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• W2048547613 abstract "Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4ΔN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4ΔN1 increased the p53 protein level, but decreased the p21Cip1 transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4ΔN1-binding protein. MAGE-A4ΔN1 was recruited through association with Miz-1 to the p21Cip1 promoter and down-regulated transcription of p21Cip1. In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4ΔN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4ΔN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4. Gankyrin, a recently discovered oncoprotein, is a promising target for drug therapy because it is overexpressed in most hepatocellular carcinomas. Since gankyrin interacts with MAGE-A4, we made several MAGE-A4 mutants and assessed their effects on cell growth. We found that the C-terminal 107 amino acids of MAGE-A4 (MAGE-A4ΔN1) induced p53-dependent and p53-independent apoptosis. MAGE-A4ΔN1 increased the p53 protein level, but decreased the p21Cip1 transcript and protein levels. During apoptosis Bcl-xL was down-regulated and mitochondrial integrity was disrupted. A yeast two-hybrid screen identified Miz-1 as a MAGE-A4ΔN1-binding protein. MAGE-A4ΔN1 was recruited through association with Miz-1 to the p21Cip1 promoter and down-regulated transcription of p21Cip1. In 293T cells and U-2 OS cells, full-length MAGE-A4 was processed to generate a C-terminal fragment of 104 amino acids with activities similar to MAGE-A4ΔN1. Processing was inhibited with a broad range caspase inhibitor Z-VAD-FMK, but not by site-directed mutagenesis of aspartic acids in MAGE-A4, suggesting an indirect involvement of caspase(s) in the processing. The amount of the processed form was increased by exposure of cells to adriamycin. Transduction with a HIV Tat-MAGE-A4ΔN1 fusion protein suppressed anchorage-independent growth of gankyrin-overexpressing cells in vitro and in vivo. These results demonstrate that the C-terminal fragment of MAGE-A4 induces apoptosis at least partly by binding to Miz-1, and that the fragment may be exploited as an anticancer agent. Furthermore, the finding that a C-terminal fragment with pro-apoptotic activity is generated from full-length MAGE-A4 after genotoxic stress in human cells suggests a novel function for MAGE-A4. The melanoma antigen (MAGE) 1The abbreviations used are: MAGE, melanoma antigen; 3AT, 3-aminotriazole; Cdk, cyclin-dependent kinase; FITC, fluorescein isothiocyanate; HA, hemagglutinin; HCC, hepatocellular carcinoma; HIV, human immunodeficiency virus; hrGFP, human recombinant green fluorescent protein; RB, retinoblastoma tumor suppressor protein; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; Z, benzyloxycarbonyl; FMK, fluoromethylketone; PI, propidium iodide. 1The abbreviations used are: MAGE, melanoma antigen; 3AT, 3-aminotriazole; Cdk, cyclin-dependent kinase; FITC, fluorescein isothiocyanate; HA, hemagglutinin; HCC, hepatocellular carcinoma; HIV, human immunodeficiency virus; hrGFP, human recombinant green fluorescent protein; RB, retinoblastoma tumor suppressor protein; TRITC, tetramethylrhodamine isothiocyanate; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; Z, benzyloxycarbonyl; FMK, fluoromethylketone; PI, propidium iodide. gene family is composed of more than 25 genes in humans (1Chomez P. De Backer O. Bertrand M. De Plaen E. Boon T. Lucas S. Cancer Res. 2001; 61: 5544-5551PubMed Google Scholar). MAGE genes are highly expressed in various forms of cancer, but not in most healthy adult tissues except for the testis (2Forslund K.O. Nordqvist K. Exp. Cell Res. 2001; 265: 185-194Crossref PubMed Scopus (73) Google Scholar). MAGE genes encode tumor-specific antigenic peptides presented by HLA class I molecules to CD8+ T lymphocytes (3van der Bruggen P. Traversari C. Chomoz P. Lurquin C. De Plaen E. van den Eynde B. Knuth A. Boon T. Science. 1991; 254: 1643-1647Crossref PubMed Scopus (3104) Google Scholar), and have therefore been extensively studied as targets for cancer immunotherapy (4Castelli C. Rivoltini L. Andreola G. Carrabba M. Renkvist N. Parmiani G. J. Cell Physiol. 2000; 182: 323-331Crossref PubMed Scopus (101) Google Scholar, 5Herin M. Lemoine C. Weynants P. Vessiere F. Van Pel A. Knuth A. Devos R. Boon T. Int. J. Cancer. 1987; 39: 390-396Crossref PubMed Scopus (188) Google Scholar). Despite recent reports that some members of the MAGE family play important roles in cell cycle control and apoptosis (6Barker P.A. Salehi A. J. Neurosci. Res. 2002; 67: 705-712Crossref PubMed Scopus (261) Google Scholar), the physiological functions of MAGE proteins remain mostly unknown. Hepatocellular carcinoma (HCC), one of the most common cancers in the world, develops from transformed hepatocytes during the course of chronic liver diseases (7Okuda K J. Hepatol. 2000; 32: 225-237Abstract Full Text PDF PubMed Google Scholar). When transformed hepatocytes expand in vivo, they encounter various microenvironmental stresses, such as hypoxia, decreased growth factor availability, and lack of nutrient supply, all of which activate apoptosis (8Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1624) Google Scholar, 9Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2702) Google Scholar). In addition, activated oncogenes also trigger apoptosis (10Hanahan D. Weinberg R.A. Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (22068) Google Scholar). Therefore, resistance to apoptosis is one of the essential alterations for development of HCCs. Gankyrin (also known as PSMD10 and p28) is an oncoprotein overexpressed in most HCCs (11Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar). Gankyrin consists of seven ankyrin repeats (12Krzywda S. Brzozowski A.M. Higashitsuji H. Fujita J. Welchman R. Dawson S. Mayer R.J. Wilkinson A.J. J. Biol. Chem. 2004; 279: 1541-1545Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and binds to the retinoblastoma tumor suppressor protein (RB), the S6 ATPase subunit of the 26 S proteasome, and cyclin-dependent kinase 4 (Cdk4) (11Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar, 13Dawson S. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. J. Biol. Chem. 2002; 277: 10893-10902Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Overexpression of gankyrin increases degradation of RB in vitro and in vivo, and oncogenically transforms NIH/3T3 cells (11Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar). Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16INK4A and p18INK4C (14Tsai L.J. Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (98) Google Scholar). In a rodent model of hepatocarcinogenesis, gankyrin is overexpressed from the earliest stage of tumor development (15Park T.J. Kim H.S. Byun K.H. Jang J.J. Lee Y.S. Lim I.K. Mol. Carcinog. 2001; 30: 138-150Crossref PubMed Scopus (55) Google Scholar). These findings suggest that gankyrin is a major player in cell cycle control and tumorigenesis in HCCs. The POZ domain/zinc finger transcription factor Miz-1 binds to the core promoter of the Cdk inhibitor p21Cip1 gene (16Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (291) Google Scholar, 17Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell Biol. 2001; 3: 392-399Crossref PubMed Scopus (450) Google Scholar). Although the p21Cip1 promoter is highly responsive to p53, both p53 and Miz-1 are necessary for up-regulation of p21Cip1 and cell cycle arrest upon UV irradiation (18Herold S. Wanzel M. Beuger V. Frohme C. Beul D. Hillukkala T. Syvaoja J. Saluz H.P. Haenel F. Eilers M. Mol. Cell. 2002; 10: 509-521Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Activation of p53 induces either cell cycle arrest mediated by transcriptional activation of p21Cip1 or apoptotic cell death (9Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2702) Google Scholar, 19Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6713) Google Scholar). The transactivation activity of Miz-1 has been shown to be inhibited by interaction with Myc (17Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell Biol. 2001; 3: 392-399Crossref PubMed Scopus (450) Google Scholar, 20Seoane J. Pouponnot C. Staller P. Schader M. Eilers M. Massague J. Nat. Cell Biol. 2001; 3: 400-408Crossref PubMed Scopus (406) Google Scholar), and the recruitment of Myc to the p21Cip1 promoter by Miz-1 results in repression of p21Cip1 transcription, even in the presence of activated p53 (21Seoane J. Le H.V. Massague J. Nature. 2002; 419: 729-734Crossref PubMed Scopus (563) Google Scholar). Thus, Myc switches the p53-dependent response of cells from cell cycle arrest to apoptosis. Previously, we have found that MAGE-A4 specifically interacts with gankyrin and suppresses its tumorigenic activity (22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In this study, to further characterize the antitumorigenic activity of MAGE-A4 and to facilitate development of a therapeutic approach against HCCs, we made several mutants of MAGE-A4 and analyzed their effects on cell growth. We found that a C-terminal fragment of MAGE-A4 has strong pro-apoptotic activity, at least partly, by binding to Miz-1, and that a similar fragment is generated from full-length MAGE-A4 in human cells. Plasmids—Wild-type MAGE-A4 cDNA and mutant derivatives tagged with HA or FLAG cDNA, and gankyrin cDNA and Miz-1 cDNA tagged with HA cDNA were cloned into the eukaryotic expression vector pMKit-neo as previously described (11Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar, 22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Point mutations were introduced into plasmid DNA by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The bicistronic pIRES-hrGFP-1a vector (Stratagene) was used to express human p53, MAGE-A4 and MAGE-A4 mutants together with GFP. The pact-p53 plasmid expressing human wild-type p53 was kindly provided by Dr. S. Ishii, Tsukuba, Japan. A cDNA encoding 6 N-terminal histidines and the 11-amino acid protein transduction domain from the HIV tat protein (YGRKKRRQRRR) (23Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (885) Google Scholar) was fused with a cDNA encoding the full-length, N-terminal 210 or C-terminal 107 amino acids of MAGE-A4 in the pTAT vector (kindly provided by Dr. Steven F. Dowdy, La Jolla, CA). Cell Culture—Mouse NIH/3T3 cells and derivative GK-S25 cells overexpressing gankyrin, human U-2 OS cells, HuH-7 cells, PLC/PRF/5 cells, H1299 cells, HeLa cells, 293T cells, and monkey COS-7 cells were cultured and transfected with plasmid DNAs as described (22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). For some experiments, cells were treated with the following chemicals (Calbiochem, San Diego, CA) or vehicle alone: adriamycin (0.1 and 1.0 μm), Z-VAD-FMK (20 and 50 μm), Ac-LDESD-CHO (50 μm), Z-SEVD-FMK (50 μm), and Z-LEHD-FMK (50 μm). The numbers of surviving cells were counted by the trypan blue dye exclusion method using a hemocytometer under a microscope. To assess the anchorage dependence of growth in vitro, 5 × 102 cells were incubated with recombinant proteins and assayed as previously described (22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Statistical differences between sample means were calculated by analysis of variance, followed by the unpaired Student's t test. Western Blot Analysis, Immunoprecipitation, and Northern Blot Analysis—Western blot analysis and immunoprecipitation were performed as described (11Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (278) Google Scholar, 24Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Mouse monoclonal antibodies were anti-HA antibody (Roche Applied Science), anti-FLAG antibody (Sigma Aldrich, Tokyo, Japan), anti-SC35 antibody (Sigma Aldrich), anti-α-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-p53 antibody (BD Pharmingen, San Diego, CA), anti-His antibody (BD Biosciences Clontech, Palo Alto, CA), and anti-Bcl-xL antibodies (Santa Cruz Biotechnology, and BD Transduction Laboratories, Lexin, KY). Polyclonal antibodies were mouse anti-Miz-1 antibody (Santa Cruz Biotechnology), rabbit anti-β-actin antibody (Chemicon International, Inc., Temecula, CA), and horseradish peroxidase-conjugated goat anti-mouse antibody (DAKO, Kyoto, Japan) and anti-rabbit antibody (DAKO). Interaction of transiently expressed exogenous MAGE-A4 or its mutants with exogenous Miz-1 was examined by co-transfecting COS-7 or HeLa cells with plasmids expressing FLAG-tagged MAGE-A4 or MAGE-A4ΔN1, FLAG alone, HA-tagged Miz-1, or HA alone in various combinations. Preparation of nuclear and cytoplasmic extracts and Northern blot analysis were performed as described (24Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Measurement of Cellular DNA Content—Cells were harvested with 4 mm EDTA/PBS, fixed in 70% ethanol and stained with 5 μg/ml propidium iodide (PI, Sigma Aldrich). The DNA content of the cells was analyzed with a flow cytometer (EPICS XL; Beckmann Coulter, Tokyo, Japan). To selectively analyze cells expressing transfected cDNA, only cells manifesting a high level of GFP fluorescence was analyzed. TUNEL Assay—U-2 OS cells and HuH-7 cells were transfected with cDNAs encoding the C-terminal 107 amino acids of MAGE-A4 in the expression vector or vector alone using LipofectAmine Plus (Invitrogen, Carlsbad, CA). 36 h after transfection, the cells were rinsed three times with PBS and were fixed with 2% paraformaldehyde. The cells were then labeled by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction using the In Situ Cell Death Detection kit (Roche Applied Science). The same field of cells was examined under a confocal laser microscope (Olympus, Tokyo, Japan) for 4′, 6′-diamidino-2-phenylindole (DAPI) (Sigma Aldrich) staining and immunofluorescence. Measurement of Caspase Activities—Approximately 4 × 105 cells were used for measurement of caspase activities. Caspase-8- and caspase-9-like activities were measured using IETD-p-nitroanilide (MBL, Nagoya, Japan) and LEHD-p-nitroanilide (MBL) as substrates, respectively. The fluorescence of the released p-nitroanilide was measured at an excitation wavelength of 360 nm and an emission wavelength of 530 nm. Measurement of Mitochondrial Membrane Potential—To assess mitochondrial membrane potential, H1299 cells were transfected with plasmids expressing MAGE-A4ΔN1-IRES-hrGFP or vector alone. 48 h after transfection, cells were incubated in the medium containing 500 nm of MitoTracker Red CMXRos (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C. They were then trypsinized and resuspended in PBS. The red X-rosamine fluorescence of the GFP-positive cells was analyzed by flow cytometry. Immunofluorescence Staining—Immunofluorescence staining was performed using mouse monoclonal anti-FLAG antibody (Sigma Aldrich), rabbit polyclonal anti-HA antibody (Berkeley Antibody, Richmond, CA) or anti-cytochrome c antibody (Santa Cruz Biotechnology) as described previously (24Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The bound antibodies were reacted with FITC-linked anti-mouse and TRITC-linked anti-rabbit IgGs (Amersham Biosciences) and observed under a confocal laser microscope (Olympus). Yeast Two-hybrid Assay—Using the C-terminal 107 amino acids of MAGE-A4 as bait, a U-2 OS cell cDNA library was screened by the yeast two-hybrid method as described (22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Gel Shift Experiments—COS-7 cells were transfected with plasmids expressing HA-tagged Miz-1 and FLAG-tagged MAGE-A4ΔN1, and nuclear extracts were prepared as described (24Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Binding was carried out for 10 min at room temperature in 20 mm HEPES pH 7.8, 5 mm MgCl2, 5 mm KCl, 0.5% Nonidet P-40, 500 mm NaCl, 0.1% deoxycholate, and protease inhibitors. 32P-labeled oligonucleotides spanning the site of the human p21Cip1 gene (-46 to -32) was used as a probe (21Seoane J. Le H.V. Massague J. Nature. 2002; 419: 729-734Crossref PubMed Scopus (563) Google Scholar). Reporter Assays—Luciferase reporter plasmids containing the -518 to +32 region of the p21Cip1 gene as a promoter were co-transfected with pRL-TK (Promega, Madison, WI) and increasing amounts of plasmids expressing MAGE-A4ΔN1 onto HeLa cells by the calcium phosphate method. After 30 h, cells were lysed and luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer's protocol. Preparation of Wild-type MAGE-A4 and Mutants Fused to HIV Tat Protein—The His-tagged Tat-fused proteins were expressed in and purified from BL21(DE3)pLysS cells (Novagen, Darmstadt, Germany) as described (23Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (885) Google Scholar). BL21(DE3)pLysS cells from overnight culture were spun down and then sonicated in buffer Z (8 m urea, 100 mm NaCl, 20 mm Hepes, pH 8.0). The supernatant of the centrifuged sonicate was loaded onto an Econocolumn (Bio-Rad Laboratories, Hercules, CA) packed with a 3-ml Ni-NTA column (Qiagen, Tokyo, Japan). Eluted proteins were then desalted on a PD-10 column (Amersham Biosciences, Piscataway, NJ) in PBS, aliquoted, and flash frozen in 10% glycerol, and stored at -80 °C. Bovine serum albumin passed through a PD-10 column in PBS was used as a control. Tumorigenicity in Nude Mice—12 female BALB/c Slc-nu/nu athymic mice (4 weeks old) were injected s.c with GK-S25 cells (8 × 106 cells each) suspended in PBS. The cells were pre-incubated either with 500 nm Tat-MAGE-A4ΔN1 or bovine serum albumin (six mice each) in PBS for 60 min, washed in PBS, and then injected. Tumor size was calculated by measuring the length, width, and thickness with calipers. This work was carried out under the Japanese Law Concerning the Care and Control of Animals, and has been approved by the Animal Research Committee, Graduate School of Medicine, Kyoto University. Pro-apoptotic Activity of N-terminally Truncated MAGE-A4—MAGE-A4 directly interacts with gankyrin and suppresses its tumorigenic activity, although it shows no effect on cell cycle progression or apoptosis (22Nagao T. Higashitsuji H. Nonoguchi K. Sakurai T. Dawson S. Mayer R.J. Itoh K. Fujita J. J. Biol. Chem. 2003; 278: 10668-10674Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). While investigating the effects of MAGE-A4 and its deletion mutants on cell growth, we noticed that a C-terminal fragment of MAGE-A4 (residues 211-317, MAGE-A4ΔN1) induced cell death (Fig. 1A). At 48 h after transfection, about 25% of U-2 OS cells expressing MAGE-A4ΔN1 together with GFP were positively stained with PI. The effect was comparable to that of p53. As expected, the percentage of PI-positive cells expressing GFP and full-length MAGE-A4 (residues 1-317) or the N terminus of MAGE-A4 (residues 1-210) were not different from that expressing GFP alone (Fig. 1A). To assess whether MAGE-A4ΔN1 induced cell death by apoptosis, we measured the DNA content of cells by flow cytometry. As shown in Fig. 1B, a significant increase in the percentage of cells with sub-G1 DNA content was observed in U-2 OS cells expressing MAGE-A4ΔN1 compared with those expressing full-length or N terminus of MAGE-A4 or GFP alone. The kinetics of the change in the DNA content of cells expressing MAGE-A4ΔN1 was similar to that induced by p53 (Fig. 1C). At 45 and 60 h after transfection, 20-25 and 55-60%, respectively, of transfectants showed sub-G1 DNA content. Essentially similar results were obtained when MAGE-A4ΔN1 was expressed together with GFP in HuH-7 hepatoma cells (data not shown). We next examined the pro-apoptotic activities of the C termini of other MAGE-A family proteins structurally similar to MAGE-A4, i.e. MAGE-A1, MAGE-A2, and MAGE-A12 (1Chomez P. De Backer O. Bertrand M. De Plaen E. Boon T. Lucas S. Cancer Res. 2001; 61: 5544-5551PubMed Google Scholar) in U2-OS cells. As shown in Fig. 1D, MAGE-A4ΔN1 showed the strongest pro-apoptotic activity among MAGE-A family members studied. When we performed TUNEL staining of HuH-7 cells after transfection with plasmids expressing MAGE-A4ΔN1, TUNEL positive HuH-7 cells displayed chromatin condensation (Fig. 1E). Significantly, more HuH-7 cells expressing MAGE-A4ΔN1 were TUNEL positive compared with those transfected with the empty vector (Fig. 1F). Taken together, these results demonstrated that MAGE-A4ΔN1 induces apoptosis in human cancer cells, while full-length MAGE-A4 does not. The apoptosis-inducing effects of various MAGE-A4 mutants were next examined by selectively analyzing U-2 OS transfectants bicistronically expressing GFP. As shown in Fig. 2, MAGE-A4ΔN1 induced apoptosis as efficiently as p53. Further deletion of N-terminal or C-terminal amino acids from MAGE-A4ΔN1 decreased its pro-apoptotic activity, indicating that both N-terminal (residues 211-226) and C-terminal (residues 288-317) amino acids were essential for maximal activity. Induction of p53-dependent and p53-independent Apoptosis by MAGE-A4ΔN1—Most apoptotic mechanisms are dependent on caspase activities (25Nicholson D.W. Thornberry N.A. Science. 2003; 299: 214-215Crossref PubMed Scopus (212) Google Scholar). The broad range caspase inhibitor Z-VAD-FMK prevented apoptosis induced by MAGE-A4ΔN1 in U-2 OS cells (Fig. 3A). Two caspase-activating pathways that lead to apoptosis are well characterized, i.e. the caspase-8-activating pathway and the caspase-9-activating pathway (8Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1624) Google Scholar, 25Nicholson D.W. Thornberry N.A. Science. 2003; 299: 214-215Crossref PubMed Scopus (212) Google Scholar, 26Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar). The former is initiated from cell surface death receptors, while the latter is triggered by various stress signals and involves changes in mitochondrial integrity. A significant increase in caspase-9 activity, but not caspase-8 activity, was detected in U-2 OS cells expressing MAGE-A4ΔN1 (Fig. 3B). Within the mitochondrial apoptotic pathway, the ratio of Bcl-2 family death agonists to antagonists dictates the susceptibility of cells to an apoptotic stimulus (27Cory S. Adams J.M. Nat. Rev. Cancer. 2002; 2: 647-656Crossref PubMed Scopus (3307) Google Scholar). Bcl-xL and Bcl-2 works as death antagonists either by preventing the release of mitochondrial caspase-activating factors such as cytochrome c or by sequestration and inhibition of caspases via interaction with APAF-1 (apoptotic protease-activating factor-1) (8Igney F.H. Krammer P.H. Nat. Rev. Cancer. 2002; 2: 277-288Crossref PubMed Scopus (1624) Google Scholar, 26Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1073) Google Scholar, 28Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (93) Google Scholar). When we examined the level of Bcl-2 family members in U-2 OS cells overexpressing MAGE-A4ΔN1, a decrease in the level of Bcl-xL, but no change in the levels of other Bcl-2 family members including Bax and Bid were observed (Fig. 3C and data not shown). The tumor suppressor p53 induces mitochondria-mediated apoptosis in many experimental systems and increases caspase-9 activity (9Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2702) Google Scholar). Thus, we examined whether p53 is involved in MAGE-A4ΔN1-induced apoptosis. We first determined the levels of p53 in U-2 OS cells after transfection with cDNAs expressing MAGE-A4 or its various mutants (Fig. 3D). Plasmids expressing luciferase were co-transfected as a control for transfection efficiency. An increase in the p53 protein level was observed in MAGE-A4ΔN1 transfectants. MAGE-A4ΔN1ΔC2 and MAGE-A4ΔN1ΔC3, which showed pro-apoptotic activities (Fig. 2B) also increased p53 levels. These findings are consistent with the notion that p53 is involved in the MAGE-A4ΔN1-induced apoptosis. p21Cip1 is known to protect cells from p53-induced cell death in addition to inhibiting cell proliferation (20Seoane J. Pouponnot C. Staller P. Schader M. Eilers M. Massague J. Nat. Cell Biol. 2001; 3: 400-408Crossref PubMed Scopus (406) Google Scholar, 29Gartel A.L. Tyner A.L. Mol. Cancer Ther. 2002; 1: 639-649PubMed Google Scholar). As shown in Fig. 3E, MAGE-A4ΔN1 decreased the levels of p21Cip1 transcript and protein. Taken together, these results strongly suggest that MAGE-A4ΔN1 induces apoptosis in a p53-dependent manner. However, despite low p53 levels apoptosis was observed in MAGE-A4ΔN2 and MAGE-A4ΔN3 transfectants (Figs. 2B and 3D). Furthermore, MAGE-A4ΔN1 showed more potent pro-apoptotic activity than that expected from the up-regulated level of p53 (Fig. 4A). These results suggest that some of the observed pro-apoptotic activities of MAGE-A4ΔN1 are p53-independent. To assess this possibility, we examined the sensitivity of p53-defective cells, i.e. HuH-7 and PLC/PRF/5 hepatoma cell lines containing mutant p53 (30Hsu I.C. Tokiwa T. Bennet W. Metcalf R.A. Welsh J.A. Sun T. Harris C.C. Carcinogenesis. 1993; 14: 987-992Crossref PubMed Scopus (217) Google Scholar) and H1299 lung cancer cell line deficient in p53 (31Mitsudomi T. Steinberg S.M. Nau M.M. Carbone D. D'Amico D. Bodner S. Oie H.K. Linnoila R.I. Mulshine J.L. Minna J.D. Gazder A.F. Oncogene. 1992; 7: 171-180PubMed Google Scholar) to MAGE-A4ΔN1-induced apoptosis. As shown in Fig. 4B, MAGE-A4ΔN1 induced apoptosis in the absence of wild-type p53. The p53-independent MAGE-A4ΔN1-induced apoptosis was also dependent on a caspase(s) (Fig. 4C), and was accompanied by increased caspase-9, but not caspase-8, activity (Fig. 4D). In H1299 cells, expression of MAGE-A4ΔN1 disrupted mitochondrial integrity (Fig. 4E), and induced release of cytochrome c from mitochondria (Fig. 4F). These results indicate that p53-independent apoptosis caused by MAGE-A4ΔN1 is mediated via a mitochondrial pathway. Interaction of MAGE-A4ΔN1 with Miz-1 Resulting in Suppression of the p21Cip1 Expression—To further characterize the pro-apoptotic activity of MAGE-A4ΔN1, we performed a yeast two-hybrid assay using MAGE-A4ΔN1 as bait. We identified 10 clones of 3.5 × 106 yeast clon" @default.
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• W2048547613 title "A Cleaved Form of MAGE-A4 Binds to Miz-1 and Induces Apoptosis in Human Cells" @default.
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