The Cytoprotective Aminothiol WR1065 Activates p53 through a Non-genotoxic Signaling Pathway Involving c-Jun N-terminal Kinase
2003; Elsevier BV; Volume: 278; Issue: 14 Linguagem: Inglês
10.1074/jbc.m207396200
ISSN1083-351X
AutoresOlivier Pluquet, Sophie North, Anindita Bhoumik, Konstantinos Dimas, Ze’ev A. Ronai, Pierre Hainaut,
Tópico(s)Cancer Mechanisms and Therapy
ResumoWR1065 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 507 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. 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 507 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. c-Jun N-terminal kinase phosphatidylinositol 3-kinase stress-activated protein kinase polyclonal antibody electrophoretic gel mobility shift assay phosphate-buffered saline dithiothreitol reverse transcriptase aminoguanidine cycloheximide 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 (1Calabro-Jones P.M. Fahey R.C. Smoluk G.D. Ward J.F. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1985; 47: 23-27Google Scholar). WR1065 penetrates into cells by both passive and active mechanisms (2Calabro-Jones P.M. Aguilera J.A. Ward J.F. Smoluk G.D. Fahey R.C. Cancer Res. 1988; 48: 3634-3640Google Scholar, 3Yuhas J.M. Cancer Res. 1980; 40: 1519-1524Google Scholar) and protects cells against toxicity by radiation, alkylating agents, and platinum compounds in both animals and humans (4Douay L. Hu C. Giarratana M.C. Gorin N.C. Eur. J. Cancer. 1995; 31A Suppl. 1: 14-16Google Scholar, 5List A.F. Heaton R. Glinsmann-Gibson B. Capizzi R.L. Semin. Oncol. 1996; 23: 58-63Google Scholar, 6Travis E.L. Fang M.Z. Basic I. Int. J. Radiat. Oncol. Biol. Phys. 1988; 15: 377-382Google Scholar, 7Treskes M. Nijtmans L.G. Fichtinger-Schepman A.M. van der Vijgh W.J. Biochem. Pharmacol. 1992; 43: 1013-1019Google Scholar). WR1065 is currently used as a cytoprotector in patients treated with cisplatin for ovarian cancer and, in some protocols, for colorectal or lung cancers (8Kemp G. Rose P. Lurain J. Berman M. Manetta A. Roullet B. Homesley H. Belpomme D. Glick J. J. Clin. Oncol. 1996; 14: 2101-2112Google Scholar, 9Poplin E.A. LoRusso P. Lokich J.J. Gullo J.J. Leming P.D. Schulz J.J. Veach S.R. McCulloch W. Baker L. Schein P. Cancer Chemother. Pharmacol. 1994; 33: 415-419Google Scholar, 10Schiller J.H. Storer B. Berlin J. Wittenkeller J. Larson M. Pharo L. Larson M. Berry W. J. Clin. Oncol. 1996; 14: 1913-1921Google Scholar). The administration of WR1065 in vivoprotects normal cells without impairing the efficacy of radio- and chemotherapy on tumor cells (11Capizzi R.L. Eur. J. Cancer. 1996; 32 Suppl. 4: 5-16Google Scholar, 12Treskes M. Boven E. Holwerda U. Pinedo H.M. van der Vijgh W.J. Cancer Res. 1992; 52: 2257-2260Google Scholar, 13Treskes M. Boven E. van de Loosdrecht A.A. Wijffels J.F. Cloos J. Peters G.J. Pinedo H.M. van der Vijgh W.J. Eur. J. Cancer. 1994; 30: 183-187Google Scholar). WR1065 acts primarily as a free radical scavenger with anti-clastogenic and anti-mutagenic effectsin vitro as well as in γ-irradiated CHO-AA8 cells (15Grdina D.J. Shigematsu N. Dale P. Newton G.L. Aguilera J.A. Fahey R.C. Carcinogenesis. 1995; 16: 767-774Google Scholar). In addition, WR1065 stimulates the proliferation of bone marrow progenitors in vitro, inhibits DNA topoisomerase II α (16Murley J.S. Constantinou A. Kamath N.S. Grdina D.J. Cell Proliferation. 1997; 30: 283-294Google Scholar,17Snyder R.D. Grdina D.J. Cancer Res. 2000; 60: 1186-1188Google Scholar), and represses the transcription of genes such as c-myc(18Liu S.C. Murley J.S. Woloschak G. Grdina D.J. Carcinogenesis. 1997; 18: 2457-2459Google Scholar) and thymidine kinase (19Woloschak G.E. Paunesku T. Chang-Liu C.M. Grdina D.J. Cancer Res. 1995; 55: 4788-4792Google Scholar). However, the exact contribution of these activities to the selective protection of normal cells is not understood. We have shown recently (20Maurici D. Monti P. Campomenosi P. North S. Frebourg T. Fronza G. Hainaut P. Oncogene. 2001; 20: 3533-3540Google Scholar, 21North S. Pluquet O. Maurici D. El Ghissassi F. Hainaut P. Mol. Carcinog. 2002; 33: 181-188Google Scholar, 22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar) 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 (22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar). p53 acts as a transcription regulator to arrest proliferation or to induce cell death in response to multiple types of stresses (23Levine A.J. Cell. 1997; 88: 323-331Google Scholar). 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 ॆ-catenin/c-myc pathways, acting through p14arf), and non-genotoxic stress (ribonucleotide depletion and hypoxia) (21North S. Pluquet O. Maurici D. El Ghissassi F. Hainaut P. Mol. Carcinog. 2002; 33: 181-188Google Scholar, 24Pluquet O. Hainaut P. Cancer Lett. 2001; 174: 1-15Google Scholar). Target genes of p53 include, among others, regulators of cell cycle progression in G1 and G2 (p21waf-1 and14-3-3ς), activators of apoptosis (Bax-1,Aip-1, APO-1/Fas, and Apaf-1), and genes involved in redox metabolism (PIG-3, COX-2, andNOS-2) (see review in Refs. 21North S. Pluquet O. Maurici D. El Ghissassi F. Hainaut P. Mol. Carcinog. 2002; 33: 181-188Google Scholar and 25Balint E.E. Vousden K.H. Br. J. Cancer. 2001; 85: 1813-1823Google Scholar, 26Bargonetti J. Manfredi J.J. Curr. Opin. Oncol. 2002; 14: 86-91Google Scholar, 27Hainaut P. Hollstein M. Adv. Cancer Res. 2000; 77: 81-137Google Scholar). 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 (28Fuchs S.Y. Adler V. Buschmann T. Wu X. Ronai Z. Oncogene. 1998; 17: 2543-2547Google Scholar, 29Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Google Scholar, 30Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Google Scholar, 31Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Google Scholar). 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. 24Pluquet O. Hainaut P. Cancer Lett. 2001; 174: 1-15Google Scholar, 26Bargonetti J. Manfredi J.J. Curr. Opin. Oncol. 2002; 14: 86-91Google Scholar, and 32Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Google Scholar, 33Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Google Scholar, 34Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Google Scholar) include phosphorylations at Ser-15, -20, -33, -37, and -392 (32Appella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Google Scholar, 33Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Google Scholar, 35Takenaka I. Morin F. Seizinger B.R. Kley N. J. Biol. Chem. 1995; 270: 5405-5411Google Scholar, 36Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Google Scholar, 37Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref Google Scholar), acetylation of lysine residues in the C terminus by co-activators of transcription such as p300/CBP or PCAF (38Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Google Scholar, 39Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Google Scholar), as well as sumoylation at Lys-386 (40Gostissa M. Hengstermann A. Fogal V. Sandy P. Schwarz S.E. Scheffner M. Del Sal G. EMBO J. 1999; 18: 6462-6471Google Scholar, 41Rodriguez M.S. Desterro J.M. Lain S. Midgley C.A. Lane D.P. Hay R.T. EMBO J. 1999; 18: 6455-6461Google Scholar). 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 (36Bulavin D.V. Saito S. Hollander M.C. Sakaguchi K. Anderson C.W. Appella E. Fornace Jr., A.J. EMBO J. 1999; 18: 6845-6854Google Scholar,42Oda K. Arakawa H. Tanaka T. Matsuda K. Tanikawa C. Mori T. Nishimori H. Tamai K. Tokino T. Nakamura Y. Taya Y. Cell. 2000; 102: 849-862Google Scholar). Transduction of DNA-damage signals involves kinases of the PI 3-kinase superfamily (ATM, ATR, and DNA-PK) (43Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Google Scholar, 44Smith G.C. Jackson S.P. Genes Dev. 1999; 13: 916-934Google Scholar), the cell cycle kinases Chk1 and Chk2 (45Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref Google Scholar, 46Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Google Scholar), and kinases of the mitogen-activated protein kinase/SAPK family including p38 and JNK (47Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Google Scholar, 48Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Google Scholar). 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 (37Shieh S.Y. Ahn J. Tamai K. Taya Y. Prives C. Genes Dev. 2000; 14: 289-300Crossref Google Scholar, 49Banin S. Moyal L. Shieh S. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Google Scholar). These serines are located within the domain of interaction with Mdm2, and their phosphorylations allow p53 to escape Mdm2-mediated degradation (50Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Google Scholar). Escape from degradation can also occur through the binding of Mdm2 by p14arf, the alternative product of the CDKN2a locus (24Pluquet O. Hainaut P. Cancer Lett. 2001; 174: 1-15Google Scholar, 25Balint E.E. Vousden K.H. Br. J. Cancer. 2001; 85: 1813-1823Google Scholar). 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 (29Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Google Scholar). After kinase activation, this complex dissociates, and active JNK may further participate in p53 induction by phosphorylation of Thr-81 (51Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Google Scholar). 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 (52Fuchs S.Y. Dolan L. Davis R.J. Ronai Z. Oncogene. 1996; 13: 1531-1535Google Scholar, 53Fuchs S.Y. Xie B. Adler V. Fried V.A. Davis R.J. Ronai Z. J. Biol. Chem. 1997; 272: 32163-32168Google Scholar). 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 (47Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Google Scholar). 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. (54Shen H. Chen Z.J. Zilfou J.T. Hopper E. Murphy M. Tew K.D. J. Pharmacol. Exp. Ther. 2001; 297: 1067-1073Google Scholar) 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. The breast carcinoma cell line MCF-7 (expressing wild-type functional p53) and MN1 and MDD2 cells (55Bacus S.S. Yarden Y. Oren M. Chin D.M. Lyass L. Zelnick C.R. Kazarov A. Toyofuku W. Gray-Bablin J. Beerli R.R. Hynes N.E. Nikiforov M. Haffner R. Gudkov A. Keyomarsi K. Oncogene. 1996; 12: 2535-2547Google Scholar,56Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Google Scholar) derived from MCF-7 cells, mouse 3T3, and 10.1 fibroblasts (57Harvey D.M. Levine A.J. Genes Dev. 1991; 5: 2375-2385Google Scholar) were cultured in Dulbecco's modified Eagle's medium (Invitrogen). Cells were maintained at 37 °C with 107 fetal calf serum (PAA, Linz, Austria), 2 mm l-glutamine and antibiotics, 107 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 (58Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Google Scholar). Cells were selected in 0.4 mg/ml G418. Cells were treated with different drugs at 50–657 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 ॖm. 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. Cells were seeded into 96-well microtiter plates in 100 ॖl at a density of 1·104 cells/well. After cell inoculation, the microtiter plates were incubated at 37 °C with 107 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 (59Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1107-1112Google Scholar), 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 ॖm(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)×100Equation 1 where Ti represents the absorbance of the treated cells and C the absorbance of the untreated (control) cells (59Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1107-1112Google Scholar, 60Rubinstein L.V. Shoemaker R.H. Paull K.D. Simon R.M. Tosini S. Skehan P. Scudiero D.A. Monks A. Boyd M.R. J. Natl. Cancer Inst. 1990; 82: 1113-1118Google Scholar). For WR1065, C represents the absorbance of the WR1065-treated cells. 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). Cells were washed twice with ice-cold PBS and collected by scraping. Protein extracts were prepared as described previously (61Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Google Scholar). Briefly, cells were lysed for 15 min on ice in 100 ॖl (per million cells) of buffer A (20 mm HEPES (pH 7.6), 207 glycerol, 10 mm NaCl, 1.5 mmMgCl2, 0.2 mm EDTA, 1 mm DTT, 0.17 Nonidet P-40), containing protease inhibitors (0.5 mmphenylmethylsulfonyl fluoride, 0.5 ॖg/ml leupeptin, 2 ॖg/ml aprotinin, 0.7 ॖg/ml pepstatin A, 1 mm sodium fluoride, and 50 ॖm sodium orthovanadate). After centrifugation at 300 × g for 4 min, the supernatant was designated 舠cytoplasmic fraction舡 and stored at −80 °C. The pellets were further lysed for 30 min on ice in 50 ॖl (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 舠nuclear fraction舡 and stored at −80 °C. Total protein extracts were performed in buffer B, supplemented as described above. Equal amounts of proteins (quantified by Bradford assay) were mixed with Laemmli sample buffer, resolved on 107 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) (51Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Google Scholar) were used. Although the reactivity of the anti-phospho-Thr-81 antibody is weak, its specificity has been fully characterized in a previous publication (51Buschmann T. Potapova O. Bar-Shira A. Ivanov V.N. Fuchs S.Y. Henderson S. Fried V.A. Minamoto T. Alarcon-Vargas D. Pincus M.R. Gaarde W.A. Holbrook N.J. Shiloh Y. Ronai Z. Mol. Cell. Biol. 2001; 21: 2743-2754Google Scholar). 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 ॖl of IgG (whole molecule)-agarose beads (Sigma) for 1 h at 4 °C to remove unspecific binding. Precleared extracts were mixed with 1 ॖg 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 107 SDS-PAGE, and analyzed by Western blot. 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. The double-stranded p53 consensus binding sequence p53con(5′-GGACATGCCCGGGCATGTCC-3′) was end-labeled with ∼3000 Ci/mmol [γ-32P]ATP (Amersham Biosciences) as described (61Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Google Scholar). Binding reactions contained 32P-labeled double-stranded oligonucleotide (0.5 ng), sonicated herring sperm DNA (2 ॖg; Promega, Madison, WI), bovine serum albumin (5 ॖg), DTT (4 mm), and nuclear protein extracts (10 ॖg). Reactions were adjusted to a final volume of 30 ॖl 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 (61Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Google Scholar). Binding reactions were incubated for 30 min at 20 °C. A 15-ॖl aliquot of each reaction was loaded onto a 47 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 (61Verhaegh G.W. Richard M.J. Hainaut P. Mol. Cell. Biol. 1997; 17: 5699-5706Google Scholar). Proteasome activity was evaluated as described (62Grune T. Reinheckel T. Davies K.J. J. Biol. Chem. 1996; 271: 15504-15509Google Scholar). Briefly, cells were washed in PBS after treatment, and protein extracts were prepared in 50 mm Tris-HCl (pH 7.8), 20 mmKCl, 5 mm MgOAc, 0.5 mm DTT. Then 50 ॖm Suc-LLVY-AMC (dissolved in 107 Me2SO) was incubated with 100 ॖg of protein extract in the same buffer supplemented with 5 mm MgCl2, 5 mmATP in a total volume of 200 ॖl. After 1 h of incubation at 37 °C, the reaction was terminated by adding 200 ॖl 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. Total RNA was extracted from treated and control MCF-7 cells using Trizol (Invitrogen), and 4 ॖg of RNA were used for reverse transcription. The cDNA corresponding to the TP53 gene was co-amplified with a cDNA from theॆ-actin gene. The annealing temperature of co-amplification was 56 °C (25 cycles). Co-amplified PCR products were analyzed on a 27 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′. 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'st test. Probabilities of p < 0.05 were regarded as statistically significant. We have shown previously (22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar) that WR1065 induced the accumulation and activation of wild-type p53 in cultured cells. Fig. 1shows 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 (22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar,63Meier T. Issels R.D. Biochem. Pharmacol. 1995; 50: 489-496Google Scholar). 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. In a previous study (22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar), 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 γ-irradiation than those with disrupted p53 (10.1), as detected by a reduced sub-G1peak, corresponding to apoptotic cells. In the presence of WR1065, cell death induced by irradiation was strongly reduced in 3T3 cells (from 18 to 47) but not in 10.1 cells (from 43 to 377). 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 (22North S. El Ghissassi F. Pluquet O. Verhaegh G. Hainaut P. Oncogene. 2000; 19: 1206-1214Google Scholar) 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. 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 t
Referência(s)