The Cytoprotective Aminothiol WR1065 Activates p53 through a Non-genotoxic Signaling Pathway Involving c-Jun N-terminal Kinase*

Olivier PluquetDagger , Sophie NorthDagger §, Anindita Bhoumik, Konstantinos DimasDagger ||, Ze'ev Ronai, and Pierre HainautDagger **

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma -irradiated CHO-AA8 cells (15). In addition, WR1065 stimulates the proliferation of bone marrow progenitors in vitro, inhibits DNA topoisomerase II alpha  (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.

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 beta -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 14-3-3sigma ), 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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 -20 °C.

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,


(Ti−Tz)/(C−Tz)×100 (Eq. 1)
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.

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 -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.

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 [gamma -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).

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 beta -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: beta -actin, 5'-GTGGGCCGCCCTAGGCACCA-3'/5'-CGGTTGGCCTTAGGGTTCAGGGGGG-3'; TP53, 5'-AACCTACCAGGGCAGCTACG-3'/5'-TTCCTCTGTGCGCCGGTCTC-3'.

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.

    RESULTS
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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.


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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.

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 gamma -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.


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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 gamma -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.

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.


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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.

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).


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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), gamma -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.

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).


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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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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.

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.


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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

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 gamma -irradiation in MCF-7 cells exposed to WR1065 (22).

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-kappa B, with possible implication on the mechanisms of cytoprotection (54).

    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.

    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

    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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