Sperm-Associated Antigen 4, a Novel Hypoxia-Inducible Factor 1 Target, Regulates Cytokinesis, and Its Expression Correlates with the Prognosis of Renal Cell Carcinoma
2013; Elsevier BV; Volume: 182; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.02.024
ISSN1525-2191
AutoresKumi Shoji, Takashi Murayama, Imari Mimura, Takehiko Wada, Haruki Kume, Akiteru Goto, Takamoto Ohse, Tetsuhiro Tanaka, Reiko Inagi, Frans A. van der Hoorn, Ichiro Manabe, Yukio Homma, Masashi Fukayama, Takashi Sakurai, Takeshi Hasegawa, Hiroyuki Aburatani, Tatsuhiko Kodama, Masaomi Nangaku,
Tópico(s)Epigenetics and DNA Methylation
ResumoHypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1–specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis. Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1–specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis. Hypoxia plays a crucial role in a number of physiological and pathophysiological phenomena, including cancer biology. Because the abnormal tumor microvasculature cannot provide a sufficient blood supply, hypoxia is a common characteristic of all solid tumors.1Brown J.M. Giaccia A.J. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.Cancer Res. 1998; 58: 1408-1416PubMed Google Scholar Although hypoxia is inevitably deleterious to cells, including those in tumors, cancer cells show a range of genetic changes that enable them to adapt to hypoxic conditions and improve their survival. A key transcription factor that regulates these responses to hypoxia is hypoxia-inducible factor (HIF), which composed of an α subunit (HIF-1α; HIF-2α, alias EPAS-1; or HIF-3α) and a β subunit (HIF-1β; alias ARNT).2Semenza G.L. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1.Annu Rev Cell Dev Biol. 1999; 15: 551-578Crossref PubMed Scopus (1672) Google Scholar Although HIF-1β is constitutively present, the HIF-α subunits are highly unstable, except under low oxygen tension. The stability of an HIF-α subunit is determined mainly by its degradation by von Hippel-Lindau protein (VHL) in an oxygen-dependent manner.3Berra E. Benizri E. Ginouvès A. Volmat V. Roux D. Pouysségur J. HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia.EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1091) Google Scholar, 4Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel-Lindau tumor suppressor protein.J Biol Chem. 2000; 275: 25733-25741Crossref PubMed Scopus (921) Google Scholar Under hypoxic conditions, HIF-α escapes degradation and HIF activates the transcription of target genes with hypoxia-responsive elements. HIF plays a crucial role in tumorigenesis, and HIF-1α and HIF-2α are overexpressed in many human cancers, including renal cell carcinoma (RCC), as a result of intratumoral hypoxia and genetic alterations.5Zhong H. De Marzo A.M. Laughner E. Lim M. Hilton D.A. Zagzag D. Buechler P. Isaacs W.B. Semenza G.L. Simons J.W. Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases.Cancer Res. 1999; 59: 5830-5835PubMed Google Scholar, 6Talks K.L. Turley H. Gatter K.C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Harris A.L. The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.Am J Pathol. 2000; 157: 411-421Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 7Klatte T. Seligson D.B. Riggs S.B. Leppert J.T. Berkman M.K. Kleid M.D. Yu H. Kabbinavar F.F. Pantuck A.J. Belldegrun A.S. Hypoxia-inducible factor 1 alpha in clear cell renal cell carcinoma.Clin Cancer Res. 2007; 13: 7388-7393Crossref PubMed Scopus (165) Google Scholar Furthermore, these elevated levels correlate with cancer-related mortality and treatment failure.8Semenza G.L. Targeting HIF-1 for cancer therapy.Nat Rev Cancer. 2003; 3: 721-732Crossref PubMed Scopus (5312) Google Scholar To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing (ChIP-Seq) in combination with microarray analysis, and identified a number of novel HIF targets. One of the targets, SPAG4, was originally identified as a testis-specific protein localized to microtubule-containing spermatid structures.9Shao X. Tarnasky H.A. Lee J.P. Oko R. van der Hoorn F.A. Spag4, a novel sperm protein, binds outer dense-fiber protein Odf1 and localizes to microtubules of manchette and axoneme.Dev Biol. 1999; 211: 109-123Crossref PubMed Scopus (134) Google Scholar, 10Tarnasky H. Gill D. Murthy S. Shao X. Demetrick D.J. van der Hoorn F.A. A novel testis-specific gene, SPAG4, whose product interacts specifically with outer dense fiber protein ODF27, maps to human chromosome 20q11.2.Cytogenet Cell Genet. 1998; 81: 65-67Crossref PubMed Scopus (25) Google Scholar SPAG4 belongs to the family of SUN domain-containing proteins, and SUN is a conserved domain in eukaryotes.11Tzur Y.B. Wilson K.L. Gruenbaum Y. SUN-domain proteins: ‘Velcro’ that links the nucleoskeleton to the cytoskeleton.Nat Rev Mol Cell Biol. 2006; 7: 782-788Crossref PubMed Scopus (193) Google Scholar, 12Razafsky D. Hodzic D. Bringing KASH under the SUN: the many faces of nucleo-cytoskeletal connections.J Cell Biol. 2009; 186: 461-472Crossref PubMed Scopus (189) Google Scholar, 13Hasan S. Güttinger S. Mühlhäusser P. Anderegg F. Bürgler S. Kutay U. Nuclear envelope localization of human UNC84A does not require nuclear lamins.FEBS Lett. 2006; 580: 1263-1268Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Recent studies demonstrated the ubiquitous expression of human SPAG4 in various normal tissues and neoplastic tissues at the mRNA level.14Siliņa K. Zayakin P. Kalniņa Z. Ivanova L. Meistere I. Endzeliņš E. Abols A. Stengrēvics A. Leja M. Ducena K. Kozirovskis V. Linē A. Sperm-associated antigens as targets for cancer immunotherapy: expression pattern and humoral immune response in cancer patients.J Immunother. 2011; 34: 28-44Crossref PubMed Scopus (62) Google Scholar, 15Kennedy C. Sebire K. de Kretser D.M. O’Bryan M.K. Human sperm associated antigen 4 (SPAG4) is a potential cancer marker.Cell Tissue Res. 2004; 315: 279-283Crossref PubMed Scopus (29) Google Scholar, 16Condomines M. Hose D. Rème T. Requirand G. Hundemer M. Schoenhals M. Goldschmidt H. Klein B. Gene expression profiling and real-time PCR analyses identify novel potential cancer-testis antigens in multiple myeloma.J Immunol. 2009; 183: 832-840Crossref PubMed Scopus (24) Google Scholar Little, however, is known about its function. We investigated SPAG4 expression in RCC. We also performed functional analyses to reveal a biological function of SPAG4. Human umbilical vascular endothelial cells (HUVECs; ATCC, Manassas, VA) were cultured in EGM-2 MV complete medium (Lonza Japan, Tokyo, Japan). HeLa cells and human kidney-2 (HK-2) cells were cultured in Dulbecco’s modified Eagle’s medium (Nissui Seiyaku, Tokyo, Japan) containing 10% fetal bovine serum. Cells were grown in a humidified, 5% CO2–enriched atmosphere at 37°C. Hypoxic conditions were established by exposure to 1.0% O2 and 5% CO2 in an multigas incubator (APM-30D; Astec, Fukuoka, Japan). The duration of hypoxic incubation was 24 hours, except as otherwise specified. We performed ChIP-Seq analysis using anti–HIF-1α antibody (NB 100-134; Novus Biologicals, Littleton, CO) in HUVECs, as described previously.17Mimura I. Nangaku M. Kanki Y. Tsutsumi S. Inoue T. Kohro T. Yamamoto S. Fujita T. Shimamura T. Suehiro J. Taguchi A. Kobayashi M. Tanimura K. Inagaki T. Tanaka T. Hamakubo T. Sakai J. Aburatani H. Kodama T. Wada Y. Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A.Mol Cell Biol. 2012; 32: 3018-3032Crossref PubMed Scopus (174) Google Scholar A next-generation sequencer (Genome Analyzer II; Illumina, San Diego, CA) was used, and all mapped sequences were analyzed using QuEST software version 2.4 (http://mendel.stanford.edu/SidowLab/downloads/quest).18Valouev A. Johnson D.S. Sundquist A. Medina C. Anton E. Batzoglou S. Myers R.M. Sidow A. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data.Nat Methods. 2008; 5: 829-834Crossref PubMed Scopus (548) Google Scholar Human Genome U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA) were used. At 48 hours after transfection of siRNA, the treated cells were incubated for a further 24 hours under normoxia or hypoxia, and their gene expression profiles were examined as described previously.17Mimura I. Nangaku M. Kanki Y. Tsutsumi S. Inoue T. Kohro T. Yamamoto S. Fujita T. Shimamura T. Suehiro J. Taguchi A. Kobayashi M. Tanimura K. Inagaki T. Tanaka T. Hamakubo T. Sakai J. Aburatani H. Kodama T. Wada Y. Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A.Mol Cell Biol. 2012; 32: 3018-3032Crossref PubMed Scopus (174) Google Scholar Preparation of cRNA and hybridization of probe arrays were performed according to the manufacturer’s instructions. The raw data in the Affymetrix Human Genome U133 Plus 2.0 array were exported to GeneSpring GX software version 11.5 (Agilent Technologies, Santa Clara, CA) and were normalized by robust multiarray average (RMA) normalization algorithms. For identification of genes significantly altered by hypoxia, total detected entities were filtered by signal intensity value (upper cutoff, 100th percentile; lower cutoff, 20th percentile) and error (SD < 0.1) to remove very low signal entities and to select reproducible signal values of entities among the experiments. To investigate biological functions associated with the gene lists, we used the Database for Annotation, Visualization and Integrated Discovery (DAVID),19Huang D.W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25470) Google Scholar, 20Huang D.W. Sherman B.T. Lempicki R.A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists.Nucleic Acids Res. 2009; 37: 1-13Crossref PubMed Scopus (10315) Google Scholar an online gene database provided by the NIH (version 6.7; http://david.abcc.ncifcrf.gov). Benjamini and Hochberg false discovery rates (5% false positive discovery rate) were used to correct for multiple testing. siRNAs were obtained from Life Technologies–Invitrogen (Carlsbad, CA). Transfection was performed with Lipofectamine RNAiMax reagent in HUVECs and with Lipofectamine 2000 reagent in HeLa cells, according to the manufacturer’s protocols (Life Technologies–Invitrogen). At 48 hours after transfection, siRNA-treated cells were processed for further experiments. The target sequences of siRNA were as follows: human HIF-1α, forward 5′-AUAUGAUUGUGUCUCCAGCGGCUGG-3′ and reverse 5′-CCAGCCGCUGGAGACACAAUCAUAU-3′; human HIF-2α, forward 5′-UACAUUUGCGCUCAGUGGCUUGUCC-3′ and reverse 5′-GGACAAGCCACUGAGCGCAAAUGUA-3′; human SPAG4-1, forward 5′-CCUCCAGGUUUAUGAUGAAACUGA-3′ and reverse 5′-UUCAGUUUCAUCAUAAACCUGGAGG-3′; and human SPAG4-2, forward 5′-ACUCCACAAGGAGGUGUCCACUGUU-3′ and reverse 5′-AACAGUGGACACCUCCUUGUGGAGU-3′. Stealth RNAi Negative Control Med GC Duplex 2 or 3 (Life Technologies–Invitrogen) was used as control. RNA extraction, RT-PCR, and real-time quantitative RT-PCR (RT-qPCR) were performed as described previously.21Chiang C.K. Tanaka T. Inagi R. Fujita T. Nangaku M. Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent manner.Lab Invest. 2011; 91: 1564-1571Crossref PubMed Scopus (119) Google Scholar Primer sequences were as follows: human SPAG4, forward 5′-ATGAGGATTTTGTGCGGAAG-3′ and reverse 5′-GCGTAATCGTGGGATGTCTT-3′; human HIF-1α, forward 5′-CGAGATGCAGCCAGATCTCGGCGAAGTAA-3′ and reverse 5′-CTCTCATTTCCTCATGGTCACATGGATG-3′; human HIF-2α, forward 5′-GAAGTCCCGGGATGCTGCGCG-3′ and reverse 5′-ACTATGTCCTGTTAGCTCCACCTGTG-3′; and human β-actin (internal control), forward 5′-TCCCCCAACTTGAGATGTATGAAG-3′ and reverse 5′-AACTGGTCTCAAGTCAGTGTACAGG-3′. To generate the human SPAG4-promoter-luc plasmid and SPAG4-enhancer-luc plasmid, we isolated fragments from the human SPAG4 promoter from −1000 to +118 relative to the transcription start site, and from the enhancer from +1633 to +892 relative to the transcription start site by PCR, using human genomic DNA as template. We cloned these fragments into the pGL3-basic vector (Promega, Madison, WI). Mutagenesis was conducted using mutated primer pairs. HUVECs were transiently transfected with plasmid DNA using FuGENE HD reagent (Roche Applied Science, Mannheim, Germany; Indianapolis, IN), and luciferase activity was measured using a dual-luciferase assay kit by cotransfection with pRL-CMV (Promega) to normalize transfection efficiency, as described previously.17Mimura I. Nangaku M. Kanki Y. Tsutsumi S. Inoue T. Kohro T. Yamamoto S. Fujita T. Shimamura T. Suehiro J. Taguchi A. Kobayashi M. Tanimura K. Inagaki T. Tanaka T. Hamakubo T. Sakai J. Aburatani H. Kodama T. Wada Y. Dynamic change of chromatin conformation in response to hypoxia enhances the expression of GLUT3 (SLC2A3) by cooperative interaction of hypoxia-inducible factor 1 and KDM3A.Mol Cell Biol. 2012; 32: 3018-3032Crossref PubMed Scopus (174) Google Scholar, 22Tanaka T. Miyata T. Inagi R. Fujita T. Nangaku M. Hypoxia in renal disease with proteinuria and/or glomerular hypertension.Am J Pathol. 2004; 165: 1979-1992Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Immunoblotting was performed as described previously.23Tanaka T. Yamaguchi J. Shoji K. Nangaku M. Anthracycline inhibits recruitment of hypoxia-inducible transcription factors and suppresses tumor cell migration and cardiac angiogenic response in the host.J Biol Chem. 2012; 287: 34866-34882Crossref PubMed Scopus (34) Google Scholar The primary antibodies used for immunoblotting were anti-SPAG4 (1:1000; sc-85927; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin antibody (1:2000; A2066; Sigma-Aldrich, St. Louis, MO). Densitometry analysis was performed using ImageJ software version 1.47b (NIH, Bethesda, MD). The clinical and pathological data of 190 adult patients who had been diagnosed with sporadic clear cell RCC (ccRCC) and had been treated with partial or radical nephrectomy between 1998 and 2003 at the University of Tokyo Department of Urology were reviewed. The diagnosis of ccRCC was based on the evaluation of H&E-stained sections by pathologists at the University of Tokyo in accordance with the World Health Organization classification.24Eble J.N. Sauter G. Epstein J.I. Sesterhenn I.A. Pathology & Genetics of Tumours of the Urinary System and Male Genital Organs. World Health Organization Classification of Tumours. IARC Press, Lyon2004: 23-25Google Scholar Tumor staging was revised according to the 2009 TNM staging system.25Sobin L. Gospodarowicz M. Wittekind C. TNM Classification of Malignant Tumors. Wiley-Blackwell, Chichester, UK2009Google Scholar Nuclear grading was based on the appearance of the nuclei of tumor cells and was categorized as grade 1 (nuclei of tumor cells were smaller than those of normal renal tubular cells), grade 2 (nuclei were the same size), or grade 3 (nuclei were larger, and sometimes pleomorphic and bizarre).26The Japanese Urological Association, The Japanese Society of Pathology, Japan Radiological SocietyGeneral Rule for Clinical and Pathological Studies on Renal Cell Carcinoma.ed 4. Kanehara, Tokyo, Japan2011Google Scholar The Eastern Co-operative Oncology Group (ECOG) scale was used to measure performance status (PS).27Oken M.M. Creech R.H. Tormey D.C. Horton J. Davis T.E. McFadden E.T. Carbone P.P. Toxicity and response criteria of the Eastern Cooperative Oncology Group.Am J Clin Oncol. 1982; 5: 649-655Crossref PubMed Scopus (8258) Google Scholar Incidentaloma was defined as a renal tumor detected during evaluation or surveillance of an unrelated medical condition, and symptomatic tumor was defined as a renal tumor detected on evaluation of hematuria, ipsilateral flank pain, a mass, or systemic symptoms such as loss of appetite.28Lee C.T. Katz J. Fearn P.A. Russo P. Mode of presentation of renal cell carcinoma provides prognostic information.Urol Oncol. 2002; 7: 135-140Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Written informed consent was obtained from all participants, and institutional review board approval was obtained from the University of Tokyo. Tissue microarrays (TMAs) consisting of 190 sporadic ccRCC samples were constructed for the immunohistochemical analysis of SPAG4. All tissue samples were fixed in formalin and embedded in paraffin. Representative tissue areas were punched out of the paraffin block using a 2.0-mm punch, and duplicate cores per sample were arrayed on a recipient paraffin block, to decrease sampling error. Indirect immunoperoxidase staining was performed as described previously.29Ohse T. Pippin J.W. Vaughan M.R. Brinkkoetter P.T. Krofft R.D. Shankland S.J. Establishment of conditionally immortalized mouse glomerular parietal epithelial cells in culture.J Am Soc Nephrol. 2008; 19: 1879-1890Crossref PubMed Scopus (68) Google Scholar The primary and secondary antibodies used in this analysis were anti-SPAG4 rabbit polyclonal antibody (1:3000)9 and biotinylated goat anti-rabbit IgG antibody (1:1000; BA-1000; Vector Laboratories, Burlingame, CA). TMAs were scored in a masked manner by two independent observers (K.S. and M.N.). For the few instances of discrepant scoring, a consensus score was determined. cDNA encoding human SPAG4 was obtained from HUVECs by RT-PCR and was subcloned into pEGFP-N1 (Clontech Laboratories, Mountain View, CA) and pcDNA4/TO/myc-HisA (Life Technologies–Invitrogen). cDNA encoding human calnexin was amplified from human embryonic kidney (HEK 293) cells by RT-PCR, and subcloned into the modified pIREShyg2 (Clontech) carrying PhiC31 attB sequence that had been excised from pJTI Fast DEST (Life Technologies–Invitrogen). mCherry (the generous gift of Dr. Roger Tsien)30Shu X. Shaner N.C. Yarbrough C.A. Tsien R.Y. Remington S.J. Novel chromophores and buried charges control color in mFruits.Biochemistry. 2006; 45: 9639-9647Crossref PubMed Scopus (379) Google Scholar was fused to the C terminus of calnexin or SPAG4. Deletion mutants of human SPAG4 were constructed by PCR using pEGFP-N1-human SPAG4 as a template. Transient transfection was performed with Lipofectamine 2000 reagent (Life Technologies–Invitrogen). Stable transfectants of HeLa cells expressing tagged SPAG4 were generated using the Jump-In system (Life Technologies–Invitrogen) according to the manufacturer’s protocol. To generate double stable transfectants, HeLa cells stably expressing SPAG4-EGFP were cotransfected with the modified pIREShyg2 carrying calnexin-mCherry and pJTI PhiC31 (Life Technologies–Invitrogen). Live-cell imaging was performed with a high-speed confocal laser scanning microscope system, as described previously.31Kobayashi T. Murayama T. Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.PLoS One. 2009; 4: e7827Crossref PubMed Scopus (38) Google Scholar HeLa cells were grown on collagen-coated glass-bottom dishes and were observed using a Nipkow-disk confocal laser scanning unit (CSU22; Yokogawa Electric, Tokyo, Japan) equipped with an argon-krypton ion laser (488 and 568 nm excitation). Images were acquired with an electron-multiplying charge-coupled device camera (EMCCD C9100; Hamamatsu Photonics, Shizuoka, Japan) at a rate of 466 ms per image. For multicolor observations, cells were observed using a W-view system (model 8509; Hamamatsu Photonics). For long-term live-cell imaging, cells were observed using a CellVoyager CV1000 confocal scanner system (Yokogawa). Images were acquired every 5 minutes at a rate of 200 ms per image. To visualize tubulin structures during live-cell imaging, HeLa cells were transduced with CellLight Tubulin-RFP BacMam 2.0 (Life Technologies–Invitrogen) according to the manufacturer’s protocol. Cells were collected by trypsinization and were fixed with 70% ethanol overnight at −20°C. The cells were then stained with a solution containing 50 μg/mL propidium iodide (Dojindo Molecular Technologies, Kumamoto, Japan) and 5 mg/mL RNase A (Sigma-Aldrich) in 0.1% bovine serum albumin in PBS for 20 minutes in the dark at room temperature. For each sample, 10,000 cells were analyzed using a flow cytometer (BD Biosciences, San Jose, CA), and data were collected using Cell Quest software (Becton Dickinson). The population of tetraploid cells was quantitated based on scatter analysis using ModFIT LT software version 3.1 (Verity Software House, Topsham, ME). Cells were fixed with methanol/acetone (1:1) or 2% paraformaldehyde, followed by permeabilization with 0.3% Triton X-100 surfactant in PBS. After a washing, the cells were incubated with anti–c-Myc antibody (1:200; sc-40; Santa Cruz Biotechnology), anti–α-tubulin antibody (1:1000; T6199; Sigma-Aldrich), or anti–β-catenin antibody (1:200; 610154; BD Biosciences). For c-Myc and α-tubulin staining, anti-mouse IgG1-FITC antibody (1:200; 1070-02; SouthernBiotech, Birmingham, AL) was applied. For β-catenin, biotinylated anti-mouse IgG antibody (1:1000; BA-2001; Vector Laboratories) was applied, followed by Alexa Fluor 594–conjugated streptavidin (1:500; S-32356; Life Technologies–Invitrogen). Nuclei were stained with Hoechst 33342 dye (Sigma-Aldrich). An Eclipse E600 microscope (Nikon, Tokyo, Japan) was used for observation. siRNA-treated HeLa cells were plated in 100 μL of medium on 96-well plates. Forty-eight hours after plating, cell incorporation of bromodeoxyuridine (BrdU) was measured by enzyme-linked immunosorbent assay with a BrdU Labeling and Detection Kit III (Roche Applied Science) according to the manufacturer’s protocol. Absorbance at 405 nm was measured using a Biochrom plate reader (Asys Hitech, Eugendorf, Austria; Cambridge, UK). siRNA-treated HeLa cells were plated in 100 μL of medium on 96-well plates. At 1, 24, 48, and 72 hours after plating, we performed CellTiter 96 AQueous one-solution cell proliferation assay (Promega) as described previously.21Chiang C.K. Tanaka T. Inagi R. Fujita T. Nangaku M. Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent manner.Lab Invest. 2011; 91: 1564-1571Crossref PubMed Scopus (119) Google Scholar Absorbance at 490 nm was measured using a Biochrom plate reader (Asys Hitech). All data are reported as means ± SD. Data for two groups were analyzed with an unpaired t-test, and those for more than two groups were compared by analysis of variance with Tukey’s post hoc test. Nonparametric data were analyzed with the U-test or Kruskal-Wallis test when appropriate. Cancer-specific survival was estimated using the Kaplan-Meier method. The log-rank test was used to compare survival between different groups. The Cox proportional hazards regression model was used to determine the effect of clinicopathological parameters on survival. The hazard ratio was used as a measure of risk of death in different categories. Differences with a P value of 1.0 log2-fold up-regulation in the presence of control siRNA under hypoxia, compared with normoxia. To identify genes regulated by HIF-1, we set the following criteria for downstream HIF-1 targets: the base-2 logarithm of fold-change value was >1.6 in normoxia, compared with hypoxia, and the base-2 logarithm of fold-change value was <1.2 in control siRNA, compared with HIF-1 siRNA, under hypoxia. Using these criteria, we identified 276 genes that are regulated by HIF-1 directly or indirectly under hypoxia (Supplemental Table S2). The overlap of the HIF-1 target genes identified in ChIP-Seq and microarray analysis should correspond to genes that are regulated by HIF-1 directly, and we identified 78 such genes (Figure 1A and Supplemental Table S3). Among these targets, we focused on a novel HIF-1 target, SPAG4, because SPAG4 was the sixth highest gene in the increase rate of mRNA expression after 24 hours of hypoxia and because the function of the SPAG4 product was unknown. We confirmed up-regulation of SPAG4 under hypoxia in HUVECs by RT-qPCR (Figure 1B). Our ChIP-Seq analysis showed HIF-1 binding in the enhancer of human SPAG4 gene (Figure 1C), and we investigated the HIF-1 dependency of SPAG4 regulation by luciferase reporter assay using this enhancer region. Addition of the SPAG4 enhancer to the SPAG4 promoter-luc resulted in the 3.6-fold up-regulation of reporter activity under hypoxia, compared with normoxia (Figure 1D). Furthermore, mutation of the hypoxia-responsive element motif of the SPAG4 enhancer (ACGTG→AAATG) resulted in a 76.5% reduction in reporter activity. These results demonstrated that a hypoxia-responsive element in the enhancer of SPAG4 is responsible for hypoxic induction. To investigate whether SPAG4 is a specific target of HIF-1, we evaluated SPAG4 mRNA levels using siRNA targeting HIF-1α or HIF-2α under hypoxia. The knockdown efficiencies of HIF-1α and HIF-2α at the mRNA level were 83% and 90%, respectively. SPAG4 mRNA levels were decreased only when HIF-1α mRNA levels were suppressed (Figure 1E). These results suggest that SPAG4 expression is regulated by HIF-1, but not by HIF-2. An online database search using Gene Expression Omnibus profiles (GEO Profiles; http://www.ncbi.nlm.nih.gov/geoprofiles) revealed the same results in MCF-7 breast canc
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