E2F Mediates Sustained G2 Arrest and Down-regulation of Stathmin and AIM-1 Expression in Response to Genotoxic Stress
2003; Elsevier BV; Volume: 278; Issue: 3 Linguagem: Inglês
10.1074/jbc.m210327200
ISSN1083-351X
AutoresShirley Polager, Doron Ginsberg,
Tópico(s)DNA Repair Mechanisms
ResumoExposure of cells to genotoxic agents results in activation of checkpoint pathways leading to cell cycle arrest. These arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the G1/S checkpoint function. In addition, regulation of the G2 checkpoint by the tumor suppressor p53 is RB-dependent. However, the molecular mechanism underlying the involvement of RB and its related proteins p107 and p130 in the G2 checkpoint is not fully understood. We show here that sustained G2/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from G2. Expression of the E7 papilloma virus protein, which dissociates complexes containing E2F and RB family members, also prevents the down-regulation of these mitotic genes and leads to premature exit from G2 after genotoxic stress. Furthermore, genotoxic stress increases the levels of nuclear E2F-4 and p130 as well as their in vivo binding to the Stathmin promoter. Thus, functional complexes containing E2F and RB family members appear to be essential for repressing expression of critical mitotic regulators and maintaining the G2/M checkpoint. Exposure of cells to genotoxic agents results in activation of checkpoint pathways leading to cell cycle arrest. These arrest pathways allow repair of damaged DNA before its replication and segregation, thus preventing accumulation of mutations. The tumor suppressor retinoblastoma (RB) is required for the G1/S checkpoint function. In addition, regulation of the G2 checkpoint by the tumor suppressor p53 is RB-dependent. However, the molecular mechanism underlying the involvement of RB and its related proteins p107 and p130 in the G2 checkpoint is not fully understood. We show here that sustained G2/M arrest induced by the genotoxic agent doxorubicin is E2F-dependent and involves a decrease in expression of two mitotic regulators, Stathmin and AIM-1. Abrogation of E2F function by dominant negative E2F abolishes the doxorubicin-induced down-regulation of Stathmin and AIM-1 and leads to premature exit from G2. Expression of the E7 papilloma virus protein, which dissociates complexes containing E2F and RB family members, also prevents the down-regulation of these mitotic genes and leads to premature exit from G2 after genotoxic stress. Furthermore, genotoxic stress increases the levels of nuclear E2F-4 and p130 as well as their in vivo binding to the Stathmin promoter. Thus, functional complexes containing E2F and RB family members appear to be essential for repressing expression of critical mitotic regulators and maintaining the G2/M checkpoint. retinoblastoma estrogen receptor reverse transcription glyceraldehyde-3-phosphate dehydrogenase phosphate-buffered saline chromatin immunoprecipitation wild type fluorescence-activated cell sorter Cell cycle arrest in response to DNA damage is an important mechanism for maintaining genomic integrity. This cell cycle arrest provides time for DNA repair to prevent replication or segregation of damaged DNA. Induction of growth arrest by DNA damage occurs mainly through the activation of checkpoint pathways that delay cell cycle progression at G1, S, and G2(1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2637) Google Scholar, 2Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Crossref PubMed Scopus (1925) Google Scholar). Growth arrest at both G1 and G2 is believed to occur in two steps, resulting in a rapid arrest that is followed by a more sustained arrest. Initial arrest at G1 involves phosphorylation and degradation of both the protein phosphatase cdc25A and cyclin D1, resulting in inhibition of G1cyclin-dependent kinases (3Agami R. Bernards R. Cell. 2000; 102: 55-66Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 4Costanzo V. Robertson K. Ying C.Y. Kim E. Avvedimento E. Gottesman M. Grieco D. Gautier J. Mol. Cell. 2000; 6: 649-659Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 5Mailand N. Falck J. Lukas C. Syljuasen R.G. Welcker M. Bartek J. Lukas J. Science. 2000; 288: 1425-1429Crossref PubMed Scopus (651) Google Scholar). Initial arrest at G2 involves phosphorylation and inhibition of the protein phosphatase cdc25C, leading to inhibition of cdc2 activity (6Peng C.Y. Graves P.R. Thoma R.S., Wu, Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1189) Google Scholar, 7Sanchez Y. Wong C. Thoma R.S. Richman R., Wu, Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1124) Google Scholar). Through transactivation of p21, the tumor suppressor p53 is one of the essential mediators of sustained arrest at both G1 and G2 in response to DNA damage (8Brugarolas J. Moberg K. Boyd S.D. Taya Y. Jacks T. Lees J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1002-1007Crossref PubMed Scopus (223) Google Scholar, 9Deng C. Zhang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1946) Google Scholar, 10Waldman T. Kinzler K.W. Vogelstein B. Cancer Res. 1995; 55: 5187-5190PubMed Google Scholar, 11Bunz 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 (2537) Google Scholar). p21 binds to cyclin-cyclin-dependent kinase complexes and inhibits their ability to phosphorylate the retinoblastoma tumor suppressor, RB,1 and its related proteins, p107 and p130 (12Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5238) Google Scholar). Cells deficient of RB fail to arrest at G1 after DNA damage, indicating that RB plays an essential role in this arrest (8Brugarolas J. Moberg K. Boyd S.D. Taya Y. Jacks T. Lees J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1002-1007Crossref PubMed Scopus (223) Google Scholar, 13Harrington E.A. Bruce J.L. Harlow E. Dyson N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11945-11950Crossref PubMed Scopus (209) Google Scholar). In addition, p53 regulation of DNA damage-induced G2 arrest was shown to be RB-dependent (14Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (146) Google Scholar). RB, p107, and p130 play a key role in negative regulation of cell cycle progression, and their growth inhibitory activity is largely attributed to their association with members of the E2F family of transcription factors (15Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar, 16Nevins J.R. Cell Growth Differ. 1998; 9: 585-593PubMed Google Scholar). The E2F family is composed of six members, E2F-1–E2F-6, which heterodimerize with the DP proteins, DP-1 or DP-2, to form the DNA-binding, active transcription factor (15Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar, 16Nevins J.R. Cell Growth Differ. 1998; 9: 585-593PubMed Google Scholar). E2F plays a crucial and well established role in the control of cell cycle progression mainly by up-regulating expression of genes required for the G1/S transition as well as for DNA replication (15Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar,16Nevins J.R. Cell Growth Differ. 1998; 9: 585-593PubMed Google Scholar). This transcriptional activity of E2F is inhibited by its interaction with RB, p107, and p130 (15Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1971) Google Scholar, 16Nevins J.R. Cell Growth Differ. 1998; 9: 585-593PubMed Google Scholar). In addition, the complex containing E2F and RB family members also actively represses transcription. Assembly of such repressive complexes, containing E2F and RB family members (referred to herein as E2F-RB, although they may contain p107 or p130), on promoters that have E2F-binding sites is critical for growth suppression by RB family members (17Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (958) Google Scholar, 18Zhang H.S. Dean D.C. Oncogene. 2001; 20: 3134-3138Crossref PubMed Scopus (84) Google Scholar). The combination of cessation of repression of some E2F-regulated genes by the E2F-RB complex and the activation of others by activated E2F constitutes a major step in promoting G1 exit. E2F-1, -2, and -3 comprise a subgroup of the E2F family. These E2Fs are specifically regulated by RB and not by the RB-related proteins, p107 and p130. Their release from RB precedes the activation of E2F-responsive genes as well as S-phase entry (19Moberg K. Starz M.A. Lees J.A. Mol. Cell. Biol. 1996; 16: 1436-1449Crossref PubMed Scopus (304) Google Scholar), and their overexpression induces quiescent cells to enter S-phase (20Johnson D.G. Schwarz J.K. Cress W.D. Nevins J.R. Nature. 1993; 365: 349-352Crossref PubMed Scopus (834) Google Scholar, 21Qin X.Q. Livingston D.M. Kaelin W.G., Jr. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar, 22Lukas J. Petersen B.O. Holm K. Bartek J. Helin K. Mol. Cell. Biol. 1996; 16: 1047-1057Crossref PubMed Scopus (266) Google Scholar, 23DeGregori J. Leone G. Miron A. Jakoi L. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7245-7250Crossref PubMed Scopus (607) Google Scholar, 24Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (285) Google Scholar). In addition, E2F-1, and possibly also E2F-2 and -3, induce apoptosis (21Qin X.Q. Livingston D.M. Kaelin W.G., Jr. Adams P.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10918-10922Crossref PubMed Scopus (691) Google Scholar,24Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (285) Google Scholar, 25Kowalik T.F. DeGregori J. Leone G. Jakoi L. Nevins J.R. Cell Growth Differ. 1998; 9: 113-118PubMed Google Scholar). E2F-4 and -5 constitute another subgroup of the E2F family. They interact also with p107 and p130 and are implicated mainly in repression of gene expression (26Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar, 27Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Crossref PubMed Scopus (965) Google Scholar). Unlike E2F-1, -2, and -3, which are constitutively nuclear, E2F-4 and -5 are found in the nucleus only in G0 and early G1, when many of the E2F-regulated genes are repressed (28Magae J., Wu, C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar, 29Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 30Muller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar, 31Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (181) Google Scholar). Furthermore, binding of E2F-4 to promoters is associated with gene repression (26Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816PubMed Google Scholar, 32Tommasi S. Pfeifer G.P. Mol. Cell. Biol. 1995; 15: 6901-6913Crossref PubMed Scopus (158) Google Scholar). Recent studies indicate that E2F regulates the expression of genes involved not only in cell cycle progression but also in other biological processes (33Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (497) Google Scholar, 34Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Crossref PubMed Scopus (630) Google Scholar, 35Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar, 36Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Crossref PubMed Scopus (912) Google Scholar). Many of these novel E2F targets function in various cellular responses to DNA damage, including activation of checkpoints, DNA repair, and apoptosis, thus implicating E2F in the DNA damage response. Further support to this notion comes from recent reports demonstrating that the E2F-1 protein is stabilized and its levels are increased following DNA damage (37Huang Y. Ishiko T. Nakada S. Utsugisawa T. Kato T. Yuan Z.M. Cancer Res. 1997; 57: 3640-3643PubMed Google Scholar, 38Blattner C. Sparks A. Lane D. Mol. Cell. Biol. 1999; 19: 3704-3713Crossref PubMed Scopus (197) Google Scholar, 39Hofferer M. Wirbelauer C. Humar B. Krek W. Nucleic Acids Res. 1999; 27: 491-495Crossref PubMed Scopus (63) Google Scholar, 40O'Connor D.J. Lu X. Oncogene. 1999; 19: 2369-2376Crossref Scopus (67) Google Scholar). This stabilization is due to phosphorylation of E2F-1 by the protein kinase ATM, one of the master controllers of the response to DNA damage (41Lin W.C. Lin F.T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar). We and others have shown that E2F up-regulates expression of a number of genes involved in entry to and progression through mitosis (33Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (497) Google Scholar, 35Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar). However, the mode of regulation of these mitotic genes by E2F and the biological consequences of this regulation are not fully understood. We show here that expression of two of these mitotic genes, AIM-1 and Stathmin, is also elevated by transcriptionally inactive E2F-1, indicating that they are subjected to E2F-dependent repression. Furthermore, we show that E2F-containing complexes are required for DNA damage-induced down-regulation of AIM-1 and Stathmin. This repression of gene expression is correlated with E2F-dependent maintenance of DNA damage-induced growth arrest at G2. NIH3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum. Rat-1a-MT-wtE2F-1 and Rat-1a-MT-E2F-1dlTA, which are the Rat1 cell lines transfected with an inducible plasmid expressing either wild type of a dominant negative mutant of E2F-1, were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and G418 (500 μg/ml). For zinc induction, cells were maintained for 48 h in medium with 0.1% serum, and then 100 μmZnCl2 were added to the medium. 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All cells were maintained at 37 °C in a humidified 8% CO2- containing atmosphere. Activation of the E2F-1 fused to estrogen receptor ligand-binding domain (ER-E2F-1) was induced by addition of 4-hydroxytamoxifen to a final concentration of 30 nm. Cycloheximide was added to a final concentration of 10 μg/ml. Doxorubicin was added to a final concentration of 0.2 μg/ml. The pSV-ψ-E-MLV packaging plasmid and pBABE-ER-E2F-1 were described previously (24Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (285) Google Scholar, 42Muller A.J. Young J.C. Pendergast A.M. Pondel M. Landau N.R. Littman D.R. Witte O.N. Mol. Cell. Biol. 1991; 11: 1785-1792Crossref PubMed Scopus (354) Google Scholar). pBABE-E2F-1dlTA was generated by inserting the E2F-1dlTA XbaI-HindIII fragment from pRcCMV-E2F-1-(1–363) (43Hofmann F. Martelli F. Livingston D.M. Wang Z. Genes Dev. 1996; 10: 2949-2959Crossref PubMed Scopus (214) Google Scholar) into the pBABE-puro vector. Cells of the packaging cell line 293 were co-transfected with 10 μg of ψ ecotropic packaging plasmid, pSV-ψ-E-MLV, and 10 μg of the relevant plasmid using the calcium phosphate method in the presence of chloroquin (25 μmfinal concentration, Sigma C6628). After 8 h, the transfection medium was replaced with fresh medium, and 5 ml of retroviral-containing cell supernatant was collected at 6-h intervals. Five collections were pooled together and frozen in aliquots. For infection, NIH3T3 cells were incubated for 5 h at 37 °C in 3 ml of retroviral supernatant, supplemented with 8 μg/ml polybrene (Sigma H9268). Then, 7 ml of medium was added, and after 24 h the medium was replaced with fresh medium containing 10% serum and 2 μg/ml puromycin (Sigma P7130). Reverse transcription-PCR (RT-PCR) was performed on total RNA prepared by the Tri Reagent method. For this assay, 7.5 μg of RNA was employed for cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Promega, 200 u) and oligo(dT) (Amersham Biosciences, 0.5 μg). Following are the number of cycles, annealing temperature, and the sequences of 5′ and 3′ primers used for each of the tested genes, respectively: for the gene encoding AIM-1, 28 cycles, 58 °C using 5′-AGATTGGGCGTCCTCTGGG and 5′-TCAATCATCTCTGGGGGCAG; for the gene encoding Stathmin, 35 cycles, 58 °C using 5′-GGTGAAAGAACTGGAGAAGCG and 5′-GTGCTTATCCTTCTCTCGC; for the gene encoding ARPP-PO, 19 cycles, 58 °C using 5′-GTGGGAGCAGACAATGTGG and 5′-CAGCTGCACATCGCTCAGG; for the gene encoding GAPDH, 20 cycles, 58 °C using 5′-ACCACAGTCCATGCCATCAC and 5′-TCCACCACCCTGTTGCTGTA. For nuclear and cytoplasmic fractions, cell pellets were resuspended in four packed cell volumes of hypotonic buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 1 mm EDTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 10 μg/ml leupeptin) and incubated for 15 min on ice. Cells were then lysed by adding Nonidet P-40 to a final concentration of 0.6% and vortexing. After a short centrifugation, the cytoplasmic supernatant was taken out, and the nuclear pellet was lysed in two packed cell volumes of lysis buffer (20 mm HEPES, pH 7.9, 400 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 10 μg/ml leupeptin). Protein concentrations were determined by Bradford assay. The ratio of loaded volumes of cytoplasmic and nuclear extracts from a given cell population was equivalent to the ratio of volumes of hypotonic and lysis buffers, respectively, and therefore it is considered as the per cell ratio of cytoplasmic and nuclear proteins. For whole cell extrcats, cells were lysed in lysis buffer (20 mm HEPES, pH 7.8, 450 mm NaCl, 25% glycerol, 0.2 mm EDTA, 0.5 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 1 μg/ml leupeptin). Equal amounts of protein from each lysate, as determined by Bradford assay, were resolved by electrophoresis through an SDS 7.5–12.5% polyacrylamide gel and transferred to a filter (Protran BA 85, Schleicher & Schüll). Filters were incubated with a primary antibody for 2 h, after 1 h blocking in PBS with 0.05% Tween 20 and 5% dry milk. Primary antibodies used were as follows: anti E2F-1 (sc-251, Santa Cruz), anti-RB (14001A, PharMingen), anti-p130 (sc-317, Santa Cruz), anti-AIM-1 (BD Biosciences), anti-Stathmin (STC, gift of Andre Sobel), anti-E2F-4 (sc-866 Santa Cruz), and anti-B23 (sc-6013, Santa Cruz). Binding of the primary antibody was detected using an enhanced chemiluminescence kit (ECL, Amersham Biosciences). Cells were trypsinized and fixed with methanol (−20 °C). After fixation, cells were centrifuged for 5 min at 1200 rpm, resuspended in PBS and incubated for 30 min at 4 °C. After recentrifugation cells were resuspended in PBS containing 5 μg/ml propidium iodide and 50 μg/ml RNase A and incubated for 30 min at room temperature. Fluorescence intensity was analyzed using a BD Biosciences flow cytometer. Approximately 108 cells were cross-linked by addition of formaldehyde directly to the growth medium (final concentration 1%). Cross-linking was stopped after 10 min at room temperature by the addition of glycine (final concentration: 0.125 m). Cross-linked cells were washed with PBS, trypsinized, scrapped, washed with PBS, and then resuspended in buffer I (10 mm HEPES, pH 6.5, 10 mm EDTA, 0.5 mm EGTA, and 0.25% Triton X-100). Cells were pelleted by microcentrifugation and then resuspended in buffer II (10 mm HEPES, pH 6.5, 1 mm EDTA, 0.5 mm EGTA, and 200 mm NaCl). After microcentrifugation, nuclei were resuspended in lysis buffer (50 mm Tris, pH 8.1, 10 mm EDTA, 1% SDS, and protease inhibitors). The resulting chromatin was sonicated to an average size of 1000 bp and then microcentrifuged. The supernatant was diluted 1:10 with dilution buffer (10 mm Tris, pH 8.1, 150 mm NaCl, 2 mm EDTA, and 1% Triton X-100) and divided into aliquots. After preclearing with blocked protein A-Sepharose beads, 1 μg of antibody was added to each aliquot of chromatin and incubated on a rotating platform overnight at 4 °C. Immunocomplexes were recovered with blocked protein A-Sepharose beads. Following extensive washing, bound DNA fragments were eluted and analyzed by subsequent PCR. Antibodies used were as follows: anti-E2F-4 (sc-866, Santa Cruz) and anti-p130 (sc-317, Santa Cruz). Primers used for PCR were for Stathmin promoter (forward) 5′-ACAAGCTGCCGTGTGTCCG-3′ and (reverse) 5′-CTGGAGAGAAGCATTTCGGG-3′ and for β-actin (forward) 5′-ACTCTTCCAGCCTTCCTTCC-3′ and (reverse) 5′-TCCTTCTGCATCCTGTCAGC-3′. Our initial studies aimed at understanding the regulation of mitotic genes by E2F focused on one of these E2F-regulated mitotic genes, Stathmin (also known as oncoprotein 18), which encodes a protein involved in microtubule dynamics and spindle assembly (44Cassimeris L. Curr. Opin. Cell Biol. 2002; 14: 18-24Crossref PubMed Scopus (363) Google Scholar). To determine whether Stathmin is a direct target of E2F, we infected NIH3T3 cells with a retrovirus carrying E2F-1 fused to the estrogen receptor ligand-binding domain (ER-E2F-1). The ER-E2F-1 is expressed as an inactive fusion protein, which is activated upon addition of the ligand 4-hydroxytamoxifen (24Vigo E. Muller H. Prosperini E. Hateboer G. Cartwright P. Moroni M.C. Helin K. Mol. Cell. Biol. 1999; 19: 6379-6395Crossref PubMed Scopus (285) Google Scholar). As was previously reported by Ishidaet al. (33Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Crossref PubMed Scopus (497) Google Scholar) induction of E2F-1 led to an increase in Stathmin mRNA levels (Fig. 1 A). Interestingly, a similar E2F1-induced increase in Stathmin mRNA levels was detected in the presence of the protein synthesis inhibitor, cycloheximide (CHX) (Fig. 1 A). These data indicate thatde novo protein synthesis is not required for E2F1-induced up-regulation of the Stathmin mRNA, suggesting that Stathmin is a direct target of E2F. The observed E2F1-induced up-regulation of Stathmin may be due to either activation or derepression. To distinguish between these two possibilities we analyzed the expression of Stathmin in cells containing an inducible wild type E2F-1, or mutant E2F-1, E2F-1dlTA. This truncated E2F-1dlTA lacks both the transactivation and the RB-binding domains, and when overexpressed it can negate both activation of gene expression by E2F and repression by E2F-RB complexes. Once again, induction of wtE2F-1 resulted in an increase in Stathmin mRNA levels (Fig. 1 B). Importantly, induction of E2F-1dlTA led to a similar increase in Stathmin mRNA levels (Fig. 1 B). wt and mutant E2F-1 were expressed at comparable levels (Fig. 1 C). Expression of other E2F-regulated mitotic genes, including Cdc2, BUB1b, EB1, and SAK-a, was similarly up-regulated by both wtE2F-1 (35Polager S. Kalma Y. Berkovich E. Ginsberg D. Oncogene. 2002; 21: 437-446Crossref PubMed Scopus (222) Google Scholar) and E2F-1dlTA (data not shown). These data strongly suggest that Stathmin, as well as other mitotic genes, is under E2F-dependent negative regulation. A physiological setting in which such negative regulation may be important is during growth arrest in response to DNA damage. Low doses of DNA-damaging agents induce cellular growth arrest, and we analyzed the effects of such a treatment on expression of both Stathmin and another E2F-regulated mitotic gene, AIM-1. AIM-1(aurora and Ipl-1-like midbody-associated protein kinase, also called aurora1 or STK12) is a serine/threonine kinase required for cytokinesis (reviewed in Refs. 45Bischoff J.R. Plowman G.D. Trends Cell Biol. 1999; 9: 454-459Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar and 46Terada Y. Cell Struct. Funct. 2001; 26: 653-657Crossref PubMed Scopus (73) Google Scholar). Following addition of the chemotherapeutic agent doxorubicin, NIH3T3 cells underwent growth arrest at the G1 and G2/M phases of the cell cycle (Fig. 2 A). This DNA damage-induced growth arrest was already apparent 24 h after doxorubicin addition and persisted for at least 72 h. The growth arrest was accompanied by accumulation of hypophosphorylated RB and p130 (Fig. 2 B). In addition, treatment with doxorubicin led to a significant decrease in mRNA levels of Stathmin and AIM-1 (Fig. 2 C). Similar results were obtained using HCT116 human colorectal carcinoma cells (data not shown). The decrease in mRNA levels of the studied genes could be detected 24 h after treatment and was most evident at 48 h (Fig. 2 C). Thus, the increase in hypophosphorylated RB and p130, which are the growth repressive forms, coincided with or preceded the down-regulation of Stathmin and AIM-1. This result raises the possibility that complexes containing either RB or p130 mediate the repression of Stathmin and AIM-1 in response to DNA damage. Next we tested whether E2F mediates the decrease in expression of these mitotic genes in response to DNA damage. To this end, NIH3T3 cells were infected with retroviruses containing either E2F-1dlTA or an empty vector and then treated with doxorubicin. As shown earlier for uninfected cells (Fig. 2), in vector-infected NIH3T3 cells, the doxorubicin-induced growth arrest was accompanied by a significant increase in levels of hypophosphorylated RB and p130 (Fig. 3 B). In addition, as shown earlier for uninfected cells, treatment of empty vector-infected NIH3T3 cells with doxorubicin led to a decrease in mRNA levels of the E2F-regulated genes, AIM-1 and Stathmin (Fig. 3 A). A similar doxorubicin-induced decrease was detected in protein levels of AIM-1 and Stathmin (Fig. 3 B). Expression of E2F1-dlTA did not significantly affect the doxorubicin-induced accumulation of hypophosphorylated RB and p130; however, it abolished the decrease in mRNA and protein levels of AIM-1 and Stathmin (Fig. 3, Aand B). Thus, in cells expressing E2F1-dlTA, levels of AIM-1 and Stathmin remained unchanged throughout the experiment. Following doxorubicin addition, cells infected with an empty vector, similarly to uninfected cells, arrested at the G1 and G2/M phases of the cell cycle. This arrest at G1 and G2/M persisted for at least 96 h (Fig. 3 C). Expression of E2F-1dlTA did not result in noticeable changes in cell cycle distribution of untreated cells; however, it had profound effects on cell cycle distribution after treatment with doxorubicin. Cells expressing E2F-1dlTA failed to arrest at G1 and accumulated at G2/M. Interestingly, growth arrest at G2/M was not maintained, the percentage of cells at G2/M gradually decreased, and a concomitant increase in cells with 4 n DNA content was detected (Fig. 3 C). These data suggest that while E2F is not essential for the initiation of G2/M arrest, it is required for its maintenance. To study more directly the role of E2F-RB complexes in the response to DNA damage, we tested the effect of their dissociation by the papilloma virus E7 protein. To this end, NIH3T3 cells were infected with a retrovirus containing either an empty vector, the wtE7 gene of HPV16, or a mutated E7, E7Δ21–35, which does not bind RB family members. The wt and mutated E7 were expressed at similar levels (data not shown); however, expression of wtE7, but not E7Δ21–35, inhibited the doxorubicin-induced decrease in protein levels of AIM-1 and Stathmin (Fig. 4 A). In addition, expression of wtE7 led to profound changes in cell cycle distribution after doxorubicin treatment (Fig. 4 B). The effects of wtE7 expression were highly similar to those of E2F-1dlTA and included: 1) failure of cells to arrest at G1 and their accumulation at G2/M; 2) inability to maintain growth arrest at G2/M, with a gradual decrease in the percentage of cells at G2/M and an increase in cells with 4 n DNA content (Fig. 4 B). Expression of E7Δ21–35 had no apparent effect on cell cycle distribution after DNA damage (Fig. 4 B). These findings suggest that endogenous E2F-RB complexes mediate repression of mitotic genes and sustained G2/M arrest in response to DNA damage. To identify the distinct members of the E2F and RB families that mediate the repression of E2F-regulated genes in response to DNA damage we performed a ChIP using antibodies directed against specific family members. The human Stathmin promoter contains three putative E2F-binding sites at positions −28, −577, and −701 upstream to the transcription start site (47Melhem R.F. Zhu X.X. Hailat N. Strahler J.R. Hanash S.M. J. Biol. Chem. 1991; 266: 17747-17753Abstract Full Text PDF PubMed Google Scholar), and our analysis of the murine Stathmin promoter indicates that it too contains three putative E2F-binding sites. Taken together with our observation that E2F-induced up-regulation of Stathmin does not require de novo protein synthesis (Fig. 1 A), this sequence information suggests that E2Fs may interact with the St
Referência(s)