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The Activation of c-Jun NH2-terminal Kinase (JNK) by DNA-damaging Agents Serves to Promote Drug Resistance via Activating Transcription Factor 2 (ATF2)-dependent Enhanced DNA Repair

2003; Elsevier BV; Volume: 278; Issue: 23 Linguagem: Inglês

10.1074/jbc.m210992200

ISSN

1083-351X

Autores

Jun Hayakawa, Chantal Depatie, Masahide Ohmichi, Dan Mercola,

Tópico(s)

PARP inhibition in cancer therapy

Resumo

The activating transcription factor 2 (ATF2) is a member of the ATF/cAMP-response element-binding protein family of basic-leucine zipper proteins involved in cellular stress response. The transcription potential of ATF2 is enhanced markedly by NH2-terminal phosphorylation by c-Jun NH2-terminal kinase (JNK) and mediates stress responses including DNA-damaging events. We have observed that four DNA-damaging agents (cisplatin, actinomycin D, MMS, and etoposide), but not the cisplatin isomer, transplatin, which does not readily damage DNA, strongly activate JNK, p38, and extracellular signal-regulated kinase (ERK), and strongly increase phosphorylation and ATF2-dependent transcriptional activity. Selective inhibition studies with PD98059, SB202190, SP600125, and the dominant negative JNK indicate that activation of JNK but not p38 kinase or ERK kinase is required for the phosphorylation and transcriptional activation of ATF2. Stable expression of ATF2 in human breast carcinoma BT474 cells increases transcriptional activity and confers resistance to the four DNA-damaging agents, but not to transplatin. Conversely, stable expression of a dominant negative ATF2 (dnATF2) quantitatively blocks phosphorylation of endogenous ATF2 leading to a marked decrease in transcriptional activity by endogenous ATF2 and a markedly increased sensitivity to the four agents as judged by decreased cell viability. Similarly, application of SB202190 at 50 μm or SP600125 inhibited JNK activity, blocked transactivation, and sensitized parental cells to the four DNA-damaging drugs. Moreover, the wild type ATF2-expressing clones exhibited rapid DNA repair after treatment with the four DNA-damaging agents but not transplatin. Conversely, expression of dnATF2 quantitatively blocks DNA repair. These results indicate that JNK-dependent phosphorylation of ATF2 plays an important role in the drug resistance phenotype likely by mediating enhanced DNA repair by a p53-independent mechanism. JNK may be a rational target for sensitizing tumor cells to DNA-damaging chemotherapy agents. The activating transcription factor 2 (ATF2) is a member of the ATF/cAMP-response element-binding protein family of basic-leucine zipper proteins involved in cellular stress response. The transcription potential of ATF2 is enhanced markedly by NH2-terminal phosphorylation by c-Jun NH2-terminal kinase (JNK) and mediates stress responses including DNA-damaging events. We have observed that four DNA-damaging agents (cisplatin, actinomycin D, MMS, and etoposide), but not the cisplatin isomer, transplatin, which does not readily damage DNA, strongly activate JNK, p38, and extracellular signal-regulated kinase (ERK), and strongly increase phosphorylation and ATF2-dependent transcriptional activity. Selective inhibition studies with PD98059, SB202190, SP600125, and the dominant negative JNK indicate that activation of JNK but not p38 kinase or ERK kinase is required for the phosphorylation and transcriptional activation of ATF2. Stable expression of ATF2 in human breast carcinoma BT474 cells increases transcriptional activity and confers resistance to the four DNA-damaging agents, but not to transplatin. Conversely, stable expression of a dominant negative ATF2 (dnATF2) quantitatively blocks phosphorylation of endogenous ATF2 leading to a marked decrease in transcriptional activity by endogenous ATF2 and a markedly increased sensitivity to the four agents as judged by decreased cell viability. Similarly, application of SB202190 at 50 μm or SP600125 inhibited JNK activity, blocked transactivation, and sensitized parental cells to the four DNA-damaging drugs. Moreover, the wild type ATF2-expressing clones exhibited rapid DNA repair after treatment with the four DNA-damaging agents but not transplatin. Conversely, expression of dnATF2 quantitatively blocks DNA repair. These results indicate that JNK-dependent phosphorylation of ATF2 plays an important role in the drug resistance phenotype likely by mediating enhanced DNA repair by a p53-independent mechanism. JNK may be a rational target for sensitizing tumor cells to DNA-damaging chemotherapy agents. Activating transcription factor 2 (ATF2) 1The abbreviations used are: ATF2, activating transcription factor-2; CBP, CREB-binding protein; cisplatin, cis-diaminodichloroplatinum; CMV, cytomegalovirus; CREB, cyclic AMP-responsive element-binding protein; dnATF2, dominant negative ATF2; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; Luc, luciferase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Me2SO, dimethyl sulfoxide; MMS, methionine-S-methylsulfonium chloride; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; SAPK, SAPK, stress-activated protein kinase; tk, thymidine kinase; TNF-α, tumor necrosis factor-α; transplatin, trans-diaminodichloroplatinum; wtATF2, wild type ATF2. 1The abbreviations used are: ATF2, activating transcription factor-2; CBP, CREB-binding protein; cisplatin, cis-diaminodichloroplatinum; CMV, cytomegalovirus; CREB, cyclic AMP-responsive element-binding protein; dnATF2, dominant negative ATF2; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; Luc, luciferase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; Me2SO, dimethyl sulfoxide; MMS, methionine-S-methylsulfonium chloride; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt; SAPK, SAPK, stress-activated protein kinase; tk, thymidine kinase; TNF-α, tumor necrosis factor-α; transplatin, trans-diaminodichloroplatinum; wtATF2, wild type ATF2./cyclic AMP-responsive element-binding protein-1 is a member of the leucine zipper protein family which regulates gene transcription by interacting with ATF/cAMP-response elements of genes. ATF2 commonly plays an important role in the cellular stress responses (1Abdel-Hafiz H.A. Heasley L.E. Kyriakis J.M. Avruch J. Kroll D.J. Johnson G.L. Hoeffler J.P. Mol. Endocrinol. 1992; 6: 2079-2089Crossref PubMed Scopus (85) Google Scholar, 2Hai T.W. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (751) Google Scholar, 3Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (291) Google Scholar, 4Karin M. Hunter T. Curr. Biol. 1995; 5: 747-757Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Various forms of cellular stress including genotoxic agents, inflammatory cytokines, and UV irradiation stimulate the transcriptional activity of ATF2 (5Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1333) Google Scholar, 6Livingstone C. Patel G. Jones N. 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Silva E. Gershenson D. Hung M.C. Oncogene. 1989; 4: 985-989PubMed Google Scholar). Also, similar to many human breast cancers, BT474 cells express a mutated p53 gene product (37Elstner E. Linker-Israeli M. Said J. Umiel T. de Vos S. Shintaku I.P. Heber D. Binderup L. Uskokovic M. Koeffler H.P. Cancer Res. 1995; 55: 2822-2830PubMed Google Scholar, 38Davis P.L. Shaiu W.L. Scott G.L. Iglehart J.D. Hsieh T.S. Marks J.R. Anticancer Res. 1998; 18: 2919-2932PubMed Google Scholar), thereby providing a means of examining the DNA damage response pathway without the complicating effects of p53-induced apoptosis or cell cycle arrest. Here, we provide evidence that DNA-damaging agents such as cisplatin, MMS, etoposide, but not the cisplatin isomer, transplatin, which does not readily damage DNA, lead to the phosphorylation and enhanced transactivation potential of ATF2 in a JNK-dependent manner. Further, cells modified to express even low levels of wild type ATF2 exhibit accelerated DNA repair as judged by quantitative PCR and are resistant to the DNA-damaging agents. Conversely, cells modified to express stably a similar level of a nonphosphorylatable dominant negative inhibitor of ATF2 (dnATF2) or cells treated with a pyridinyl imidazole inhibitor, SB202190, or an anthrapyraxolone, SP600125, exhibit complete inhibition of phosphorylation of endogenous ATF2, blocked DNA repair, and a markedly decreased cell viability after treatment with the four different DNA-damaging agents. These results suggest that ATF2 plays an important role in the modulation of DNA repair and in determination of the drug-resistant phenotype. Cell Cultures and Transfections—The human breast cancer BT474 cell line was obtained from American Type Culture Collection. The cells were cultured at 37 °C in Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a water-saturated atmosphere of 95% O2 and 5% CO2. BT474 cells were transfected using LipofectAMINE Plus (Invitrogen). The total amount of DNA was kept constant at 2 μg by adding the empty vector plasmid DNA to the transfection mixtures. The experiments were repeated at least three times. DNA Constructs—Wild type ATF2 (wtATF2), pLHCATF2, and a nonphosphorylatable dominant negative ATF2 (dnATF2) pLHCdn-ATF2(T69A,T71A) were constructed by insertion of the cDNA for wtATF2 and dnATF2 into the retroviral plasmid pLHCX, where L is the retroviral long terminal repeat, H is the hygromycin phosphotransferase gene for resistance to hygromycin B, C is an abbreviated human cytomeglia virus promoter, and X is a polylinker thereby creating pLHCwtATF2 and pLHCdnATF2, respectively. The vector pLHCX itself was constructed as described previously (22Potapova O. Basu S. Mercola D. Holbrook N.J. J. Biol. Chem. 2001; 276: 28546-28553Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 39Potapova O. Fakhrai H. Baird S. Mercola D. Cancer Res. 1996; 56: 280-286PubMed Google Scholar). The cDNAs for wtATF2 and dnATF2 were excised from pECE-ATF2 or pECE-ATF2(T69A,T71A), kindly provided by Dr. Z. Ronai and Dr. M. Green. The resulting retroviral vectors were characterized by restriction enzyme digests and by the ability to impart hygromycin B resistance and by confirmation of plasmid protein expression (see below). The plasmids encoding the dominant negative SAPK/JNK (pcDL-SRα-SAPK-VPF) and the wild type SAPK/JNK (pcDL-SRα-wt-SAPK) (40Toyoshima F. Moriguchi T. Nishida E. J. Cell Biol. 1997; 139: 1005-1015Crossref PubMed Scopus (142) Google Scholar) were kind gifts from Dr. E. Nishida. The reporter constructs p5xjun2tk-Luc and control vector ptk-Luc (7van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar, 41van Dam H. Huguier S. Kooistra K. Baguet J. Vial E. van der Eb A.J. Herrlich P. Angel P. Castellazzi M. Genes Dev. 1998; 12: 1227-1239Crossref PubMed Scopus (99) Google Scholar) were kindly provided by Dr. P. Angel. Clone Selection—BT474 cells were transfected for 12 h in six-well tissue culture plates with 2 μg of pLHCdnATF2(T69A,T71A), pLH-CATF2, pLHCcJun(S63A,S73A), or the empty vector pLHCX with LipofectAMINE Plus. The preparation of pLHCX and pLHCcJun-(S63A,S73A) has been described (51Han Z. Boyle D.L. Chang L. Bennett B. Karin M. Yang L. Manning A.M. Firestein G.S. J. Clin. Invest. 2001; 108: 73-81Crossref PubMed Scopus (713) Google Scholar, 52Iordanov M.S. Pribnow D. Magun J.L. Dinh T.H. Pearson J.A. Chen S.L. Magun B.E. Mol. Cell. Biol. 1997; 17: 3373-3381Crossref PubMed Google Scholar). The vector pLHCc-Jun(S63A,S73A) has been used previously for the stable expression of a nonphosphorylatable form of c-Jun in various human tumor lines (19Potapova O. Haghighi A. Bost F. Liu C. Birrer M.J. Gjerset R. Mercola D. J. Biol. Chem. 1997; 272: 14041-14044Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 42Bost F. McKay R. Dean N. Mercola D. J. Biol. Chem. 1997; 272: 33422-33429Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Clone selection was performed by adding hygromycin B to the medium to a 400 μg/ml final concentration 2 days after the transfection. After 3 weeks several clones were isolated using cloning rings. Selected clones were then maintained in medium supplemented with 400 μg/ml hygromycin B, and only low passage cells (p < 10) were used for the experiments described here. Western Analysis—Cells were lysed in a solution containing 150 mm NaCl, 10 mm Tris-HCl, pH 7.4, 5 mm EDTA, 1% Triton X-100, and protease inhibitors phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin. Equal amounts of lysates (50 μg) were size fractionated in 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Proteins were detected using an enhanced chemiluminescence system (Amersham Biosciences) after incubation of the polyvinylidene difluoride membranes with specific antibodies. In Vitro Kinase Assay—JNK assay to c-Jun and p38 MAPK kinase assays to ATF2 were performed with assay kits for the respective kinases (Cell Signaling Technology Inc.) following the company's instructions. Briefly, treated cells were lysed in buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm EDTA, 1 mm EGTA, 1 mm sodium orthovanadate, 1 mm α-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml leupeptin) for 15 min on ice. The cell lysates (250 μg) were incubated overnight at 4 °C with immobilized c-Jun (Cell Signaling Technology Inc.) or antiphospho-p38 MAPK (Cell Signaling Technology Inc.) for JNK and p38 MAPK, respectively. In preliminary experiments, it was confirmed that anti-phospho-p38 reliably and uniformly precipitated p38, and this was confirmed for all conditions (lanes) of Fig. 1C. The immunoprecipitated products were washed twice with the cell lysis buffer and twice with kinase buffer with 25 mm Tris, pH 7.5, 5 mm glycerophosphate, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate, and 10 mm MgCl2. The pellets were suspended in the kinase buffer containing 100 μm ATP for the JNK assay, 200 μm ATP, and 2 μg of ATF2 for the p38 MAPK assay. Each reaction was then incubated for 30 min at 30 °C. Western blotting was performed with the phospho-c-Jun and phospho-ATF2 antibodies, respectively. JNK assay for the phosphorylation of ATF2 was performed using an ATF2 fusion protein (Cell Signaling Technology Inc.) as a substrate. Briefly, The cell lysates (250 μg) were incubated with JNK antibody (Cell Signaling Technology Inc.) overnight at 4 °C. The immunoprecipitated products were washed twice with the cell lysis buffer and twice with kinase buffer. In preliminary studies, it was confirmed that anti-JNK reliably precipitated JNK, and this was confirmed for all conditions (lanes) examined here (i.e.Fig. 1C). The pellets were suspended in the kinase buffer containing 200 μm ATP and 2 μg of ATF2. Each reaction was then incubated for 30 min at 30 °C. Western blotting was performed with the phospho-ATF2 antibodies. In some of the experiments, BT474 cells cultured in 100-mm dishes were transfected with hemagglutinin-tagged wild type SAPK/JNK expression plasmid (1 μg of pcDL-SRα-wt-SAPK) or hemagglutinin-tagged dominant negative SAPK/JNK expression plasmid (1 μg of pcDL-SRα-SAPK-VPF) using LipofectAMINE Plus. At 72 h after transfection, treated cells were lysed, and 250 μg of cell lysates were immunoprecipitated with anti-hemagglutinin antibody (Santa Cruz Biotechnology), and the JNK activity was measured as described above. Cytotoxicity—To assess viability, cells were seeded at a density of 1,000 cells/well in 96-well tissue culture plates, and on the following day, they were treated in the same medium with various cytotoxic agents for 1 h. After all treatments, the cells were washed with phosphate-buffered saline and supplemented with fresh complete medium. In some of the experiments, the cells were treated with SB202190, SP600125, or Me2SO (mock) for 30 min before the addition of cytotoxic agents. The measurements of viable cell mass were performed 5 days later using a colorimetric based reaction after the addition of the dye MTS in accordance with the manufacturer's protocol (Promega), which is reduced in proportion to the amount of intact mitochondria, i.e. viable cell mass. All determinations were carried out with eight samples for each condition. Cell viability was expressed as viable cell mass after a given treatment by normalizing the averaged value to that of parallel cultures of untreated cells × 100 (viability, %) (21Hayakawa J. Ohmichi M. Kurachi H. Ikegami H. Kimura A. Matsuoka T. Jikihara H. Mercola D. Murata Y. J. Biol. Chem. 1999; 274: 31648-31654Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 22Potapova O. Basu S. Mercola D. Holbrook N.J. J. Biol. Chem. 2001; 276: 28546-28553Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The chemicals and inhibitors used were purchased as follows: LatinolR®AQ cisplatin was used as aqueous Platinol® (Bristol-Myers Squibb Laboratories); etoposide was from Sigma; MMS was from Aldrich; and actinomycin D, SB202190, SP600125, and PD98059 were from Calbiochem. Analysis of Transcription—BT474 cells cultured for 1 day in 24-well tissue culture plates were transfected with reporter constructs (p5xjun2tk-Luc or vector ptk-Luc) (7van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (569) Google Scholar, 41van Dam H. Huguier S. Kooistra K. Baguet J. Vial E. van der Eb A.J. Herrlich P. Angel P. Castellazzi M. Genes Dev. 1998; 12: 1227-1239Crossref PubMed Scopus (99) Google Scholar) and pCMV-β-galactosidase plasmid (to normalize for cell viability and transfection efficiency) in combination with the indicated plasmids using LipofectAMINE Plus. At 24 h after transfection, the serum-deprived cells were incubated with various cytotoxic agents or in buffer alone (phosphate-buffered saline) for 1 h. In some of the experiments, the cells were treated with 10 or 50 μm SB202190 or 30 μm SP600125 for 30 min before the addition of 100 μm cisplatin. After 16 h, the cells were lysed by exposure to three sequential freeze-thaw cycles in 100 mm potassium phosphate, pH 7.8, and 10 mm dithiothreitol. The frozen/thawed cells were vortexed vigorously to enhance cell lysis. The lysates were clarified by centrifugation (microfuge) at 10,000 rpm for 10 min at 4 °C. The aliquots of the supernatants were used in the subsequent luciferase and β-galactosidase assays. Luciferase activity was assayed using the luciferase assay mixture contained 20 mm NaOH, pH 7.8, 1 mm dithiothreitol, 3.7 mm MgSO4, 270 μm coenzyme A, 530 μm ATP, and 470 μm luciferin. A 100-μl portion of the luciferase assay mixture was added to a 20-μl aliquot of cell extract just before recording the intensity of phosfluorescent light emission, which was measured in duplicate during the first 20 s of the reaction at 25 °C in a luminometer (EG & G Berthhold, LB96V luminometer, Bundoora, Australia). β-Galactosidase was assayed using the β-galactosidase buffer containing 60 mm sodium phosphate, pH 7.5, 1 mm MgCl2, 0.80 mg/ml O-nitrophenyl-β-d-galactopyranoside, and 40 mm β-mercaptoethanol. A standard curve for reactions containing 100–2 microunits of β-galactosidase was made with each assay. A 30-μl aliquot of cell extract prepared as described above was incubated with assay buffer until color developed (30–120 min), and the reaction was then stopped by adding 150 μl of 1 m sodium bicarbonate. The absorbance at 420 nm was determined using a spectrophotometer (Molecular Devices, SPECTRA Max Plus 384). Luciferase-catalyzed light emission measured in arbitrary units was normalized to the activity of β-galactosidase observed for control samples. The resulting normalized values were averaged and expressed as average -fold stimulation relative to the control values ± S.E. Analysis of DNA Damage and Repair—Cisplatin adduct formation and repair were analyzed by a PCR-based DNA damage assay (PCR stop assay) as described previously (19Potapova O. Haghighi A. Bost F. Liu C. Birrer M.J. Gjerset R. Mercola D. J. Biol. Chem. 1997; 272: 14041-14044Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 20Gjerset R.A. Lebedeva S. Haghighi A. Turla S.T. Mercola D. 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