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• W1983761036 abstract "HSCO (hepatoma subtracted-cDNA library clone one, also called ETHE1) was originally identified by its frequent overexpression in hepatocellular carcinomas. HSCO inhibits function of NF-κB by binding to RelA and accelerating its export from the nucleus. We show here that HSCO exhibits anti-apoptotic activity in cells exposed to DNA-damaging agents by suppressing transcriptional activity of p53. Induction of pro-apoptotic genes, Noxa, Perp, PIG3, and Bax were suppressed in cells over-expressing HSCO. By increasing ubiquitylation and degradation of p53, HSCO reduces p53 protein levels. HSCO specifically associates with histone deacetylase 1 (HDAC1) independently of Mdm2 and facilitates deacetylation of p53 at Lys-373/382 by HDAC1. The metallo-β-lactamase family consensus sequence in HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies suggested that HSCO, HDAC1, and p53 form a complex in the nucleus. Thus, HSCO is a cofactor that increases the deacetylase activity of HDAC1 toward p53, leading to suppression of apoptosis. Treatment of hepatocellular carcinomas that retain wild-type p53 and overexpress HSCO with anti-HSCO agents might re-establish the p53 response and revert chemoresistance. HSCO (hepatoma subtracted-cDNA library clone one, also called ETHE1) was originally identified by its frequent overexpression in hepatocellular carcinomas. HSCO inhibits function of NF-κB by binding to RelA and accelerating its export from the nucleus. We show here that HSCO exhibits anti-apoptotic activity in cells exposed to DNA-damaging agents by suppressing transcriptional activity of p53. Induction of pro-apoptotic genes, Noxa, Perp, PIG3, and Bax were suppressed in cells over-expressing HSCO. By increasing ubiquitylation and degradation of p53, HSCO reduces p53 protein levels. HSCO specifically associates with histone deacetylase 1 (HDAC1) independently of Mdm2 and facilitates deacetylation of p53 at Lys-373/382 by HDAC1. The metallo-β-lactamase family consensus sequence in HSCO is important for its effect on p53 deacetylation. Co-immunoprecipitation and immunofluorescence studies suggested that HSCO, HDAC1, and p53 form a complex in the nucleus. Thus, HSCO is a cofactor that increases the deacetylase activity of HDAC1 toward p53, leading to suppression of apoptosis. Treatment of hepatocellular carcinomas that retain wild-type p53 and overexpress HSCO with anti-HSCO agents might re-establish the p53 response and revert chemoresistance. The p53 tumor suppressor is mutated in ∼50% of many different cancers (1Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5744) Google Scholar) and is probably rendered inactive by a range of indirect mechanisms such as Mdm2 amplification and loss of p14ARF in the remaining 50% (2Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1011) Google Scholar). In unstressed cells, p53 is normally maintained at low levels by continuous ubiquitylation catalyzed by E3 3The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HSCO, hepatoma subtracted-cDNA library clone one; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; TSA, trichostatin A; HEK, human embryonic kidney; DKO-MEF, doubly knocked-out mouse embryonic fibroblast; shRNA, short hairpin RNA; HA, hemagglutinin; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; TRITC, tetramethylrhodamine isothiocyanate; ActD, Actinomycin D; WB, Western blot; IP, immunoprecipitation. 3The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; HSCO, hepatoma subtracted-cDNA library clone one; HCC, hepatocellular carcinoma; HDAC, histone deacetylase; TSA, trichostatin A; HEK, human embryonic kidney; DKO-MEF, doubly knocked-out mouse embryonic fibroblast; shRNA, short hairpin RNA; HA, hemagglutinin; CMV, cytomegalovirus; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; TRITC, tetramethylrhodamine isothiocyanate; ActD, Actinomycin D; WB, Western blot; IP, immunoprecipitation. ubiquitin ligases such as Mdm2, COP1, Pirh2, TOPORS, and ARF-BP1/Mule, and subsequent degradation by the 26 S proteasome (3Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2690) Google Scholar, 4Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (640) Google Scholar, 5Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar). In response to genotoxic stress, p53 is rapidly stabilized and activated. The activated p53 mainly functions as a sequence-specific DNA-binding transcription factor to activate or repress a large number of target genes, which mediate cell-cycle arrest, apoptosis, senescence, differentiation, DNA repair, and inhibition of angiogenesis and metastasis (6Liu G. Chen X. J. Cell. Biochem. 2006; 15: 448-458Crossref Scopus (75) Google Scholar). The activity of p53 is largely controlled by the cellular p53 level, its DNA-binding activity, subcellular localization, and recruitment of transcriptional co-activators or corepressors. Although the precise mechanisms of p53 activation are not fully elucidated, accumulating evidence indicates that post-translational modifications of p53, including phosphorylation of Ser and Thr residues and ubiquitylation, acetylation, and sumoylation of Lys residues, play important roles in regulating its stability and transcriptional activity (5Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar, 7Xu Y. Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar). Furthermore, these modifications are interrelated. For example, phosphorylation of Ser-15 or Ser-33/37 increases the affinity of p53 for p300 and promotes acetylation of p53 at Lys-373/382 (7Xu Y. Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar, 8Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar). Because the Lys residues acetylated in p53 overlap with those that are ubiquitylated, p53 acetylation has been considered to be important for p53 degradation as well as transcriptional activation (2Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1011) Google Scholar, 5Brooks C.L. Gu W. Mol. Cell. 2006; 21: 307-315Abstract Full Text Full Text PDF PubMed Scopus (683) Google Scholar).Histone deacetylase (HDAC) activity has been linked to diet, premalignant cell changes, aging, and development of diseases, including cancer. Eighteen potential HDACs have been identified in humans (9de Ruijter A.J. van Gennip A.H. Caron H.N. Kemp S. van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2421) Google Scholar, 10Glozak M.A. Sengupta N. Zhang X. Seto E. Gene (Amst.). 2005; 363: 15-23Crossref PubMed Scopus (1263) Google Scholar), which are classified into three groups based on homology to yeast proteins. The enzymatic activities of HDACs in Class I (Rpd3-like) such as HDAC-1, -2, and -3, and Class II (Hda1-like) are zinc-dependent and sensitive to the inhibitor trichostatin A (TSA). Class I HDACs are ubiquitously expressed small nuclear proteins, whereas Class II HDACs are larger proteins that shuttle between the cytoplasm and the nucleus. The NAD-dependent enzymatic activities of Class III (Sir2-like) HDACs are inhibited by nicotinamide but not by TSA (11Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (802) Google Scholar). Histone acetylation can be reversed by HDACs. HDAC recruitment to promoter regions by p53 through interaction with mSin3A, which directly binds to HDACs, down-regulates gene expression by core histone deacetylation (6Liu G. Chen X. J. Cell. Biochem. 2006; 15: 448-458Crossref Scopus (75) Google Scholar). Thus, HDACs act as p53 co-repressors. HDACs can deacetylate non-histone proteins as well (10Glozak M.A. Sengupta N. Zhang X. Seto E. Gene (Amst.). 2005; 363: 15-23Crossref PubMed Scopus (1263) Google Scholar). HDAC1 interacts with p53 possibly through mSin3A or PID/MTA2, deacetylates p53 in vitro and in vivo, and down-regulates p53 transcriptional activity (12Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar, 13Juan L.J. Shia W.J. Chen M.H. Yang W.M. Seto E. Lin Y.S. Wu C.W. J. Biol. Chem. 2000; 275: 20436-20443Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 14Luo J. Su F. Chen D. Shiloh A. Gu W. Nature. 2000; 408: 377-381Crossref PubMed Scopus (673) Google Scholar). Deacetylation of p53 is required for its effective degradation mediated by the ubiquitin ligase Mdm2, and Mdm2 can promote p53 deacetylation by recruiting a complex containing HDAC1 (15Ito A. Kawaguchi Y. Lai C.H. Kovacs J.J. Higashimoto Y. Appella E. Yao T.P. EMBO J. 2002; 21: 6236-6245Crossref PubMed Scopus (442) Google Scholar). In addition to HDAC1, a Class III HDAC, SIRT1, interacts with p53 and deacetylates it at Lys-382 (2Bode A.M. Dong Z. Nat. Rev. Cancer. 2004; 4: 793-805Crossref PubMed Scopus (1011) Google Scholar).Hepatocellular carcinoma (HCC) is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death (16Thomas M.B. Zhu A.X. J. Clin. Oncol. 2005; 23: 2892-2899Crossref PubMed Scopus (372) Google Scholar). Although screening of high risk populations by ultrasonography and measurement of the serum α-fetoprotein level has facilitated the early detection of HCC, a majority of patients present with advanced disease. Even for those patients who undergo surgical resection, the recurrence rates are as high as 50% at 2 years, and nonsurgical treatments are ineffective or minimally effective at best (16Thomas M.B. Zhu A.X. J. Clin. Oncol. 2005; 23: 2892-2899Crossref PubMed Scopus (372) Google Scholar, 17Treiber G. Dig. Dis. 2001; 19: 311-323Crossref PubMed Scopus (31) Google Scholar). It is therefore important to identify molecules that can be used to develop novel diagnostic, preventive, or therapeutic strategies.By constructing subtracted cDNA libraries, we have previously identified 19 genes overexpressed in HCCs, including two novel genes (18Higashitsuji 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, 19Gotoh K. Nonoguchi K. Higashitsuji H. Kaneko Y. Sakurai T. Sumitomo Y. Itoh K. Subjeck J.R. Fujita J. FEBS Lett. 2004; 560: 19-24Crossref PubMed Scopus (41) Google Scholar). One of these genes was named HSCO (hepatoma subtracted-cDNA library clone one) (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar). HSCO mRNA was overexpressed in 20 of 30 HCCs analyzed. Overexpression of HSCO inhibits caspase 9 activation and apoptosis induced by DNA-damaging agents such as adriamycin and etoposide, whereas it augments apoptosis induced by tumor necrosis factor-α. HSCO is a nuclear-cytoplasmic shuttling protein that binds to RelA and sequesters it in the cytoplasm by accelerating its export from the nucleus, resulting in inhibition of NF-κB activity. This activity of HSCO underlies the abrogation of p53-induced apoptosis in Saos-2 cells. In addition to our discoveries, Oue et al. (21Oue N. Hamai Y. Mitani Y. Matsumura S. Oshimo Y. Aung P.P. Kuraoka K. Nakayama H. Yasui W. Cancer Res. 2004; 64: 2397-2405Crossref PubMed Scopus (259) Google Scholar) used serial analysis of gene expression and found that HSCO (also called YF13H12) was overexpressed in 52% of 46 gastric carcinomas. Tiranti et al. (22Tiranti V. D'Adamo P. Briem E. Ferrari G. Mineri R. Lamantea E. Mandel H. Balestri P. Garcia-Silva M.T. Vollmer B. Rinaldo P. Hahn S.H. Leonard J. Rahman S. Dionisi-Vici C. Garavaglia B. Gasparini P. Zeviani M. Am. J. Hum. Genet. 2004; 74: 239-252Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), via positional cloning, identified HSCO as the gene responsible for ethylmalonic encephalopathy. Ethylmalonic encephalopathy is characterized by neuro-developmental delay and regression, prominent pyramidal and extrapyramidal signs, recurrent petechiae, orthostatic acrocyanosis, and chronic diarrhea, leading to death in the first decade of life. They proposed the name of the gene be changed from HSCO to ETHE1 and suggested that its product is a mitochondrial protein. Here, we show that HSCO can control the transcriptional activity of p53 by enhancing its deacetylation in association with HDAC1.EXPERIMENTAL PROCEDURESCell Culture and Transfection—Adenovirus-transformed human embryonic kidney (HEK) 293 cells (p53 wild type), SV40 large T antigen-expressing 293T cells (p53 wild type), human osteosarcoma U-2 OS cells (p53 wild type), human non-small cell lung carcinoma A549 cells (p53 wild type), and p53-/-/mdm2-/- doubly knocked-out mouse embryonic fibroblasts (DKO-MEFs, kindly provided by Dr. D. P. Lane) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Human lung adenocarcinoma H1299 cells (p53-null) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum. Mouse NIH/3T3 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum. All cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air. DNA transfection was performed by using the calcium phosphate method or FuGENE6 reagent (Roche Applied Science).Induction of Apoptosis and Assays for Caspase Activity—Exponentially proliferating cells (2 × 104) were plated into a 35-mm plate and treated with actinomycin D (7.5 nm), Adriamycin (1 μg/ml), etoposide (50 μg/ml), or tumor necrosis factor-α and cycloheximide (50 ng/ml and 100 μg/ml, respectively), or transfected with p53 cDNA in pcDNA3.1(+)-Neo or vector alone. Cell numbers were determined in triplicates at different time points. Viability of cells was determined by staining with trypan blue, and cell numbers were counted under a microscope. Caspase 3 activity and caspase 9 activity were determined by using the Caspase-3 Colorimetric Assay Kit and Caspase-9 Colorimetric Assay Kit (Medical & Biological Laboratories), respectively.Reporter Gene Assays—Luciferase reporter plasmids, p53-Luc or pAP-1-Luc, containing p53- or AP-1-binding sites, respectively (Stratagene), and pRL-TK (Promega, Madison, WI) were co-transfected with wild-type or mutant (H79N or R159H) HSCO cDNA fused to HA or FLAG tag in expression vector pCMV4-3HA or pcDNA3.1(+)-Neo as described (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar). 24 or 48 h later, cells were assayed for luciferase activity or treated with actinomycin D and assayed 24 h later. Luciferase activity was measured by the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. In some experiments, plasmids expressing p53 were co-transfected as well. pFC-MEKK plasmid (Stratagene) served as a positive control for the AP-1 reporter assay.Analyses of Gene Expression and Protein-Protein Interactions—RNA extraction, Northern blot analysis, immunoprecipitation, and Western blot analysis were performed as described (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar). For immunoprecipitation, anti-p53 antibody (DO-1 and FL-393, Santa Cruz Biotechnology), anti-HA antibody (12CA5, Roche Applied Science), anti-FLAG antibody (M2, Sigma), anti-GFP antibody (Nacalai), agarose-immobilized anti-p53 antibody (DO-1, Santa Cruz Biotechnology), and agarose-immobilized anti-FLAG antibody (M2, Sigma) were used. About 5-10% (10-25 μg of protein) of total cellular lysates used for immunoprecipitation was also analyzed by Western blotting. Protein bands were visualized by using the Enhanced Chemiluminescence kit (Amersham Biosciences). Western blot analysis was performed using the antibody against p53 (DO-1, Santa Cruz Biotechnology, DO-7, BD Pharmingen), HA, FLAG, β-actin (C4, Chemicon), ubiquitin (FL-76, Santa Cruz Biotechnology), Mdm2 (SMP14, Santa Cruz Biotechnology), HDAC1 (Santa Cruz Biotechnology), HDAC2 (Santa Cruz Biotechnology), HDAC3 (Santa Cruz Biotechnology), SIRT1 (Santa Cruz Biotechnology), acetylated Lys (Cell Signaling Technology), acetylated histone H4 (Upstate Biotechnology), acetylated p53 (Lys-320, Upstate), and acetylated p53 (Lys-373/382, Upstate). In some experiments, biotinylated anti-GFP antibody (B-2, Santa Cruz Biotechnology) and anti-FLAG antibody (BioM2, Sigma) were used. Histones were isolated from cells by HCl extraction and acetone precipitation (23Zweidler A. Methods Cell Biol. 1978; 17: 223-233Crossref PubMed Scopus (312) Google Scholar).For GST pulldown assays, full-length p53, HSCO, RelA, and HDAC1 cDNAs were cloned into the expression vector pGEX-4T or pGEX-6P-1 (Amersham Biosciences) and expressed as proteins fused to GST in Escherichia coli (DH5α or BL21 strain). The fusion proteins and GST were immobilized on glutathione-Sepharose and incubated with recombinant HSCO or HDAC1 protein from which GST had been removed after cleavage with PreScission protease (Amersham Biosciences), or immunoprecipitates prepared from cell lysates. After incubation at 4 °C for 60 min, bound proteins were analyzed by SDS-PAGE and Western blotting.Electrophoretic Mobility Shift Assays—A 27-bp double-stranded oligonucleotide probe selected from the human gadd45α promoter (5′-TACAGAACATGTCTAAGCATGCTGGGG-3′) was labeled with [γ-32P]ATP and purified with MicroSpin TM G25 columns (Amersham Biosciences). As a control, Sp1 binding-site probe (5′-GGATAGGGGCGGGGCGAGG-3′) was used. Nuclear extracts (10 μg) from HEK293 cells were incubated with 5 ng of labeled probe in a 20-μl reaction buffer (10 mm Tris-HCl, pH 7.5, 10 mm EDTA, 0.5 mm dithiothreitol, 50 mm NaCl, and 5% glycerol) containing 10 μg of bovine serum albumin and 1 μg of poly(dI-dC):poly(dI-dC) for 20 min at room temperature. For competition tests, 50-fold excess (250 ng) of unlabeled wild-type or mutant (5′-TACAGAATCGCTCTAAGCATGCTGGGG-3′) gadd45α probes were added to each reaction. The reaction mixture was electrophoresed in 4% polyacrylamide gel. The gels were dried and exposed to film at -80 °C with an intensifying screen.Analysis of p53 Stability and Ubiquitylation in Vivo—For in vivo p53 degradation assays, U-2 OS cells stably expressing HSCO-FLAG or FLAG alone (three clones each) and H1299 cells co-transfected with plasmids expressing p53 alone or in combination with HSCO-FLAG were used and analyzed as described (24Higashitsuji H. Higashitsuji H. Itoh K. Sakurai T. Nagao T. Sumitomo H. Masuda T. Dawson S. Shimada Y. Mayer R.J. Fujita J. Cancer Cell. 2005; 8: 75-87Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To analyze the effects of HSCO down-regulation, U-2 OS cells transfected with HSCO-specific short hairpin RNA (shRNA) were used.For in vivo ubiquitylation assays, H1299 cells or DKO-MEFs in 60-mm dish were co-transfected with plasmids expressing p53 (1.0 μg), HSCO-FLAG (1.0 μg), HA-Mdm2 (1.0 μg), and His-tagged ubiquitin in various combinations. Prior to collection after 48 h, cells were treated with proteasome inhibitors, MG115 (10 μm) and MG132 (10 μm), for 6 h. Cells were then lysed in buffer (6.0 m guanidium-HCl, 0.1 m Na2HPO4/NaH2PO4, 10 mm Tris-HCl, pH 8.0, 5 mm imidazole, and 10 mm 2-mercaptoethanol), and sonicated. The lysates were incubated with nickel-nitrilotriacetic acid-agarose beads. After extensive washing, bound proteins were eluted and analyzed by Western blotting.In Vivo p53 Acetylation and Deacetylation Assays—For in vivo p53 acetylation assays, 293T cells were transfected with plasmids expressing wild-type or mutant (H79N or R159H) HSCO, Mdm2, and p300. The cells were cultured in the presence or absence of 5 μm TSA and lysed in buffer (20 mm Tris-HCl, pH 7.6, 170 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 μm dithiothreitol) supplemented with proteinase inhibitors and TSA. To detect acetylated p53, cell lysates (250-500 μg of proteins) were incubated with 1 μg of agarose-immobilized antibody specific to human p53 (DO-1) for 4 h at 4 °C. The p53 content in each precipitated sample was equalized and subjected to Western blotting to assess the amount of acetylated p53.For in vivo p53 deacetylation assays, p53-null H1299 cells were co-transfected with plasmids expressing p53, wild-type or mutant (H141A) HDAC1, HSCO, and p300. In some experiments, H1299 and U-2 OS cells were co-transfected with plasmids expressing p53, p300, FLAG-tagged HSCO, and HDAC1-specific shRNA. The cells were lysed and analyzed by Western blotting as described above.In Vitro p53 Acetylation Assays—The cDNA for human HSCO was cloned into pGEX6P-1 (Amersham Biosciences) and expressed as GST fusion protein in E. coli strain BL21. The GST tag was cleaved using PreScission protease (Amersham Biosciences). p300 HAT domain fused to GST (1.0 μg, Upstate) was preincubated with the purified HSCO or Mdm2 for 10 min at room temperature. Substrate (1.0 μg of GST-p53) was added, and the mixture was incubated with 50 nCi of [14C]acetyl-CoA in 20 μl of reaction buffer (50 mm Tris-HCl, pH 8.0, 10% glycerol, 1.0 mm dithiothreitol, 0.1 mm EDTA, 1.0 mm phenylmethylsulfonyl fluoride) for another 60 min at 37 °C. Acetylation was analyzed by SDS-PAGE followed by autoradiography.In Vitro p53 Deacetylation Assays—The expression vector for wild-type or mutant (H141A) HDAC1 tagged with FLAG was co-transfected with HSCO cDNA in expression vector pcDNA3.1(+)-Neo or vector alone onto 293T cells. The cells were lysed in low stringency buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 0.5 mm EDTA, 0.5% Nonidet P-40) in the presence of protease inhibitors. After pre-clearing with protein A beads, the extracts were immunoprecipitated with anti-FLAG antibody in the presence of protein A beads for 2 h at 4°C. Then, the beads were washed twice with low stringency buffer, twice with low stringency buffer containing 0.5 m NaCl, and twice with deacetylation buffer (10 mm Tris-HCl, pH 8.0, 10 mm NaCl, 10% glycerol). The immune complexes were then incubated with acetylated p53 in 20 μl of deacetylation buffer for 2 h at 37 °C and analyzed by Western blotting. Acetylated p53 was prepared via an in vitro p53 acetylation reaction (25Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Kouzarides T. EMBO J. 2002; 21: 2383-2396Crossref PubMed Scopus (744) Google Scholar).Inhibition of Endogenous Gene Expression by shRNA—For production of small interfering RNA in the cells, we used the pSuper vector expressing shRNA as described (24Higashitsuji H. Higashitsuji H. Itoh K. Sakurai T. Nagao T. Sumitomo H. Masuda T. Dawson S. Shimada Y. Mayer R.J. Fujita J. Cancer Cell. 2005; 8: 75-87Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). To suppress expression of endogenous HSCO, the pSuper plasmid was digested with BglII and HindIII, and the annealed oligonucleotides corresponding to human HSCO (wt1: 5′-CTCTATGCTGTGAATACCC-3′, wt2: 5′-CAGGCTGACTTACACATTG3′, and mutant: 5′-CAGGTCGACTATCCAATGT-3′) were cloned into it. To suppress expression of endogenous HDAC1 and p53, oligonucleotides corresponding to human HDAC1 (5′-CTATGGTCTCTACCGAAAA-3′) and p53 (5′-GACTCCAGTGGTAATCTAC-3′), respectively, were cloned into the pSuper plasmid. Transfection of pSuper plasmids was performed using FuGENE6 Reagent (Roche Applied Science).Immunofluorescence Staining—Immunofluorescence staining was performed as described (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar). FLAG-tagged HSCO was detected with anti-FLAG antibody (BioM2, Sigma) and streptavidin-allophycocyanin (BD Pharmingen). HA-tagged p53 was detected with anti-HA antibody (Sigma) and TRITC-conjugated anti-rabbit IgG (DAKO). EGFP-tagged HDAC1 was detected by GFP fluorescence. They were observed using a confocal laser microscope (Olympus). In some experiments, cells stably expressing HSCO N- or C-terminally tagged with FLAG were treated with MitoTracker (Molecular Probes). After fixation, FLAG was detected with anti-FLAG antibody (M2, Sigma), fluorescein isothiocyanate-conjugated anti-mouse IgG (DAKO), and MitoTracker with its fluorescence using a confocal microscope.RESULTSHSCO Increases Resistance to p53-dependent Apoptosis—In human U-2 OS cells, HSCO confers resistance to apoptosis induced by DNA-damaging agents but not tumor necrosis factor-α (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar). DNA damage induces p53-dependent apoptosis, whereas tumor necrosis factor-α triggers apoptosis p53-independently (26Varfolomeev E.E. Ashkenazi A. Cell. 2004; 116: 491-497Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). As shown in Fig. 1A, expression of HSCO increased the survival of U-2 OS cells exposed to actinomycin D. The reduced activation of caspases 3 and 9 indicated that the increased survival was due to suppression of apoptosis (Fig. 1B). The anti-apoptotic effect of HSCO was also observed in actinomycin D-treated A549 cells, a human lung carcinoma cell line expressing wild-type p53, but not in actinomycin D-treated HLE cells, a human HCC cell line expressing mutant p53 (data not shown). In p53-null human H1299 cells, apoptosis and activation of caspase 9 induced by introduction of p53 were also inhibited by overexpression of HSCO (Fig. 1A and data not shown).When HSCO-specific shRNA was expressed in U-2 OS cells, the cytotoxic effect of actinomycin D and activation of caspase 9 were enhanced (Fig. 1, C and D). The effect of HSCO-specific shRNA on caspase-9 activity was reduced by concomitant suppression of p53 expression. Expression of shRNAs specific to HSCO, its mutant, or p53 did not affect the caspase-9 activity without actinomycin D treatment (data not shown). Taken together, these results suggest that HSCO suppresses the proapoptotic signaling pathway mediated by p53.HSCO Suppresses Transcriptional Activity of p53—After genotoxic stress, p53 transactivates many genes involved in apoptotic pathways (3Vousden K.H. Lu X. Nat. Rev. Cancer. 2002; 2: 594-604Crossref PubMed Scopus (2690) Google Scholar). To analyze the effects of HSCO on the transcriptional activity of p53, we transfected U-2 OS cells with a luciferase p53-cis reporter plasmid. Actinomycin D induced luciferase activity, which was suppressed dose dependently by HSCO (Fig. 2A). Interestingly, the mutant (H79N) HSCO having a missense mutation within the consensus sequence conserved throughout the metallo-β-lactamase family (20Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 333-346Abstract Full Text Full Text PDF Scopus (61) Google Scholar) did not suppress the induction of luciferase. In the p53-null H1299 and Saos-2 cells, expression of HSCO suppressed the p53-cis reporter activity induced by exogenous p53 (Fig. 2B, and data not shown). The inhibition was specific to the p53 transactivation, since HSCO did not affect the luciferase AP-1-cis reporter activity (Fig. 2C). Essentially similar results were obtained using mdm2-/-/p53-/- DKO-MEFs (Figs. 2D and 2E), indicating that the observed effect is independent of Mdm2. In U-2 OS cells exposed to actinomycin D, induction of p53-inducible genes involved in apoptosis, such as Noxa, Perp, PIG3, and Bax was suppressed by HSCO (Fig. 2F).FIGURE 2Effects of HSCO on transcriptional activity of p53. A, reduced transcriptional activity of p53. U-2 OS cells were co-transfected with a p53-responsive luciferase reporter, pRL-TK, and plasmids expressing wild-type (-wt) HSCO, mutant (H79N) HSCO, or vector alone. 24 h after exposure to actinomycin D (ActD), luciferase activity was assayed (upper panel). The results were normalized to Renilla luciferase activity and represent the mean ± S.D. of triplicates. Cell lysates were also analyzed by Western blotting using the indicated antibodies (lower panels). B, p53-null H1299 cells were co-transfected and analyzed as in A except that p53-expressing plasmids were co-transfected instead of ActD treatment. C, U-2 OS cells were co-transfected and analyzed as in A except that an AP-1-responsive luciferase reporter was used instead of a p53-responsive reporter. pFC-MEKK served as a positive control. D, mdm2-/-/p53-/- DKO-MEFs were co-transfected and analyzed as in B. E, mdm2-/-/p53-/- DKO-MEFs were co-transfected and analyzed as in C. F, reduced induction of p53-inducible genes. U-2 OS cells were transfected with plasmids expressing HSCO-FLAG or FLAG alone (Mock). After incubation with (+) or without (-) ActD, gene expression was analyzed by Northern blotting using the indicated cDNA probes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HSCO Reduces the Amount of p53 Bound to the p53-responsive Element—To clarify the mechanisms by which HSCO decreases the transcriptional activity of p53, we analyzed the effect of HSCO on binding of p53 to the p53-responsive element in vitro. As shown in Fig. 3A, inc" @default.
• W1983761036 created "2016-06-24" @default.
• W1983761036 creator A5008327100 @default.
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• W1983761036 title "Enhanced Deacetylation of p53 by the Anti-apoptotic Protein HSCO in Association with Histone Deacetylase 1" @default.
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