Abstract: ATF3 is a stress-inducible gene that encodes a member of the ATF/CREB family of transcription factors. Current literature indicates that ATF3 affects cell death and cell cycle progression. However, controversies exist, because it has been demonstrated to be a negative or positive regulator of these processes. We sought to study the roles of ATF3 in both cell death and cell cycle regulation in the same cell type using mouse fibroblasts. We show that ATF3 promotes apoptosis and cell cycle arrest. Fibroblasts deficient in ATF3 (ATF3-/-) were partially protected from UV-induced apoptosis, and fibroblasts ectopically expressing ATF3-/- under the tet-off system exhibited features characteristic of apoptosis upon ATF3 induction. Furthermore, ATF3-/- fibroblasts transitioned from G2 to S phase more efficiently than the ATF3+/+ fibroblasts, suggesting a growth arrest role of ATF3. Consistent with the growth arrest and pro-apoptotic roles of ATF3, ATF3- fibroblasts upon Ras transformation exhibited higher growth rate, produced more colonies in soft agar, and formed larger tumor upon xenograft injection than the ATF3+/+ counterparts. ATF3-/- cells, either with or without Ras transformation, had increased Rb phosphorylation and higher levels of various cyclins. Significantly, ATF3 bound to the cyclin D1 promoter as shown by chromatin immunoprecipitation (ChIP) assay and repressed its transcription by a transcription assay. Taken together, our results indicate that ATF3 promotes cell death and cell arrest, and suppresses Ras-mediated tumorigenesis. Potential explanations for the controversy about the roles of ATF3 in cell cycle and cell death are discussed. ATF3 is a stress-inducible gene that encodes a member of the ATF/CREB family of transcription factors. Current literature indicates that ATF3 affects cell death and cell cycle progression. However, controversies exist, because it has been demonstrated to be a negative or positive regulator of these processes. We sought to study the roles of ATF3 in both cell death and cell cycle regulation in the same cell type using mouse fibroblasts. We show that ATF3 promotes apoptosis and cell cycle arrest. Fibroblasts deficient in ATF3 (ATF3-/-) were partially protected from UV-induced apoptosis, and fibroblasts ectopically expressing ATF3-/- under the tet-off system exhibited features characteristic of apoptosis upon ATF3 induction. Furthermore, ATF3-/- fibroblasts transitioned from G2 to S phase more efficiently than the ATF3+/+ fibroblasts, suggesting a growth arrest role of ATF3. Consistent with the growth arrest and pro-apoptotic roles of ATF3, ATF3- fibroblasts upon Ras transformation exhibited higher growth rate, produced more colonies in soft agar, and formed larger tumor upon xenograft injection than the ATF3+/+ counterparts. ATF3-/- cells, either with or without Ras transformation, had increased Rb phosphorylation and higher levels of various cyclins. Significantly, ATF3 bound to the cyclin D1 promoter as shown by chromatin immunoprecipitation (ChIP) assay and repressed its transcription by a transcription assay. Taken together, our results indicate that ATF3 promotes cell death and cell arrest, and suppresses Ras-mediated tumorigenesis. Potential explanations for the controversy about the roles of ATF3 in cell cycle and cell death are discussed. During cancer development, the cells encounter many stress signals, including genotoxic damages, inappropriate activation of oncogenes, telomere erosion, hypoxia, and nutrient deprivation in the tumor microenvironment (for review, see Ref. 1Evan G.I. Vousden K.H. Nature. 2001; 411: 342-348Crossref PubMed Scopus (2711) Google Scholar). All along, the cells have built-in mechanisms to restrain or eliminate themselves (for review, see Ref. 2Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22406) Google Scholar). A prominent example is p53, which upon stress induction either arrests or kills cells (for reviews, see Refs. 3Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2292) Google Scholar and 4White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1325) Google Scholar). Another example is oncogene-induced killing: oncogenic stress, such as inappropriate activation of the E2F1 and c-Myc oncogenes, triggers apoptosis (for reviews, see Refs. 1Evan G.I. Vousden K.H. Nature. 2001; 411: 342-348Crossref PubMed Scopus (2711) Google Scholar and 5Sherr C.J. Genes Dev. 1998; 12: 2984-2991Crossref PubMed Scopus (663) Google Scholar). Therefore, the successful cancer cells are those that manage to foil the hardwired stress response to eliminate themselves during the process of transformation from normal cells to cancerous cells. Thus, to understand cancer development, it is important to study the stress response genes that may play an important role in this selfeliminating safeguard process. Activating transcription factor 3 (ATF3) 3The abbreviations used are: ATF3, activating transcription factor 3 gene; CREB, cAMP responsive element-binding protein; RT, reverse transcription; ChIP, chromatin immunoprecipitation; Cdk, cyclin-dependent kinase; Rb, retinoblastoma; PARP, poly-(ADP-ribose) polymerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling; BrdU, 5-bromo-2-deoxyuridine; FBS, fetal bovine serum; UV, ultraviolet light; MEF, mouse embryonic fibroblast; MTT, methylthiazolyldiphenyl-tetrazolium; H&E, hematoxylin and eosin; s.c., subcutaneous; i.v., intravenous; JNK, c-Jun N-terminal kinase; PI, propidium iodide. is a member of the ATF/CREB family of transcription factors. Overwhelming evidence indicates that ATF3 is a stress-inducible gene: its mRNA level is low or not detectable in most cells, but is greatly induced by a variety of stress signals, including genotoxic agents such as ultraviolet light (UV), benzo-[a]pyrene diol epoxide (BPDE), ionizing radiation, and methyl methanesulfonate (for reviews, see Refs. 6Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar and 7Hai T. Hartman M.G. Gene (Amst.). 2001; 273: 1-11Crossref PubMed Scopus (657) Google Scholar). In addition, ATF3 is induced by ischemia (8Allen-Jennings A.E. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (258) Google Scholar) and hypoxia 4K. Ameri, C. Culmsee, M. Raida, D. M. Katschinski, R. H. Wenger, T. Hai, E. Wagner, and A. L. Harris, submitted manuscript. , conditions encountered by cancer cells in the tumor microenvironment. Emerging evidence suggests that ATF3 may play a role in cancer development. It has been reported to affect cell death and cell cycle progression, two processes that regulate the growth of cancer cells. However, controversies remain for its roles in these processes. For cell death, ATF3 has been reported to be either pro-apoptotic or anti-apoptotic. Ectopic expression of ATF3 induced apoptosis in ovarian cells (11Syed V. Mukherjee K. Lyons-Weiler J. Lau K.M. Mashima T. Tsuruo T. Ho S.M. Oncogene. 2005; 24: 1774-1787Crossref PubMed Scopus (96) Google Scholar) and enhanced the ability of etoposide or camptothecin to induce apoptosis in HeLa cells (12Mashima T. Udagawa S. Tsuruo T. J. Cell. Physiol. 2001; 188: 352-358Crossref PubMed Scopus (92) Google Scholar), suggesting a pro-apoptotic role of ATF3. Consistently, primary islets derived from ATF3 knock-out (ATF3-/-) mice were partially protected from cytokine- and nitric oxide-induced apoptosis (13Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar). Furthermore, antisense ATF3 reduced stress-induced apoptosis in endothelial cells (14Nawa T. Nawa M.T. Adachi M.T. Uchimura I. Shimokawa R. Fujisawa K. Tanaka A. Numano F. Kitajima S. Atherosclerosis. 2002; 161: 281-291Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Therefore, both gain-of-function (ectopic expression) and loss-of-function (knock-out and antisense) approaches support a pro-apoptotic role of ATF3. However, several reports suggest that ATF3 is anti-apoptotic. Adenovirus-mediated expression of ATF3 reduced nerve growth factor (NGF) withdrawal-induced apoptosis in superior cervical ganglion neurons in vitro (15Nakagomi S. Suzuki Y. Namikawa K. Kiryu-Seo S. Kiyama H. J. Neurosci. 2003; 23: 5187-5196Crossref PubMed Google Scholar), suppressed kainic acid-induced death in hippocampal neurons in vivo (16Francis J.S. Dragunow M. During M.J. Brain Res. Mol. Brain Res. 2004; 124: 199-203Crossref PubMed Scopus (42) Google Scholar), and inhibited adriamycin-induced apoptosis in primary cardiomyocytes in vitro (17Nobori K. Ito H. Tamamori-Adachi M. Adachi S. Ono Y. Kawauchi J. Kitajima S. Marumo F. Isobe M. J. Mol. Cell. Cardiol. 2002; 34: 1387-1397Abstract Full Text PDF PubMed Google Scholar). Thus far, the anti-apoptotic role of ATF3 has not been demonstrated by the loss-of-function approach. For cell cycle regulation, some reports suggest that ATF3 promotes cell proliferation. Ectopic expression of ATF3 by transient transfection moderately induced DNA synthesis in hepatic tumor cells (18Allan A.L. Albanese C. Pestell R.G. LaMarre J. J. Biol. Chem. 2001; 276: 27272-27280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Furthermore, retrovirus-mediated stable expression of ATF3 promoted the proliferation of chick embryo fibroblasts under low serum concentrations (19Perez S. Vial E. van Dam H. Castellazzi M. Oncogene. 2001; 20: 1135-1141Crossref PubMed Scopus (53) Google Scholar). In contrast, however, ectopic expression of ATF3 suppressed cell cycle progression in HeLa cells (20Fan F. Jin S. Amundson S.A. Tong T. Fan W. Zhao H. Zhu X. Mazzacurati L. Li X. Petrik K.L. Fornace Jr., A.J. Rajasekaran B. Zhan Q. Oncogene. 2002; 21: 7488-7496Crossref PubMed Scopus (151) Google Scholar). Therefore, similar to the situation in cell death, conflicting data exist for the roles of ATF3 in cell cycle regulation. One potential explanation for the above conflicting results is the diverse cell types used in the studies, ranging from primary islets or neurons to hepatic tumor cells. Other explanations include the varying levels and durations of ATF3 expression, and the differences in the approaches used in the studies; some used the gain-of-function approach whereas others used the loss-of-function approach. We sought to study the roles of ATF3 in cell death and cell cycle regulation in the same cell type using fibroblasts. In addition, we tested the hypothesis that ATF3 plays a role in cancer development using the Ras-stimulated transformation of fibroblasts as a model, a well established and widely accepted model for studying tumorigenesis. In this report, we show that ATF3 promoted apoptosis and cell cycle arrest. Its action on cell cycle arrest correlated with reduced phosphorylation of Rb and reduced protein levels of various cyclins in ATF3+/+ cells compared with that in ATF3-/- cells. Furthermore, we show that ATF3 suppressed Ras-stimulated tumorigenesis, at least in part, by inhibiting cell proliferation and promoting cell death. Cell Culture, Plasmids, and the JNK-I Inhibitor—ATF3 knock-out mice in the C57BL/6 background were described previously (13Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar). Primary mouse embryonic fibroblasts (MEFs) were isolated from day 13.5 wild-type C57BL/6 or ATF3 knock-out embryos and immortalized by the 3T9 protocol: passaged at the density of 9 × 105 cells per 6-cm plate every 3 days (21Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2004) Google Scholar). MEFs were maintained in Dulbecco's modified minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mm glutamine, 0.1 mm nonessential amino acid, and 55 μm β-mercaptoethanol. Phoenix ecotropic virus packaging cells were maintained in DMEM supplemented with 10% FBS. pRetro-off-HA-ATF3 was generated by inserting hemagglutinin (HA)-tagged human ATF3 open reading frame into the NotI site of pRetro-off vector (Clontech), pRetro-off-HA-ATF4 by inserting the HA-ATF4 open reading frame into the BamHI site of the vector, and pRetro-off-HA-ATF3-(1-100) by inserting the DNA fragment encoding HA-tagged ATF3 amino acids 1-100 into the NotI site. pBabe-puro, pBabe-hygro, and pBabe-puro-H-Ras(V12) were kindly provided by Dr. Gustavo Leone (Ohio State University). pBabe-hygro-ATF3 was generated by inserting the open reading frame of human ATF3 into the EcoRI and SalI sites of pBabe-hygro. JNK-I, a cell-permeable peptide that inhibits the activation of the JNK pathway (22Bonny C. Oberson A. Negri S. Sauser C. Schorderet D.F. Diabetes. 2001; 50: 77-82Crossref PubMed Scopus (517) Google Scholar), was from the Cleveland Clinic Foundation. Retrovirus Production and Infection—The Phoenix ecotropic virus packaging cells were transfected with pBabe constructs using the calcium phosphate method; the medium containing the viruses was collected 48 h after transfection and aliquots kept at -80 °C until use. MEFs were infected with high titer retroviruses in the presence of 4 μg/ml polybrene and selected by adding the appropriate antibiotics (puromycin 2.5 μg/ml or hygromycin 250 μg/ml) at 48 h after infection. In general, more than 50% of the cells survived selection, and by day 4 most of the non-transduced cells had died off. Transformation was judged successful if the cells displayed morphological changes characteristic of Ras transformation (highly refractile with thin and long projections). The resulting pools of transduced cells on day 7 were used in subsequent experiments. All results presented were derived from at least three repeated experiments using independently transduced cells and were reproducible using two batches of immortalized cells derived from different litters of mice. Generation of Tet-off Stable Cells and Apoptosis Assays—Stable cell lines were established by transfecting the wild-type fibroblasts with various pRetro-off constructs followed by puromycin selection. Individual colonies were selected, expanded, and maintained in the presence of tetracycline and puromycin. Each stable cell line was grown in the absence of tetracycline for the indicated times to induce the expression of the transgenes before assays. For trypan blue stain, both the floating and attached cells were collected and stained, and the blue cells scored as dead. For Annexin V stain, the cells were grown on Superfrost Plus slides (VWR Scientific); the unfixed cells were stained with 5% Annexin V-FITC (BD Pharmingen) and 5 μg/μl propidium iodide (PI) (Sigma), and visualized using a Bio-Rad MRC 1024 confocal microscope. For DAPI stain, the cells were fixed with 4% paraformaldehyde and stained with 1 μm DAPI. For TUNEL assay, the cells were fixed, permeabilized, and incubated with hydrogen peroxide before incubated with biotin-dCTP and terminal transferase (Invitrogen). The signals were detected by the ABC complex followed by the DAB substrate solution (Vector). For DNA laddering, both the floating and attached cells were collected and the genomic DNAs extracted for analysis on a 2% agarose gel. Serum Stimulation and BrdU Labeling—Cells at about 50% confluency were serum-starved with 0.1% FBS for 72 h and restimulated with 10% FBS for indicated times before harvesting for immunoblot analysis (below), transcription assay (below), or BrdU labeling (Roche Applied Science) according to the manufacturer's instructions. UV Treatment and Viability Assay—2 × 105 cells were seeded on 6-cm plates and treated with UV at the dose indicated in the Fig. 1 legend. At various times after UV treatment, cells were either harvested for immunoblot analysis (below) or assayed for viability using crystal violet stain quantified by A595 reading. The A595 reading at 48 and 72 h after UV treatment was standardized against the A595 of the respective cells upon seeding (at 4 h after seeding when the cells became attached) to reduce the artifacts caused by seeding variations. The standardized A595 reading of the wild-type cells at each time point was arbitrarily defined as 1 to obtain the relative cell viability of the knock-out cells. Chromatin Immunoprecipitation (ChIP) Assay—Cells were incubated with 1% formaldehyde at room temperature for 10 min to cross-link proteins and DNAs, followed by sonication to shear the DNAs to an average size of 500-1000 bp. Immunoprecipitation was carried out using 2 μg of ATF3 antibody (Santa Cruz Biotechnology) or IgG. After reversal of cross-linking, the DNA fragments were purified by phenol extraction and ethanol precipitation, followed by PCR analysis using primers flanking the CRE/ATF site on the cyclin D1 promoter: 5′-CGAGCGATTTGCATATCTACC-3′ (upstream) and 5′-GTAGTCCGTGTGACGTTACTG-3′ (downstream). Transcriptional Assay of Endogenous Cyclin D1 Gene—Nuclear RNAs were isolated from the nuclei of serum-starved and restimulated cells using the TRIzol method (Invitrogen) and treated with DNase I to remove the contaminating genomic DNAs. The cyclin D1 pre-mRNAs were assayed by reverse transcription coupled with polymerase chain reaction (RT-PCR) as detailed previously (8Allen-Jennings A.E. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) using a primer set targeted at the exon 2 and intron 2 of the corresponding gene: 5′-TTGACTGCCGAGAAGTTGTG-3′ (upstream) and 5′-ACAGAGGTAGAATGGGTTGG-3′ (downstream). A control RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included using the following primers: 5′-CCGGATCCTGGGAAGCTTGTCATCAACGG-3′ (upstream) and 5′-GGCTCGAGGCAGTGATGGCATGGACTG-3′ (downstream). Reactions without reverse transcriptase were included to confirm that the signals were not derived from the genomic DNAs. Cell Growth at Low Concentrations of Serum and Colony Formation in Soft Agar—For cell growth analysis, 1 × 104 cells were seeded into each well of a 6-well plate and grown in the presence of 0.1-2% FBS for 1-7 days as indicated in the Fig. 5 legend. Cells were stained by crystal violet and the A595 readings measured. For anchorage-independent growth, 5 × 104 cells were resuspended in 4 ml of growth medium containing 0.3% agarose and plated on 6-cm plates containing a solidified bottom layer made of 0.6% agarose in medium. After the 0.3% agarose solidified, 3 ml of growth medium were added to the plates and replaced every 3 days. 21 days after plating, colonies were stained with methylthiazolyldiphenyl-tetrazolium (MTT) and imaged at ×10 magnification. Each experiment was performed with duplicate plates. Xenograft Tumor Formation Assays—2 × 106 cells were resuspended in 100 μl of sterile phosphate-buffered saline and injected subcutaneously (s.c.) into the flank or intravenously (i.v.) into the tail vein of 8-10 week-old athymic NCr male mice (Taconic). For s.c. injection, ATF3+/+ and ATF3-/- cells were injected into the right and left flanks of the same mouse to eliminate the differences due to the host. Tumor size was determined at 3-day intervals by measuring the length (L) and width (W) of the tumor using a pair of calipers, and the tumor volume calculated as (L × W2)/2 (23Lin A.W. Barradas M. Stone J.C. van Aelst L. Serrano M. Lowe S.W. Genes Dev. 1998; 12: 3008-3019Crossref PubMed Scopus (766) Google Scholar). At 21 days after injection, mice were weighed to obtain their body weights and euthanized. Subcutaneous tumors and the lungs were excised and weighed, and the ratio of lung to total body weight was calculated. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the Ohio State University. Immunoblot and Immunohistochemistry Analysis—Equal amounts of whole cell lysates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by immunoblot using polyvinylidene fluoride membrane (Immobilon-P; Millipore) and various primary antibodies: ATF3, Cdk2, Cdk4, cyclin A, cyclin E, Rb, Erk (Santa Cruz Biotechnology), p-Rb, cleaved caspase 3, cleaved PARP (Cell Signaling), actin (Sigma), and cyclin D1 (Calbiochem). Bound primary antibodies were detected using the appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) and Lumi-Light Western blotting substrate (Roche Applied Science). Paraffin-embedded xenograft tumor sections were analyzed for phosphohistone H3 and cleaved caspase 3 by immunohistochemistry as described before (8Allen-Jennings A.E. Hartman M.G. Kociba G.J. Hai T. J. Biol. Chem. 2001; 276: 29507-29514Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) using the antibody against phosphohistone H3 (Upstate Biotechnologies) or cleaved caspase 3 (Cell Signaling). To quantify the phosphohistone H3-positive cells, the positive cells within the entire tumor sections were counted. The tumor area was measured by the Meta Vue program under ×40 magnification, and the positive cells per mm2 were calculated. Statistical Analysis—All numerical values are mean ± S.E. Comparison between two groups was made by two-sample Student's t test, and comparison among three groups by one-way analysis of variance (ANOVA). p < 0.05 was considered statistically significant. ATF3 Is Pro-apoptotic in Immortalized Fibroblasts—We isolated mouse embryonic fibroblasts (MEFs) from wild-type (ATF3+/+) and ATF3 knock-out (ATF3-/-) mice (13Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar), and immortalized them as detailed under "Experimental Procedures." To test whether ATF3 deficiency affects the cells in their response to stress-induced apoptosis, we used the UV-induced apoptosis as a paradigm. Fig. 1, A and B show that UV induced ATF3 in the wild-type fibroblasts but not the knock-out cells. The induction was detected 2 h after treatment, consistent with previous reports that ATF3 is induced by stress signals within 2 h in most stress paradigms (for a review, see Ref. 6Hai T. Wolfgang C.D. Marsee D.K. Allen A.E. Sivaprasad U. Gene Expr. 1999; 7: 321-335PubMed Google Scholar). Significantly, ATF3-/- cells were partially protected from UV-induced apoptosis as evidenced by increased cell viability at 48 and 72 h after treatment (Fig. 1C), decreased activation of caspase 3, and decreased cleavage of poly(ADP-ribose) polymerase (PARP) (Fig. 1D). Fig. 1C shows the averages from three experiments and Fig. 1D is a representative result. Taken together, these results suggest a pro-apoptotic role of ATF3. To confirm this conclusion by a complementary approach, we attempted to express ATF3 ectopically in the wild-type fibroblasts. Although stable cells constitutively expressing ATF3 were reported by others in HeLa cells (12Mashima T. Udagawa S. Tsuruo T. J. Cell. Physiol. 2001; 188: 352-358Crossref PubMed Scopus (92) Google Scholar), HT1080 cells (24Yan C. Wang H. Boyd D.D. J. Biol. Chem. 2002; 277: 10804-10812Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and chicken embryo fibroblasts (19Perez S. Vial E. van Dam H. Castellazzi M. Oncogene. 2001; 20: 1135-1141Crossref PubMed Scopus (53) Google Scholar), we were unable to establish such stable lines using the mouse fibroblasts. Therefore, we used the tet-off system to express ATF3 in an inducible manner. We analyzed three clones and compared them to three control cell lines generated in parallel: (a) ATF3-(1-100) line expressing a mutant ATF3 that lacks the leucine zipper domain and does not bind to DNA (25Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar), (b) ATF4 line expressing another member of the ATF/CREB family of transcription factors, (c) a vector control line. As shown in Fig. 2A, expression of ATF3 led to reduced cell viability as assayed by trypan blue exclusion test; however, expression of ATF3-(1-100) or ATF4 did not. We then analyzed one ATF3 clone (clone 38) and compared it to the vector control cells. As shown in Fig. 2, B-E, expression of ATF3 led to features characteristic of apoptosis: 1) membrane inversion by Annexin V stain (panel B), 2) pyknotic nuclei by DAPI stain (panel C), DNA fragmentation by TUNEL assay (panel D), and DNA laddering (panel E). Therefore, both loss-of-function and gain-of-function approaches demonstrated that ATF3 is pro-apoptotic in mouse fibroblasts. ATF3 Inhibits Serum Stimulation-induced Cell Cycle Progression—To determine whether ATF3 plays a role in cell cycle progression, we serum-starved the ATF3+/+ and ATF3-/- cells for 3 days and simulated them with 10% serum. As shown in Fig. 3A, serum stimulation transiently induced ATF3 expression in the wild-type cells. BrdU labeling indicated that ATF3-/- cells progressed from G1 to S phase more efficiently than the ATF3+/+ cells. At 16 h after serum stimulation, about 60% (62 ± 4%) of the ATF3-/- cells underwent DNA replication, but only about 45% (44 ± 2%) of the ATF3+/+ cells did (Fig. 3B, p < 0.05). The difference between the wild-type and knock-out cells remained at 20 h after serum stimulation (Fig. 3B, p < 0.05). At 9 and 12 h after serum stimulation, some cells had already progressed into S phase in both wild-type and knock-out cells, but there was no difference between them. Fig. 3B shows the average of three experiments and Fig. 3C shows a representative image of BrdU-stained cells at different time points. ATF3 Inhibits Ras-stimulated Cell Growth at Low Serum Concentrations and in Soft Agar—Because cell cycle arrest and cell death are important brakes for cancer cell progression, the above results suggested that ATF3 may function as a tumor suppressor. To test this hypothesis, we used the Ras-stimulated transformation of immortalized MEFs, a well established and widely accepted paradigm for studying transformation. We transduced the immortalized wild-type (ATF3+/+) and knock-out (ATF3-/-) fibroblasts with a retroviral vector expressing oncogenic H-Ras (RasV12) or an empty vector, and selected the transduced cells with appropriate antibiotics. As shown in Fig. 4A, ATF3 expression was induced in the wild-type cells upon Ras(V12) transduction. This is an important result, because ATF3, as an inducible gene, is not expressed (or expressed at a very low level) in untreated wild-type cells. If ATF3 is not induced by oncogenic Ras in the wild-type cells, the deficiency of ATF3 in the knock-out cells may not have any detectable consequences in this paradigm. Oncogenic Ras activates many signaling pathways, including the JNK pathway (for review, see Ref. 26Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (920) Google Scholar). Because JNK pathway is involved in the induction of ATF3 by various signals (13Hartman M.G. Lu D. Kim M.L. Kociba G.J. Shukri T. Buteau J. Wang X. Frankel W.L. Guttridge D. Prentki M. Grey S.T. Ron D. Hai T. Mol. Cell. Biol. 2004; 24: 5721-5732Crossref PubMed Scopus (259) Google Scholar, 27Cai Y. Zhang C. Nawa T. Aso T. Tanaka M. Oshiro S. Ichijo H. Kitajima S. Blood. 2000; 96: 2140-2148Crossref PubMed Google Scholar), we examined whether the JNK pathway is involved in the induction of ATF3 by Ras. Fig. 4B shows that treatment of the Ras-transformed cells with JNK-I, a cell-permeable inhibitor of the JNK pathway (22Bonny C. Oberson A. Negri S. Sauser C. Schorderet D.F. Diabetes. 2001; 50: 77-82Crossref PubMed Scopus (517) Google Scholar), reduced the expression of ATF3, indicating that the induction of ATF3 by H-Ras(V12) is mediated, at least in part, by the JNK pathway. For the convenience of discussion, we will refer to the Ras-transformed wild-type cells as Ras/ATF3+/+ and the Ras-transformed knock-out cells as Ras/ATF3-/- cells. All results presented below involving the Ras-transformed cells were derived from at least three independent experiments. For each experiment, pools of retrovirustransduced cells (detailed under "Experimental Procedures") were used for the assays. To rule out the possibility that the differences between Ras/ATF3+/+ and Ras/ATF3-/- cells were caused by the immortalization process rather than the ATF3 deficiency in the knock-out cells, we generated a second batch of immortalized ATF3+/+ and ATF3-/- MEFs and transformed them with H-Ras(V12). Similar results were obtained from the second batch of immortalized cells (data not shown). To examine whether ATF3 deficiency affects Ras-stimulated transformation, we examined the growth of Ras/ATF3+/+ and Ras/ATF3-/- cells at low concentrations of serum. Time course analysis showed that the cell number of Ras/ATF3-/- cells increased faster than that of Ras/ATF3+/+ cells; the difference was detectable starting at day 3 after seeding and continued to day 7 when the plates became confluent and the experiments terminated (Fig. 5A,*, p < 0.05, **, p < 0.01). Dose analysis indicated that the increase of cell number over a 4-day period (from day 2 to day 6) was statistically different (p < 0.05) between Ras/ATF3+/+ and Ras/ATF3-/- cells at 1% of serum (Fig. 5B). To test whether the difference was caused by the deficiency of ATF3 in the knock-out cells, we complemented the Ras/ATF3-/- cells with ATF3 by retroviral tran