ERK Activation Mediates Cell Cycle Arrest and Apoptosis after DNA Damage Independently of p53
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m111598200
ISSN1083-351X
AutoresDamu Tang, Dongcheng Wu, Atsushi Hirao, Jill M. Lahti, Lieqi Liu, Brie Mazza, Vincent J. Kidd, Tak W. Mak, Alistair J. Ingram,
Tópico(s)Cancer therapeutics and mechanisms
ResumoIn response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM−/− fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEFp53−/−. However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21CIP1 induction, resulting in partial release of the G2/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis. In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM−/− fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEFp53−/−. However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21CIP1 induction, resulting in partial release of the G2/M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis. phosphatidylinositol 3-kinase mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase c-Jun NH2-terminal kinase etoposide adriamycin ionizing irradiation ultraviolet irradiation fetal calf serum terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling fluorescence-activate cell sorting ataxia-telangiectasia mutant ataxia-telangiectasia and Rad-3 mouse embryonic fibroblasts Eukaryotic cells employ multiple mechanisms to ensure accurate transmission of genetic information between generations. Critical surveillance of this transmission is provided by the DNA damage response, which may arrest the cell cycle to allow damage repair or direct cells to apoptosis in situations of severe damage (1.Lowe S.W. Schmitt E.M. Smith S.W. Osborne B.A. Jacks T. Nature. 1993; 362: 847-849Crossref PubMed Scopus (2753) Google Scholar, 2.Lozano G. Elledge S.J. Nature. 2000; 404: 24-25Crossref PubMed Scopus (110) Google Scholar, 3.Tanaka H. Arakawa H. Yamaguchi T. Shiraishi K. Fukuda S. Matsui K. Takei Y. Nakamura Y. Nature. 2000; 404: 42-49Crossref PubMed Scopus (735) Google Scholar, 4.Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar). Our understanding of how cells sense DNA damage remains incomplete, but it is clear that two members of the phosphatidylinositol 3-kinase (PI3K)1 family, ATM and ATR, are major DNA damage signal transducers (4.Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar). Downstream of ATM/ATR lies p53, a central mediator of the response, which in turn induces cell cycle arrest by up-regulation of p21CIP1 and 14-3-3ς, activates DNA damage repair pathways, and induces apoptosis (5.Caspari T. Curr. Biol. 2000; 10: R315-R317Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 6.Colman M.S. Afshari C.A. Barrett J.C. Mutat. Res. 2000; 462: 179-188Crossref PubMed Scopus (149) Google Scholar). p53 is normally rapidly degraded by Mdm2-mediated ubiquitin-dependent proteolysis (7.Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3629) Google Scholar, 8.Kubbutat M.H. Jones S.N. Vousden K.H. Nature. 1997; 387: 299-303Crossref PubMed Scopus (2798) Google Scholar). In response to DNA damage, p53 is stabilized by inhibition of this proteolytic process, partly through post-translational phosphorylation. Stabilization of p53 by phosphorylation on residues Ser-15 and Ser-21 by ATM and Chk2 kinase in response to DNA damage is well described (9.Banin 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: 1674-1677Crossref PubMed Scopus (1688) Google Scholar, 10.Canman 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: 1677-1679Crossref PubMed Scopus (1684) Google Scholar, 11.Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1032) Google Scholar). Members of the mitogen-activated protein kinase (MAPK) family have also been demonstrated to phosphorylate and stabilize p53 (12.Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Crossref PubMed Scopus (278) Google Scholar, 13.Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 14.Keller D. Zeng X. Li X. Kapoor M. Iordanov M.S. Taya Y. Lozano G. Magun B. Lu H. Biochem. Biophys. Res. Commun. 1999; 261: 464-471Crossref PubMed Scopus (77) Google Scholar). To wit, activation of JNK leads to p53 phosphorylation, thus interfering with the association of Mdm2 and p53 (12.Fuchs S.Y. Adler V. Buschmann T. Yin Z. Wu X. Jones S.N. Ronai Z. Genes Dev. 1998; 12: 2658-2663Crossref PubMed Scopus (278) Google Scholar). Furthermore, activation of p38 MAPK by UV was shown to phosphorylate p53 on S389 (13.Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar,14.Keller D. Zeng X. Li X. Kapoor M. Iordanov M.S. Taya Y. Lozano G. Magun B. Lu H. Biochem. Biophys. Res. Commun. 1999; 261: 464-471Crossref PubMed Scopus (77) Google Scholar). The third member of the canonical MAPK family, ERK (extracellular signal-regulated kinase), is centered on multiple signal transduction pathways to accomplish a variety of functions. Activation of ERK through different pathways leads to fundamentally different cellular responses, including proliferation, differentiation, survival, and memory consolidation (15.Bergmann A. Agapite J. McCall K. Steller H. Cell. 1998; 95: 331-341Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 16.Impey S. Obrietan K. Storm D.R. Neuron. 1999; 23: 11-14Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, 17.Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1199) Google Scholar, 18.Kurada P. White K. Cell. 1998; 95: 319-329Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 19.Meier P. Evan G. Cell. 1998; 95: 295-298Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). ERK activation by extracellular growth signals is mediated through growth factor tyrosine phosphorylation and activation of a small G protein, Ras, and promotes cell proliferation (20.McCormick F. Nature. 1993; 363: 15-16Crossref PubMed Scopus (439) Google Scholar, 21.Moodie S.A. Wolfman A. Trends Genet. 1994; 10: 44-48Abstract Full Text PDF PubMed Scopus (144) Google Scholar). The importance of ERK in transduction of mitogenic signals is illustrated by the demonstration that activation of ERK is sufficient to transform NIH3T3 cells or MEF lacking either p53 or p16 (22.Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1844) Google Scholar, 23.Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (751) Google Scholar). In some circumstances, ligand interactions with growth factor receptors may result in ERK-mediated cell cycle exit. Indeed, ERK activation by nerve growth factor drives PC12 cell differentiation (22.Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1844) Google Scholar, 24.Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4213) Google Scholar). ERK may function in the response to DNA damage. ERK activation was observed in response to cisplatin in ovarian cancer cells (25.Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). ERK can phosphorylate p53 in vitro (25.Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), but its role in vivo is unclear (25.Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 26.Lee S.W. Fang L. Igarashi M. Ouchi T. Lu K.P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8302-8305Crossref PubMed Scopus (104) Google Scholar, 27.She Q.B. Chen N. Dong Z. J. Biol. Chem. 2000; 275: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). The role of DNA damage-induced ERK activation in mediating apoptosis has also not been clarified (28.MacKeigan J.P. Collins T.S. Ting J.P. J. Biol. Chem. 2000; 275: 38953-38956Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 29.Wang X. Martindale J.L. Holbrook N.J. J. Biol. Chem. 2000; 275: 39435-39443Abstract Full Text Full Text PDF PubMed Scopus (594) Google Scholar). In this regard, however, tumors with constitutive ERK activation undergo apoptosis when ERK activity is blocked (30.Hoshino R. Tanimura S. Watanabe K. Kataoka T. Kohno M. J. Biol. Chem. 2001; 276: 2686-2692Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Consequently, we sought to explore ERK activation in response to DNA damage, and to outline the mechanisms and consequences of such activation. We report here that multiple DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ultraviolet irradiation (UV), and ionizing radiation (IR) activate ERK in various cell lines. We show that this process depends on ATM and is independent of p53. Functionally, we demonstrate that ERK activation cooperates with p53 to lead to apoptosis or cell cycle arrest. This represents the first report to fully outline an ERK-mediated DNA damage pathway and its functional importance. Hoechst 33258, propidium iodide, ETOP, ADR, hydroxyurea, and wortmannin were from Sigma Chemical Co. The MEK1 inhibitors, PD98059 and U0126, were from Calbiochem and Promega, respectively. Stocks of ETOP, ADR, wortmannin, PD98059, and U0126 were made in Me2SO and used at the concentrations indicated. Hygromycin B was from Invitrogen. NIH3T3 and MCF7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Invitrogen) at 37 °C in a tissue culture incubator. MEF were isolated and cultured in Dulbecco's modified Eagle's medium, 10% FCS, 55 μmβ-mercaptoethanol, and 10 μg/ml gentamicin. IMR-90 was from ATCC and cultured in MEM and 10% FCS. The AT fibroblast line, GM05823, was from the NIGMS Human Genetic Cell Repository and cultured in MEM, 10% FCS, with 2× concentrated essential and non-essential amino acids and vitamins. The constitutively activated MEK1/Q56P cloned in the retroviral vector, pBabe, was kindly provided by Dr. Scott Lowe of Cold Spring Harbor Laboratories (23.Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (751) Google Scholar). pBabe/Bcl-2 and pBabe/Bcl-xL were constructed by insertion of human Bcl-2 and Bcl-xL into pBabe as we have published previously (31.Tang D. Lahti J.M. Kidd V.J. J. Biol. Chem. 2000; 275: 9303-9307Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Retroviral infection was performed as we have shown previously (31.Tang D. Lahti J.M. Kidd V.J. J. Biol. Chem. 2000; 275: 9303-9307Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 32.Tang D. Okada H. Ruland J. Liu L. Stambolic V. Mak T.W. Ingram A.J. J. Biol. Chem. 2001; 276: 30461-30466Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Constructs were subcloned into a retroviral vector. Plasmid DNA was isolated and used to generate retrovirus using phoenix packing cells. Briefly, 5 × 106 phoenix cells were seeded in a 10-cm plate overnight before transfection with 15 μg of retroviral vector for 48 h. The virus-containing medium was harvested after 48 h and filtered through a 0.45-μm filter. After addition of 10 μg/ml Polybrene (Sigma), the medium was used to infect NIH3T3 cells three times for 24 h at 8-h intervals to maximize transfection rate. Expression was confirmed with Western blot. After exposure to DNA damage agents as indicated, cells were lysed in a buffer containing 20 mm Tris (pH 7.4), 150 mm NaCl, 1 mmEDTA, 1 mm EGTA, 1% Triton X-100, 25 mm sodium pyrophosphate, 1 mm NaF, 1 mmβ-glycerophosphate, 0.1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 10 μg/ml aprotinin. 50 μg of total cell lysate was separated on SDS-PAGE gel and transferred onto Immobilon-P membranes (Millipore). Membranes were blocked with 5% skim milk and then incubated with the indicated antibodies at room temperature for 1 h. Signals were detected using an ECL Western blotting Kit (Amersham Biosciences, Inc.). Primary antibodies and concentration used were: anti-ATM (Ab-3 at 2 μg/ml, Oncogene); anti-p53 (FL-353 at 1 μg/ml, Santa Cruz Biotechnology); anti-phospho-p53(S15) (Cell Signaling, 1:1000); anti-ERK (1:500, New England BioLabs); anti-phospho-ERK (1:500 New England BioLabs); anti-Actin (Santa Cruz Biotechnology); and anti-p21CIP1 (1 μg/ml, Santa Cruz Biotechnology). DNA fragmentation and TUNEL assay were performed as we have previously published (31.Tang D. Lahti J.M. Kidd V.J. J. Biol. Chem. 2000; 275: 9303-9307Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 32.Tang D. Okada H. Ruland J. Liu L. Stambolic V. Mak T.W. Ingram A.J. J. Biol. Chem. 2001; 276: 30461-30466Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 33.Tang D. Kidd V.J. J. Biol. Chem. 1998; 273: 28549-28552Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). For the cell viability assays, 2 × 104 cells were seeded in 96-well plates and incubated at 37 °C for 4 h. Cells were then treated with DNA damage stimuli as indicated with or without PD98059 (50 μm) or U0126 (10 or 50 μm) for the indicated times. WST-1 cell proliferation reagent (Roche Diagnostics, Mannheim, Germany) 10 μl/100 μl medium was added, and cells were incubated for 30 min at 37 °C. Viable cell numbers were determined at 450 nm with a microplate reader (Fisher Diagnostics). Cell cycle distribution was determined by staining NIH3T3 cells with propidium iodide solution in the presence of RNase A overnight at 4 °C followed by detection of signals using fluorescent automated cell sorting (FACS) as we have published previously (11.Hirao A. Kong Y.Y. Matsuoka S. Wakeham A. Ruland J. Yoshida H. Liu D. Elledge S.J. Mak T.W. Science. 2000; 287: 1824-1827Crossref PubMed Scopus (1032) Google Scholar). Cells were grown to 80% confluence on a glass slip. After treatment with ETOP at the indicated concentrations for 30 min, nocodazole (200 ng/ml) was added. After 12, 18, 24, and 30 h, cells were fixed in 3.7% formaldehyde and stored at 4 °C for 24 h. Cells were then permeabilized in a Triton X-100-containing solution and stained with Hoechst 33258 DNA stain (10 μg/ml, Molecular Probes). The mitotic index was scored under a fluorescence microscope. To determine whether ERK is activated by DNA damage signals, early passage MEF cells were treated with ETOP, which induces double-strand DNA breaks by interfering with the function of DNA topoisomerase II (34.O'Dwyer P.J. Leyland-Jones B. Alonso M.T. Marsoni S. Wittes R.E. N. Engl. J. Med. 1985; 312: 692-700Crossref PubMed Scopus (335) Google Scholar). As expected, ETOP induced stabilization of p53 and up-regulation of p21CIP1 (data not shown). ETOP induced ERK activation (as measured by phosphorylation) with two phases (Fig. 1A). The first phase of ERK activation starts at 1 h and ends at 8 h of ETOP exposure, whereas the second takes place after 15 h in response to ETOP (Fig. 1A). Increasing ETOP concentrations resulted in increasing levels of ERK activation with maximal ERK activation observed at or higher than 25 μm (data not shown), indicating a correlation between intensity of DNA damage and level of ERK activation. To determine whether ERK activation is a unique property of ETOP or MEF cells, we used multiple DNA damage stimuli to initiate ERK activation. ETOP, ADR, and UV led to ERK activation in primary (MEF), immortalized (NIH3T3), and transformed (MCF7) cells (Fig. 1B). Additionally, IR and hydroxyurea also activated ERK in MEF and IMR90 (data not shown), confirming that DNA damage indeed leads to ERK activation. Because ERK activation is known to require dual phosphorylation on Thr-183 and Tyr-185 by MEKs (35.Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (835) Google Scholar, 36.Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar), we sought to determine whether DNA damage-induced ERK activation also requires MEK activity. Addition of the MEK1 inhibitors, PD98059 (Fig. 3B) and U0126 (data not shown), prevented ETOP-induced ERK activation. Complete abrogation of ERK activation was also observed when cells incubated with PD98059 or U0126 were exposed to ADR, UV, or IR (data not shown), confirming the role of MEK1 in mediating DNA damage-induced ERK activation. Taken together, these results show that ERK activation is a component of cellular DNA damage responses initiated by diverse stimuli. Because ATM plays a central role in the relay of DNA damage signals (4.Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar) and ATM has been shown to play a role in the DNA damage-induced activation of JNK and p38 (37.Shafman T.D. Saleem A. Kyriakis J. Weichselbaum R. Kharbanda S. Kufe D.W. Cancer Res. 1995; 55: 3242-3245PubMed Google Scholar, 38.Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell. Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (235) Google Scholar), we sought to determine if ATM played a role in DNA damage-induced ERK activation. As a member of the PI3K family, the kinase activity of ATM is inhibited by the PI3K inhibitor, wortmannin (39.Chan D.W. Son S.C. Block W. Ye R. Khanna K.K. Wold M.S. Douglas P. Goodarzi A.A. Pelley J. Taya Y. Lavin M.F. Lees-Miller S.P. J. Biol. Chem. 2000; 275: 7803-7810Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 40.Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar). Addition of wortmannin dose-dependently inhibited ETOP but not UV-induced ERK activation (data not shown), consistent with the fact that wortmannin inhibits the kinase activity of ATM but not ATR (40.Sarkaria J.N. Tibbetts R.S. Busby E.C. Kennedy A.P. Hill D.E. Abraham R.T. Cancer Res. 1998; 58: 4375-4382PubMed Google Scholar) and that UV-induced DNA damage responses are generally ATR-dependent. To confirm ATM dependence of ETOP-induced ERK activation, both ATM−/− (GM05823) and IMR90 (wild type) human fibroblasts were used. Expression of ATM in IMR90 but not in GM05823 was confirmed by Western blot using an anti-ATM (Ab-3) antibody (data not shown). Addition of ADR or ETOP to IMR90 cells led to p53 stabilization, associated with phosphorylation on Ser-15 and up-regulation of p21CIP1 (Fig. 2). Ser-15 phosphorylation-associated p53 stabilization was significantly reduced, and p21CIP1induction was abolished in GM05823 cells in response to ADR or ETOP (Fig. 2), further confirming the lack of ATM function in the ATM−/− fibroblasts. Importantly, ETOP-induced ERK activation was absent in GM05823 but present in IMR90 (Fig. 2), confirming a role of ATM in ETOP-induced ERK activation. However, ADR-induced ERK activation was not attenuated in the ATM−/− fibroblast line (Fig. 2), suggesting that ATR might also play an important role in this setting. Caffeine (2 mm), which inhibits both ATM and ATR (41.Sarkaria J.N. Busby E.C. Tibbetts R.S. Roos P. Taya Y. Karnitz L.M. Abraham R.T. Cancer Res. 1999; 59: 4375-4382PubMed Google Scholar), did prevent ADR-induced ERK activation in this context, supporting the supposition that ATR plays a role in the transmission of ADR-mediated DNA damage to ERK (data not shown). Because p53 is the major effector protein downstream of ATM mediating the DNA damage response, we sought to investigate if ERK activation played a role in the p53 pathway, as has been suggested by others (25.Persons D.L. Yazlovitskaya E.M. Pelling J.C. J. Biol. Chem. 2000; 275: 35778-35785Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 26.Lee S.W. Fang L. Igarashi M. Ouchi T. Lu K.P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8302-8305Crossref PubMed Scopus (104) Google Scholar, 27.She Q.B. Chen N. Dong Z. J. Biol. Chem. 2000; 275: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Kinetically, p53 stabilization by ETOP (Fig. 3A) and ADR and IR (data not shown) preceded ERK activation in all cell lines studied. Fig. 3A shows MCF-7 cells. MEK1 inhibition with PD98059 inhibited ERK activation but was without effect on p53 stabilization in response to DNA damage in MEF, NIH3T3, and MCF-7 cells (Fig. 3B, NIH3T3), suggesting that ERK activation was not upstream of p53. Conversely, pretreatment of cells with cycloheximide for 30 min to destroy endogenous p53 prevented ETOP (data not shown)- and ADR (Fig. 3C, NIH3T3)-induced p53 stabilization but not ERK activation, indicating that ERK activation is not downstream of p53. However, given conflicting data suggesting that ERK activation may be either upstream (27.She Q.B. Chen N. Dong Z. J. Biol. Chem. 2000; 275: 20444-20449Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar) or downstream (26.Lee S.W. Fang L. Igarashi M. Ouchi T. Lu K.P. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8302-8305Crossref PubMed Scopus (104) Google Scholar) of p53 after DNA damage, we wished to confirm our observations. In support of our results, a maximal level of ERK activation was not attenuated in MEFp53−/− compared with wild type MEF (Fig. 3D, inset), although the -fold increase of ERK activation is slightly lower in MEFp53−/− due to higher basal level of ERK activity in these p53-negative cells (Fig. 3D). ADR and UV also induced ERK activation in MEFp53−/− (data not shown). Taken together, these data indicate quite conclusively that under physiologic conditions, DNA damage-induced ERK activation is independent of p53. The two paradigmatic cellular responses to DNA damage are cell cycle arrest (allowing the damage to be repaired) or apoptosis in response to low or high intensities of DNA damage, respectively. The Cdk inhibitor, p21CIP1, is up-regulated in this process and plays a major role in arresting cells in either G1 or at G2/M in response to DNA damage (42.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2505) Google Scholar, 43.Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1926) Google Scholar). We have observed that wortmannin affected ERK activation and p21CIP1 induction in a more clearly dose-dependent manner than p53 stabilization after DNA damage (data not shown), suggesting that ERK activation might up-regulate p21CIP1. This is consistent with the observations that enforced ERK activation by overexpression of a constitutively activated Raf1 led to p21CIP1 induction (44.Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar,45.Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (418) Google Scholar). Consequently, we examined the effect of inhibition of ERK activation on p21CIP1 induction after DNA damage. Inhibition of ERK activation by PD98059 reduced p21CIP1 induction by half (Figs. 3B and 4) but had no effect on p53 stabilization by ETOP (Fig. 3B). PD98059 also attenuated ADR-induced p21CIP1 induction (Fig. 4). As expected, overexpression of a constitutively activated MEK1/Q56P (23.Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (751) Google Scholar) strongly activated ERK in NIH3T3 and MEF cells (data not shown) and led to p21CIP1 induction that was also inhibited by PD98059 (Fig. 4). Furthermore, expression of MEK1/Q56P in MEFp53−/− cells also resulted in p21CIP1induction (data not shown), indicating that ERK activation is capable of inducing p21CIP1 expression, consistent with prior observations with forcibly expressed Raf1 (44.Woods D. Parry D. Cherwinski H. Bosch E. Lees E. McMahon M. Mol. Cell. Biol. 1997; 17: 5598-5611Crossref PubMed Scopus (574) Google Scholar, 45.Sewing A. Wiseman B. Lloyd A.C. Land H. Mol. Cell. Biol. 1997; 17: 5588-5597Crossref PubMed Scopus (418) Google Scholar). Our data, however, is the first to demonstrate p21CIP1 induction by ERK under physiologic conditions. Because ERK activation partially contributes to p21CIP1 induction (Figs. 3B and 4), and p21CIP1 plays an important role in both induction of G1 arrest (43.Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1926) Google Scholar) and maintenance of arrest at the G2/M checkpoint (42.Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedivy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1501Crossref PubMed Scopus (2505) Google Scholar), we sought to determine whether the magnitude of p21CIP1 induction induced by ERK activation played a functional role in cell cycle arrest in response to DNA damage. Although PD98059 100 μm alone results a degree of G1 arrest (Fig. 5B), consistent with the notion that ERK activity facilitates G1 progression (46.Cheng M. Sexl V. Sherr C.J. Roussel M.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1091-1096Crossref PubMed Scopus (461) Google Scholar), addition of PD98059 to ETOP 0.5 μm (Fig. 5D) led to a marked release from the G2/M arrest seen with etoposide 0.5 μm alone (Fig. 5C). We confirmed that G2/M release was indeed occurring by mitotic labeling with nocodazole trapping and showed an increase of mitotic cells from 5 to 11% at 24 h when PD98059 is added to ETOP 0.5 μm(data not shown). To further confirm a role of ERK in maintaining G2/M arrest in response to ETOP, a more specific MEK1 inhibitor, U0126, was also used. Although 50 μm U0126 alone has a minimal effect on G1 phase (Fig. 5E), 10 and 50 μm U0126 again dramatically released ETOP (0.5 μm)-induced G2/M arrest (Fig. 5E). Similar results were observed with ADR (data not shown), although the effect of PD98059 on cell cycle arrest induced by ADR is less prominent than that induced by ETOP, consistent with the observation that inhibition of ERK activation was more effective in preventing ETOP- than ADR-mediated p21CIP1 induction (Fig. 4). Given our data for a pro-cell cycle arrest function of ERK in response to DNA damage, we wished to determine if DNA damage-induced ERK activation also mediated apoptotic responses. Addition of ETOP 10 μmfor 24 h led to DNA fragmentation in NIH3T3 cells, which was essentially prevented by ERK inhibition (Fig. 6A). Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL also prevented apoptosis in response to ETOP, indicating that the mitochondrial cellular execution pathway plays a role in DNA damage-induced apoptosis (Fig. 6A). Quantification of apoptosis using terminal deoxyuridine end-nick labeling (TUNEL) revealed that 65% of cells were apoptotic when exposed to 10 μm ETOP for 24 h, and that ERK inhibition with PD 98059 dose- dependently reduced this to less than 10%, suggesting a major role for ERK in DNA damage-induced apoptosis in response to ETOP (Fig. 6B). Similarly, Bcl-2 or Bcl-xL overexpression also reduced apoptosis to ∼10%, indicating again the importance of the mitochondrial execution pathw
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