protects murine embryo fibroblasts from paclitaxel-induced cell death in a p53-dependent
manner. It is therefore essential to determine the molecular mechanisms by which WR1065 activates p53. In this study, we show that induction of
p53 by WR1065 is not mediated through post-translational modifications that commonly occur in response to DNA damage, such as phosphorylation of Ser-15, -20, and -37. In contrast, this induction involves phosphorylation at Thr-81, the site of active JNK phosphorylation. Moreover, induction of p53 by WR1065 requires dissociation of complexes
with inactive JNK and is at least partially prevented by a dominant
negative JNK mutant. These results indicate that WR1065 induces p53 by
a stress signaling pathway that differs from the one activated by most
DNA-damaging agents.
Cell Culture and Treatments--
The breast carcinoma cell line
MCF-7 (expressing wild-type functional p53) and MN1 and MDD2 cells (55,
56) derived from MCF-7 cells, mouse 3T3, and 10.1 fibroblasts (57) were
cultured in Dulbecco's modified Eagle's medium (Invitrogen). Cells
were maintained at 37 °C with 10% fetal calf serum (PAA, Linz,
Austria), 2 mM L-glutamine and antibiotics,
10% CO2, MN1 and MDD2 were selected in 0.4 mg/ml G418
(Invitrogen). MCF-7 cells were transfected using Lipofection
(FuGENETM, Roche Molecular Biochemicals). MCF-7 cells were
stably transfected with a dominant negative JNK (JNK-APF) bearing the
FLAG epitope (pcDNA3-FLAG-JNK[T183A/Y185F]) or the empty vector
(58). Cells were selected in 0.4 mg/ml G418.
Cells were treated with different drugs at 50-65% confluency. WR2721
and WR1065 were provided by US Bioscience Inc. (West Conshohocken,
PA), dissolved in phosphate-buffered saline (PBS), and flushed with
argon to prevent oxidation. Aminoguanidine (Sigma) was dissolved in
culture medium and added to cells 10 min before WR1065. LY294002
(Sigma) was dissolved in Me2SO and used at 10 µM. Cells were pretreated with LY294002 for 1 h
before exposure to aminoguanidine and WR1065. Hydrogen peroxide and
cycloheximide (all from Sigma) were dissolved in deionized water.
Lactacystin (Calbiochem) and the fluoropeptide Suc-LLVY-AMC (Bachem
Biochemica, Heidelberg, Germany) were dissolved in
Me2SO and stored at Sulforhodamine B in Vitro Drug Assay--
Cells were seeded into
96-well microtiter plates in 100 µl at a density of
1·104 cells/well. After cell inoculation, the microtiter
plates were incubated at 37 °C with 10% CO2 for 24 h prior to addition of experimental drugs. After 24 h, a plate of
each cell line was fixed in situ with trichloroacetic acid
and sulforhodamine B staining, as described elsewhere (59), to
provide a measurement of the cell population for each cell line at the
time of drug addition (Tz). Cells were preincubated with
aminoguanidine 10 min prior to addition of WR1065. After addition of
WR1065, cells were further incubated for an additional 30 min. Then
H2O2 was added at 100 and 200 µM
(final concentration), and the plates were incubated for an
additional 24 h. Control cultures without any additive or with
only WR1065 were run in parallel. The assay was terminated by the
addition of cold trichloroacetic acid; sulforhodamine B staining
was performed, and absorbance was measured at 530 nm. Cell growth was
evaluated at each H2O2 concentration by using Equation 1,
Cell Cycle Analysis Using Flow Cytometry--
Nuclei of treated
cells were collected and stained with propidium iodate, using the cycle
TEST-PLUS DNA-staining kit, according to the manufacturer's
instructions (BD Biosciences). The DNA content of the stained nuclei
was measured on a FACSCalibur flow cytometer, and results were analyzed
using the CellQuest and ModFit LT2.0 softwares (BD Biosciences).
Protein Extraction--
Cells were washed twice with ice-cold
PBS and collected by scraping. Protein extracts were prepared as
described previously (61). Briefly, cells were lysed for 15 min on ice
in 100 µl (per million cells) of buffer A (20 mM HEPES
(pH 7.6), 20% glycerol, 10 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 1 mM DTT, 0.1%
Nonidet P-40), containing protease inhibitors (0.5 mM
phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, 2 µg/ml
aprotinin, 0.7 µg/ml pepstatin A, 1 mM sodium fluoride,
and 50 µM sodium orthovanadate). After centrifugation at
300 × g for 4 min, the supernatant was designated "cytoplasmic fraction" and stored at Western Blot, Immunoprecipitation, and Antibodies--
Equal
amounts of proteins (quantified by Bradford assay) were mixed with
Laemmli sample buffer, resolved on 10% SDS-polyacrylamide gel, and
transferred to polyvinylidene difluoride membranes (Roche Molecular
Biochemicals). Proteins were revealed by using an enhanced chemiluminescence detection system in accordance with the
manufacturer's instructions (ECL or ECL+, Amersham Biosciences). For
p53 detection, antibodies DO-7 (1:1000, DAKO, Glostrup, Denmark),
anti-phospho-Ser-15, -20, and -37 (all at 1:1000, Cell Signaling,
Beverly, MA), CM-1 (1:1000, Novocastra, Newcastle, UK), and
anti-phospho-Thr-81 (1:100) (51) were used. Although the reactivity of
the anti-phospho-Thr-81 antibody is weak, its specificity has been
fully characterized in a previous publication (51). Anti-phospho-Ser-73
c-Jun (1:1000) and c-Jun (1:1000) antibodies were from Cell Signaling.
Anti-actin monoclonal antibody (C-2, 250 ng/ml) was from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-Mdm2 monoclonal antibody (IF2,
1:1000), anti-JNK (clone 666, 1:1000), and anti-phospho-JNK (1:1000)
were obtained from Calbiochem, Pharmingen, and Cell Signaling,
respectively. Peroxidase-conjugated goat anti-mouse or anti-rabbit IgG
(250 ng/ml, Pierce) was used as secondary antibodies.
Immunoprecipitations were performed with 2 mg of whole cell extracts.
Pre-clearing was performed with 50 µl of IgG (whole molecule)-agarose
beads (Sigma) for 1 h at 4 °C to remove unspecific binding.
Precleared extracts were mixed with 1 µg of either anti-FLAG M2
antibody (Sigma), JNK monoclonal antibody (clone 666, Pharmingen), or
pAb 421 (Oncogene Science, Cambridge, MA) for 2 h at 4 °C. IgG-agarose beads were added for 1 h at 4 °C, and the mixture was centrifuged at 2500 rpm at 4 °C. Agarose beads were washed three
times, and pelleted beads were boiled in Laemmli Buffer, loaded on a
10% SDS-PAGE, and analyzed by Western blot.
SAPK/JNK Activity Assay--
The SAPK/JNK activity
was measured with a non-radioactive kit from Cell Signaling, according
to the manufacturer's instructions. Briefly, proteins lysates were
incubated with the GST-c-Jun fusion protein beads and washed to remove
non-specifically bound proteins. The kinase reaction was carried out in
the presence of cold ATP. c-Jun phosphorylations were measured by
Western blot using phospho-63 and phospho-73-c-Jun antibodies (Cell
Signaling) and chemiluminescent detection system. Densitometric
quantification of the signals was performed.
Electrophoretic Gel Mobility Shift Assay (EMSA)--
The
double-stranded p53 consensus binding sequence p53con
(5'-GGACATGCCCGGGCATGTCC-3') was end-labeled with ~3000
Ci/mmol [ Fluorescence-based Determination of Proteasome
Activity--
Proteasome activity was evaluated as described (62).
Briefly, cells were washed in PBS after treatment, and protein extracts were prepared in 50 mM Tris-HCl (pH 7.8), 20 mM
KCl, 5 mM MgOAc, 0.5 mM DTT. Then 50 µM Suc-LLVY-AMC (dissolved in 10% Me2SO) was incubated with 100 µg of protein extract in the same buffer
supplemented with 5 mM MgCl2, 5 mM
ATP in a total volume of 200 µl. After 1 h of incubation at
37 °C, the reaction was terminated by adding 200 µl of a solution
containing 0.1 M sodium borate (pH 9.0), in ethanol/water
(144:16). Degradation of the fluoropeptide Suc-VVLY-AMC was assayed by
fluorescence (Fluoroskan) at 390 nm for excitation and 460 nm for emission.
Semi-quantitative RT-PCR--
Total RNA was extracted from
treated and control MCF-7 cells using Trizol (Invitrogen), and 4 µg
of RNA were used for reverse transcription. The cDNA corresponding
to the TP53 gene was co-amplified with a cDNA from the
Statistical Evaluation--
For autoradiograms, densitometric
quantification was performed using a Bio-Rad imaging densitometer
GS-670 and Molecular Analyst software (Bio-Rad). The significance of
observed differences was evaluated using the two-tailed Student's
t test. Probabilities of p < 0.05 were
regarded as statistically significant.
Time Course of p53 Activation by WR1065--
We have shown
previously (22) that WR1065 induced the accumulation and activation of
wild-type p53 in cultured cells. Fig. 1
shows that WR1065 induced a rapid (detectable after 15 min, Fig.
1A) and long lasting (over 60 h, Fig. 1C)
accumulation of wild-type p53 in MCF-7 cells, which correlated with
enhanced DNA binding activity. As WR1065 is rapidly degraded by
copper-dependent amine oxidases in culture medium to form
toxic metabolites, all experiments were performed in the presence of
aminoguanidine (AG) at 4 mM, a concentration shown
previously to inhibit copper-dependent amine oxidases (22,
63). Fig. 1 shows that AG alone did not affect p53 levels (Western
blot, A and C) and activity (DNA binding activity, B and C). Similar results were obtained
in other cell lines expressing wild-type p53, including HCT116, A549,
and MRC-5 (data not shown). Fig. 1C compares the pattern of
p53 accumulation by WR1065 and by hydrogen peroxide, an inducer of DNA
strand breaks. These patterns were strikingly different, with a sharp
peak of p53 activation (4 h) followed by a return to basal level 8 h after treatment with hydrogen peroxide and a long, stable p53
response after treatment with WR1065. The increase in p53 levels after WR1065 treatment was not due to an increase in mRNA levels, as shown by semi-quantitative RT-PCR (Fig. 1D). Overall, these
results suggest that WR1065 induced p53 stabilization by a process that differs from the one activated by hydrogen peroxide.
Role of p53 in Cytoprotection by WR1065--
In a previous study
(22), we have shown that WR1065 protects cells against the cytotoxic
effects of H2O2 at concentrations sufficient to
induce p53 accumulation. We then evaluated whether p53 could modulate
the radioprotective effect of WR1065 in non-transformed murine
fibroblasts deficient or proficient for p53. Cells were irradiated at
15 gray in the presence or absence of WR1065 (1 mM + AG),
and cell cycle distribution was analyzed by flow cytometry (Fig.
2A). Cells with functional
wild-type p53 (3T3) were less sensitive to Accumulation of p53 in Response to WR1065 Results from
Stabilization of the Protein--
The accumulation of p53 after stress
results essentially from stabilization of the protein due to the escape
from proteasome degradation targeted by Mdm2 and/or JNK. To determine
whether the increase in p53 levels induced by WR1065 was due to protein stabilization, we evaluated the protein half-life in cells exposed to
WR1065 and cycloheximide (CHX, an inhibitor of protein synthesis). CHX
(20 µg/ml) was added to cells cultured for 2 h in presence or
absence of WR1065 (+AG). Fig. 3,
A and B, shows that, in the absence of WR1065,
60% of the p53 was degraded within 30 min after addition of CHX. With
WR1065, the half-life of the protein was at least doubled, with 50% of
the protein detectable after 60 min. AG alone had no effect. To
determine whether WR1065 had a direct effect on proteasome activity, we
monitored the degradation of the fluoropeptide Suc-LLVY-AMC in the
presence of ATP, as an indicator of the chymotrypsin-like activity of
the proteasome. Exposure of cells to the proteasome inhibitor
lactacystin resulted in 70% reduction of proteasome activity, whereas
treatment with WR1065 did not alter Suc-LLVY-AMC degradation (Fig.
3C). These data suggest that WR1065 does not induce a
general impairment of proteasome activity. Overall, these results
demonstrate that WR1065 induces accumulation of p53 by inhibiting its
degradation.
WR1065 Induces p53 by a DNA Damage-independent Pathway--
The
process of p53 activation after DNA damage involves phosphorylation of
p53 by pathways involving large proteins with PI 3-kinase domains, such
as ATM or ATR. To explore the role of PI 3-kinase activities in the
induction of p53 by WR1065, we used LY294002, an inhibitor of most PI
3-kinases. Fig. 4A shows that addition of LY294002 to cells exposed to WR1065 did not prevent the
accumulation of p53 nor the activation of its DNA binding capacity. In
contrast, LY294002 prevented the accumulation and activation of p53 in
response to hydrogen peroxide. As a control of the inhibition of PI
3-kinase activity, we verified that LY294002 prevented the
phosphorylation of the PI 3-kinase substrate Akt after stimulation with
H2O2 (data not shown). Overall, these results suggest that kinases with PI 3-kinase domains were not involved in the
pathway of induction of p53 by WR1065. These data are in agreement with
previous data showing that WR1065 is capable of inducing p53 in
lymphoblastoid cells derived from Ataxia-Telangiectasia patients, which
are deficient for p53 response to strand break damage (76).
We next examined the pattern of p53 phosphorylation at three of the
well defined regulatory serines in the N terminus of p53. One of the
hallmarks of p53 activation by DNA damage is the phosphorylation of
N-terminal residues including Ser-15, Ser-20, and Ser-37. In Fig.
4B, we show that treatment of MCF-7 cells with WR1065 did not induce phosphorylation of any of these serines. Moreover, in Fig.
4C, we show that WR1065 decreased by 90% the levels of Ser-15 and Ser-20 phosphorylation in response to hydrogen peroxide. However, the levels of p53 accumulation and of DNA binding activity were not reduced, indicating that p53 accumulated despite the absence
of phosphorylation on Ser-15 and -20 and that the effect of WR1065 was
dominant over the one of hydrogen peroxide.
Involvement of JNK in the Pathway of p53 Activation by
WR1065--
The results above indicated that activation of p53 by
WR1065 may occur by an alternative pathway, different from the one
elicited in response to DNA damage. The fact that p53 activation by
WR1065 was observed in both p14arf-deficient (MCF-7, A549) and
-proficient (HCT116, MRC-5) cells suggested that p14arf was not
an important effector in this pathway of induction (64, 65). These
observations led us to focus on the possible role of the JNK. This
kinase plays important roles in response to many types of stress and
has been shown to target p53 ubiquitination and degradation in an
Mdm2-independent pathway (see Introduction). Fig.
5A shows that WR1065 induced a
rapid increase in the levels of phospho-JNK 1 and -2 in MCF-7,
indicative of an increase of JNK activity (also demonstrated by
in vitro phosphorylation of c-Jun in Ser-73 (see Fig.
6). Co-immunoprecipitation experiments showed that JNK formed stable complexes with p53 in unstimulated MCF-7
cells but that activation by WR1065 resulted in the dissociation of
these complexes, as detected by a reduction of 50% of p53
co-precipitating with JNK (Fig. 5B). Fig. 5C
shows that WR1065 induced the phosphorylation of p53 at Thr-81, the
known site of p53 phosphorylation by JNK (51).
To assess the functional significance of JNK in the activation of p53
by WR1065, we stably transfected MCF-7 cells with a dominant negative
form of JNK, carrying two point mutations within the active kinase site
(JNK-APF) (58). Compared with cells transfected with the vector alone,
cells expressing JNK-APF showed decreased levels of Ser-73 c-Jun
phosphorylation (Fig. 6A), of JNK activity (Fig.
6B), and of JNK phosphorylation (Fig. 6C,
middle panel) after exposure to WR1065. This decrease in JNK
activity was accompanied by a reduction of 50% in the level of p53
protein accumulation (Fig. 6C, lower panel). Fig.
6D shows that, in contrast with levels of p53-JNK complexes,
the levels of p53 in complex with JNK-APF did not decrease after
exposure to WR1065. Taken together, these results show that JNK was
rapidly activated in response to WR1065 and that a dominant negative
form of JNK at least partially blocked the accumulation of p53 induced
by WR1065. These observations strongly implicate JNK as an effector in
a non-genotoxic pathway of regulation of p53 stability in response to
WR1065. However, it should be noted that the dominant negative JNK-APF
did not totally abrogate p53 induction. This observation may reflect a residual activity of JNK (as shown in Fig. 6B).
Alternatively, it is possible that others mechanisms than JNK also
contribute to p53 induction by WR1065.
Effect of WR1065 on Interactions between p53 and Mdm2--
The
long lasting accumulation of p53 induced by WR1065 is in sharp contrast
with the transient response of p53 to DNA damage (Fig. 1). Moreover,
activation by WR1065 appeared to be dominant over the one induced by
DNA damage, which is Mdm2-dependent. This observation is
consistent with the observation that phosphorylation of p53 by JNK
abrogated the association of p53 with Mdm2 (66). Fig.
7A shows that activation of
p53 by WR1065 induced an increase in Mdm2 protein expression, with a
maximum in the levels of the two proteins after 16 h. However,
despite these high levels of both proteins, co-immunoprecipitation
experiments demonstrated that after 16 h, p53-Mdm2
complexes were decreased in cells exposed to WR1065 as compared with
untreated cells. This decrease was not observed in cells expressing
JNK-APF (Fig. 7B). These results indicate that
JNK-dependent activation induces a release of p53 from
Mdm2-dependent degradation. This mechanism may account for the fact that, after exposure to WR1065, p53 cannot undergo rapid and
transient accumulation in response to DNA damage, thus preventing the
acute, p53-dependent pro-apoptotic effects of cytotoxic
drugs.
DNA damage, either by direct or indirect mechanisms, is a common
denominator of many stimuli inducing p53 (67). Here we present evidence
that WR1065, the active metabolite of the chemoprotective aminothiol
amifostine, activates p53 by an alternative pathway that is independent
of the one activated by DNA damage. Our results show that WR1065
induces p53 accumulation by post-translational stabilization and escape
from proteasome-mediated degradation, through activation of the JNK pathway.
Induction of p53 in response to WR1065 is not accompanied by the
post-translational changes generally considered as "signatures" of
DNA damage, such as phosphorylation of serines 15, 20, and 37. Inhibitors of PI 3-kinase activities failed to inhibit p53 induction by
WR1065, although they significantly reduced the activation of p53 by
hydrogen peroxide. These results are in agreement with our previous
study showing the absence of detectable DNA strand break damage and of
overall oxidative stress in cells exposed to WR1065 (76). The
stress-activated kinase JNK/SAPK is a good candidate as an effector of
p53 induction in alternative pathways to DNA damage. This kinase has
been shown to exert multiple regulatory functions in response to
various forms of pro- and anti-oxidant stress (68). A critical aspect
of these regulations is the ability of the inactive kinase to form
stable complexes with unphosphorylated proteins such as c-Jun, ATF2,
and p53, to catalyze their ubiquitination and degradation in
normal-growing cells (52, 53). JNK binds p53 at residues 97-116, in a
domain distinct from the Mdm2-binding site. We report here that WR1065
activates JNK, resulting in the phosphorylation of p53 at threonine 81 and in the decrease by over 50% the amount of p53 complexing with JNK.
Furthermore, stable transfection of a dominant negative form of JNK 1 (JNK/APF) prevents the phosphorylation of JNK 1 and -2 and reduces by
50% the accumulation of p53 in response to WR1065. These results are
in agreement with those of Fuchs et al. (66) showing that
JNK targets p53 degradation and indicate that WR1065 stabilizes p53
through a pathway that involves the activation of JNK and the
disruption of its capacity to form complexes with p53.
The biochemical mechanisms responsible for JNK activation by WR1065 are
unknown. One possibility is that JNK may become activated as a part of
a pathway of response to anti-oxidant stress induced by WR1065 (69,
70). The existence of such an anti-oxidant stress is supported by our
previous observation that levels of reduced GSH increase by about
2-fold in MCF-7 cells exposed to WR1065. This anti-oxidant pathway may
share common effectors with response to hypoxia (71, 72). JNK has been
shown to be activated in hypoxic conditions, but so far it is not clear
whether JNK is involved in the pathway of p53 induction in response to
hypoxia. A second hypothesis implicates a role for the polyamine moiety of WR1065. Several short synthetic polyamines have been shown to
stimulate p53 accumulation (73). Moreover, disruption of polyamine
metabolism through either overexpression or repression of
S-adenosylmethionine decarboxylase, the main regulatory
enzyme in the biosynthesis of higher polyamines, converge on the
activation of JNK 1 and phosphorylation of c-Jun at serine 73 (74).
However, it should be noted that many signals that induce DNA damage
can also activate the JNK pathway, making it very difficult to unravel the respective contributions of genotoxic and non-genotoxic signals in
the response to any particular stress agent. A typical example is
UV-induced damage, which triggers the activation of both
ATM/ATR-dependent pathways and of JNK (47, 75). In this
respect, response to WR1065 may represent a special case, as this drug
is apparently capable of selectively activating the JNK pathway,
without inducing DNA damage.
WR1065 has pleiotropic effects on many signaling pathways in addition
to p53, and it is likely that several of these pathways can contribute
to cytoprotection. Cytoprotection by WR1065 is generally considered as
a consequence of scavenging, which reduces the level of DNA damage by
free radicals and is apparently not dependent upon the p53 status of
the cell (14). However, our results suggest that the p53 status may
influence the response of WR1065-exposed cells to DNA-damaging agents.
Two mechanisms may account for a possible effect of p53 in
WR1065-mediated cytoprotection. First, WR1065 may preferentially elicit
a cytostatic, rather than a pro-apoptotic, p53-dependent
response. Indeed, we did not observe a significant activation of
pro-apoptotic target genes such as Bax-1 or PIG-3
in response to WR1065 (21). Second, WR1065 may neutralize the capacity
of Mdm2 to control p53 stability and therefore prevent the acute,
cytotoxic activation of p53 induced by DNA-damaging agents. This
hypothesis is supported by our results on the effect of WR1065 on
Mdm2-p53 interactions, showing a decrease in complex formation even in
the presence of high levels of both proteins. This mechanism would
result in efficient protection of normal cells against drug-induced
apoptosis. This hypothesis is in agreement with the results of Fuchs
et al. (66) who showed that Mdm2 dissociation is likely to
serve as the primary mechanism of p53 stabilization by the JNK pathway
(66). Moreover, it also explains why wild-type p53 could not be
activated in response to Despite the role for p53 demonstrated here, other factors in addition
to p53 need to be considered to fully explain cytoprotection by WR1065.
The demonstration that this drug activates the JNK/SAPK stress
signaling pathway raises the possibility that it may regulate several
other transcription factors, including in particular AP1 and NF-
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiated CHO-AA8 cells (15). In
addition, WR1065 stimulates the proliferation of bone marrow
progenitors in vitro, inhibits DNA topoisomerase II
(16,
17), and represses the transcription of genes such as c-myc
(18) and thymidine kinase (19). However, the exact contribution
of these activities to the selective protection of normal cells is not understood.
-catenin/c-myc pathways,
acting through p14arf), and non-genotoxic stress
(ribonucleotide depletion and hypoxia) (21, 24). Target genes of p53
include, among others, regulators of cell cycle progression in
G1 and G2 (p21waf-1 and
14-3-3
), activators of apoptosis (Bax-1,
Aip-1, APO-1/Fas, and Apaf-1),
and genes involved in redox metabolism (PIG-3, COX-2, and
NOS-2) (see review in Refs. 21 and 25-27).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
where Ti represents the absorbance of the treated
cells and C the absorbance of the untreated (control) cells
(59, 60). For WR1065, C represents the absorbance of the
WR1065-treated cells.
(Eq. 1)
80 °C. The pellets were further lysed for 30 min on ice in 50 µl (per million cells) of buffer B (same as buffer A, with 0.5 M instead of 10 mM NaCl) and centrifuged for 15 min at 15,000 × g and 4 °C. The supernatant was designated "nuclear
fraction" and stored at
80 °C. Total protein extracts were
performed in buffer B, supplemented as described above.
-32P]ATP (Amersham Biosciences) as described
(61). Binding reactions contained 32P-labeled
double-stranded oligonucleotide (0.5 ng), sonicated herring sperm
DNA (2 µg; Promega, Madison, WI), bovine serum albumin (5 µg), DTT (4 mM), and nuclear protein extracts (10 µg).
Reactions were adjusted to a final volume of 30 µl with buffer A. All
reactions were carried out in the presence of the monoclonal antibody
pAb 421 (100 ng/reaction). This antibody stabilizes p53-DNA complexes and is required to detect stable binding of p53 to short
oligonucleotides in cellular extracts. No band was detected in the
absence of pAb 421 (61). Binding reactions were incubated for 30 min at
20 °C. A 15-µl aliquot of each reaction was loaded onto a 4%
non-denaturing polyacrylamide gel and run in TBE buffer at 120 V for
2-3 h. Gels were fixed, dried, and exposed to Kodak x-ray films at
80 °C for 12-48 h. The specificity of the binding was controlled
by competition experiments using cold oligonucleotides and using mutant
DNA consensus sequence (61).
-actin gene. The annealing temperature of co-amplification was 56 °C (25 cycles). Co-amplified PCR products were analyzed on a 2% agarose gel with ethidium bromide.
Fluorescence was quantified using a FluorSMax apparatus (Bio-Rad). The
primers used are as follows:
-actin,
5'-GTGGGCCGCCCTAGGCACCA-3'/5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'; TP53,
5'-AACCTACCAGGGCAGCTACG-3'/5'-TTCCTCTGTGCGCCGGTCTC-3'.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
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Fig. 1.
Induction of p53 by WR1065 is
rapid and long lasting. A, MCF-7 cells were pretreated
with aminoguanidine (4 mM) and then exposed to WR1065 (1 mM) for the indicated times. Total protein extracts were
used to determine the p53 level by Western blot using DO-7 antibody.
B, analysis of p53 DNA binding activity was performed by
EMSA, using the p53 consensus binding oligonucleotide
p53con labeled with 32P. These experiments were
performed in the presence of the antibody pAb 421, which stabilizes and
supershifts p53-DNA complexes (see "Experimental Procedures"). The
right panel shows that no specific p53 binding was detected
in the absence of pAb421. The asterisk indicates the free
probe; n.s. (nonspecific band) corresponds to an already
described shifted band that does not correspond to p53 (61).
Black arrows indicate the p53-DNA-antibodies complexes.
C, MCF-7 cells were treated with
H2O2 (150 µM) or WR1065 (1 mM + AG) at different times. Analysis of the p53 DNA
binding activity and of the protein level of p53 were performed as in
A and B. Only the portion of the autoradiogram
with the specific p53-pAb 421-DNA complexes is shown. D,
MCF-7 cells were pretreated with AG (4 mM) and then exposed
for 14 h to increasing doses of WR1065. P53 mRNA levels were
determined by semi-quantitative RT-PCR.
-irradiation than those
with disrupted p53 (10.1), as detected by a reduced sub-G1
peak, corresponding to apoptotic cells. In the presence of WR1065, cell
death induced by irradiation was strongly reduced in 3T3 cells (from 18 to 4%) but not in 10.1 cells (from 43 to 37%). Furthermore, WR1065
had a marked effect in G1 phase distribution in irradiated
3T3 cells, which is increased in comparison to irradiated 3T3 cells
without WR1065. This effect was not observed in 10.1 cells. These
results clearly demonstrate that, in these experimental conditions,
WR1065 exerts a radioprotective effect mediated through p53. To
determine whether WR1065 can modulate cell proliferation in a
p53-dependent manner after exposure to H2O2, we compared the effects of WR1065 in two
isogenic cell lines derived from MCF-7, MN1 (p53 competent), and MDD2
(p53 defective) cells. Fig. 2B shows that, in both cell
lines, H2O2 had a strong inhibitory effect in
cell growth rate. However, in the presence of WR1065, this effect was
reduced in MN1 but not in MDD2 cells, indicating a protective effect in
the p53-proficient cell line. This observation is compatible with our
effect reported previously (22) of WR1065 on cell cycle arrest in these
cells. Overall, these results clearly show that p53 play a role in the
cytoprotective effects of WR1065 in vitro.
View larger version (25K):
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Fig. 2.
Role of p53 in the cytoprotective effects of
WR1065. A, non-transformed murine 3T3 (p53 competent)
and 10.1 (p53 defective) cells were treated by WR1065 (1 mM) in the presence or absence of -irradiation (15 gray). AG was included in all experiments. Cells were harvested at
48 h, and cell cycle distribution was analyzed by flow cytometry.
Results show the percentage of cells in each phase of the cell cycle,
as well as the percentage of cells with sub-G1 DNA content
(containing fragmented, apoptotic DNA). Data were from a representative
experiment. B, MN1 and MDD2 were treated by WR1065 (1 mM + AG) and then exposed to H2O2
(100 or 200 µM) for 24 h. The viability of cells was
determined by the sulforhodamine assay (see "Experimental
Procedures"). Results are expressed in % of growth rate. In each
graphs, bar represents the average ± S.D. of at least
three independent experiments.
View larger version (24K):
[in a new window]
Fig. 3.
Activation of p53 by WR1065 is due to
stabilization of the protein. A, level of p53 in MCF-7
cells treated or not (C) with WR1065 (W) (1 mM + AG) for 2 h. CHX (20 µg/ml) was then
added, and cells were harvested at 30, 60, and 90 min after treatment.
Western blot was performed on protein extracts using DO-7.
B, p53 levels from A were quantitated by
densitometry and plotted. C, the rate of Suc-LLVY-AMC
degradation was measured in MCF-7 cells after exposure to lactacystin
(L), AG, or WR1065 + AG. Fluorescence was determined as
described under "Experimental Procedures." Results were expressed
as a percentage of the fluorescence in non-treated (control) cells.
Each bar is the average ± S.D. of at least three
independent experiments.
View larger version (44K):
[in a new window]
Fig. 4.
Induction of p53 by WR1065 is not prevented
by the PI 3-kinase inhibitor LY294002 and does not involve
phosphorylation of p53 on Ser-15, -20, and -37. A,
MCF-7 cells were pretreated with LY294002 (10 µM) for
1 h and then exposed to H2O2 (150 µM), AG (4 mM), WR1065 (1 mM + AG) for 2 h. Total protein extracts were used to determine p53
level by Western blot using DO-7. P53 DNA binding activity was
determined as described in Fig. 1. B, nuclear protein
extracts were prepared at different times after treatment with WR1065
(1 mM + AG). Western blot was performed using an antibody
to phospho-Ser-15, phospho-Ser-20, or phospho-Ser-37. As positive
controls, p53 was induced by H2O2 (Ser-15),
-irradiation (Ser-20), or methyl methanesulfate (Ser-37). These
positive controls were selected in order to maximize the level of
detectable phosphorylation of each respective serine. Overall levels of
p53 (detected with DO-7) are shown as positive loading. C,
MCF-7 cells were treated with WR1065 (1 mM + AG) at times
indicated. H2O2 (150 µM) was
added after 30 min. Nuclear protein extracts were used to determine
levels of p53 by Western blot with DO-7 and levels of phospho-Ser-15
and phospho-Ser-20 antibodies. Analysis of the p53 DNA binding activity
was performed by EMSA as described in Fig. 1.
View larger version (28K):
[in a new window]
Fig. 5.
WR1065 activates JNK resulting in the
phosphorylation of p53 at threonine 81. A, MCF-7 cells
were treated or not (C) with WR1065 (1 mM + AG)
for the indicated times. Total protein extracts were used to
determine the JNK 1/2 and phospho-JNK 1/2 levels by Western blot.
B, co-immunoprecipitation was performed on total protein
extracts from WR1065-treated and control MCF-7 cells (C).
JNK was immunoprecipitated (IP) from each cell lysate after
2 h of treatment with WR1065 (WR) (1 mM + AG), followed by Western blot (IB) with the polyclonal p53
antibody CM-1 (top). The same cell lysates were then
analyzed by Western blot to determine JNK level (middle) and
p53 level (bottom). C, immunoprecipitation of p53
was performed on total protein extracts from WR1065 treated (2 h, 1 mM) and untreated MCF-7 cells. Treatment with UV (45J/m2)
was used as control for phosphorylation of Thr81. Immunoprecipitated
p53 was then blotted by the phospho-Thr-81 antibody (top).
The same cell lysates were then analyzed by Western blot to determine
p53 level (middle) and tubulin level
(bottom).
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[in a new window]
Fig. 6.
p53 activation by WR1065 is regulated by the
JNK pathway. A, MCF-7 cells stably transfected with a
dominant negative JNK (JNK-APF) or with the empty vector were exposed
to WR1065 (1 mM + AG). Total protein extracts were
used to determine the levels of c-Jun and phospho-Ser-73 c-Jun.
B, MCF-7 cells stably transfected with JNK-APF or empty
vector were exposed to WR1065 (1 mM + AG). Proteins lysates
were incubated with GST-c-Jun (see "Experimental
Procedures"), and JNK activity was measured by densitometric
quantification of Western blot using phospho-63 and phospho-73 c-Jun
antibodies. In both panels A and B, C
corresponds to the control cells. C, MCF-7 cells stably
transfected with JNK-APF or empty vector were exposed to WR1065 (1 mM + AG) for 2 h. Total protein extracts were used to
determine the levels of JNK 1/2, phospho-JNK 1/2, and p53. Variation in
levels of p53 were evaluated with respect to actin used as an internal
standard. D, JNK-APF bearing the FLAG epitope stably
transfected into MCF-7 cells was immunoprecipitated (IP) by
anti-FLAG antibody. After 2 h of WR1065 treatment,
immunoprecipitates were analyzed by Western blot (IB) with
an anti-JNK antibody (top) and with an anti-p53 antibody
(CM-1) (middle). No bands were detected after FLAG
immunoprecipitation in cells transfected with empty vector.
Bottom, the level of p53 (DO-7) expressed in whole cell
extracts.
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[in a new window]
Fig. 7.
WR1065 prevents the formation of p53-mdm2
complexes. A, MCF-7 cells were treated with WR1065 (1 mM + AG) for the indicated times. Total protein extracts
were used to determine the Mdm2 and p53 levels by Western blot
(WB). B, MCF-7 cells stably transfected with a
dominant negative JNK (JNK-APF) or with the empty vector were treated
with WR1065 (1 mM + AG) for 16 h and then exposed to
lactacystin (10 µM) for 4 h. Protein extracts were
first immunoprecipitated (IP) with antibody to p53 (pAb
421), and immunoprecipitates were analyzed by Western blot
(IB) with an anti-Mdm2 antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-irradiation in MCF-7 cells exposed to
WR1065 (22).
B,
with possible implication on the mechanisms of cytoprotection (54).
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ACKNOWLEDGEMENTS |
---|
We thank W. Oster (United States Biochemical) for providing amifostine, B. Derijard (Nice) for the pcDNA3-JNK-APF plasmid, and M. Oren for the kind gift of MN1 and MDD2 cells.
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FOOTNOTES |
---|
* This work was supported in part by grants from the French Ligue Contre le Cancer (to O. P. from the Loire Committee and to S. N. from the National Committee) and NCI Grant CA78419 from the National Institutes of Health (to A. B. and Z. R.).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.
§ Present address: Laboratoire des Facteurs de Croissance, INSERM EPI 0113, Université de Bordeaux 1, 33405 Talence, France.
Recipient of an IARC Special Training Award.
** To whom correspondence should be addressed: Unit of Molecular Carcinogenesis, International Agency for Research on Cancer, 69372 Lyon Cedex 08, France. Tel.: 33-4-72-73-85-32; Fax: 33-4-72-73-83-22; E-mail: hainaut@iarc.fr.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M207396200
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ABBREVIATIONS |
---|
The abbreviations used are: JNK, c-Jun N-terminal kinase; PI 3-kinase, phosphatidylinositol 3-kinase; SAPK, stress-activated protein kinase; pAb, polyclonal antibody; EMSA, electrophoretic gel mobility shift assay; PBS, phosphate-buffered saline; DTT, dithiothreitol; RT, reverse transcriptase; AG, aminoguanidine; CHX, cycloheximide.
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REFERENCES |
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