Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex
2005; Elsevier BV; Volume: 128; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2005.03.007
ISSN1528-0012
AutoresMasashi Idogawa, Tesshi Yamada, Kazufumi Honda, Satoshi Sato, Kohzoh Imai, Setsuo Hirohashi,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoBackground & Aims: T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by β-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/β-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/β-catenin transcriptional complex by means of proteomics. Methods & Results: A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the β-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of β-catenin and with the undifferentiated status of intestinal epithelial cells. Conclusions: In this study, we identified PARP-1 as a novel coactivator of the β-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis. Background & Aims: T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by β-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/β-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/β-catenin transcriptional complex by means of proteomics. Methods & Results: A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the β-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of β-catenin and with the undifferentiated status of intestinal epithelial cells. Conclusions: In this study, we identified PARP-1 as a novel coactivator of the β-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis. Somatic mutations associated with loss of heterozygosity of the tumor suppressor gene APC, which was originally identified as the gene responsible for the familial adenomatous polyposis (FAP) syndrome,1Nishisho I. Nakamura Y. Miyoshi Y. Miki Y. Ando H. Horii A. Koyama K. Utsunomiya J. Baba S. Hedge P. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients.Science. 1991; 253: 665-669Crossref PubMed Scopus (1618) Google Scholar are observed in more than 80% of sporadic colorectal cancers,2Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4269) Google Scholar and inactivation of the APC gene is the earliest genetic event during the adenoma-carcinoma sequence of colorectal carcinogenesis.2Kinzler K.W. Vogelstein B. Lessons from hereditary colorectal cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4269) Google Scholar, 3Powell S.M. Zilz N. Beazer-Barclay Y. Bryan T.M. Hamilton S.R. Thibodeau S.N. Vogelstein B. Kinzler K.W. APC mutations occur early during colorectal tumorigenesis.Nature. 1992; 359: 235-237Crossref PubMed Scopus (1672) Google Scholar, 4Bienz M. Clevers H. Linking colorectal cancer to Wnt signaling.Cell. 2000; 103: 311-320Abstract Full Text Full Text PDF PubMed Scopus (1305) Google Scholar A multiprotein complex consisting of the APC gene product, Axin/Axil, and glycogen synthase kinase 3β regulates the cytoplasmic content of β-catenin protein.5Kikuchi A. Tumor formation by genetic mutations in the components of the Wnt signaling pathway.Cancer Sci. 2003; 94: 225-229Crossref PubMed Scopus (200) Google Scholar Genetic inactivation of the APC gene results in the accumulation of cytoplasmic β-catenin.6Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Regulation of intracellular β-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein.Proc Natl Acad Sci U S A. 1995; 92: 3046-3050Crossref PubMed Scopus (955) Google Scholar The accumulated β-catenin protein acts as a transcriptional coactivator by forming complexes with T-cell factor (TCF)/lymphoid enhancer factor (LEF) family DNA-binding proteins.7Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Nuclear localization of β-catenin by interaction with transcription factor LEF-1.Mech Dev. 1996; 59: 3-10Crossref PubMed Scopus (782) Google Scholar, 8Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Functional interaction of β-catenin with the transcription factor LEF-1.Nature. 1996; 382: 638-642Crossref PubMed Scopus (2589) Google Scholar, 9van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF.Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar TCF/LEF proteins transactivate their target genes only when coupled with β-catenin. Aberrant transactivation of a certain set of target genes of TCF/LEF by accumulated β-catenin is considered crucial to the initiation of intestinal carcinogenesis.10Polakis P. Wnt signaling and cancer.Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 11Giles R.H. van Es J.H. Clevers H. Caught up in a Wnt storm Wnt signaling in cancer.Biochim Biophys Acta. 2003; 1653: 1-24Crossref PubMed Scopus (1329) Google Scholar TCF-4 is a member of the TCF/LEF family of transcription factors, which comprises LEF1, TCF-1, TCF-3, and TCF-4; only one of them, TCF-4, is commonly expressed in colorectal cancer cells.12Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2926) Google Scholar TCF-4 has been implicated in the maintenance of undifferentiated intestinal crypt epithelial cells, because no proliferative compartments were detected in the intestinal crypts of mice lacking TCF-4.13Korinek V. Barker N. Moerer P. van Donselaar E. Huls G. Peters P.J. Clevers H. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1320) Google Scholar Suppression of TCF-4/β-catenin-evoked gene transactivation by dominant-negative TCF-4 switches off genes involved in cell proliferation and switches on genes involved in cell differentiation.14van de Wetering M. Sancho E. Verweij C. de Lau W. Oving I. Hurlstone A. van der Horn K. Batlle E. Coudreuse D. Haramis A.P. Tjon-Pon-Fong M. Moerer P. van den Born M. Soete G. Pals S. Eilers M. Medema R. Clevers H. The β-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells.Cell. 2002; 111: 241-250Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar Induction of dominant-negative TCF-4 restored the epithelial cell polarity of a colorectal cancer cell line and converted the cell line into a single layer of columnar epithelium.15Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google Scholar The β-catenin/TCF-4 complex and its associated molecules would seem to be candidates as targets of molecular therapy against colorectal cancer. Thus far, only a few molecules, including the Groucho family,16Cavallo R.A. Cox R.T. Moline M.M. Roose J. Polevoy G.A. Clevers H. Peifer M. Bejsovec A. Drosophila Tcf and Groucho interact to repress Wingless signalling activity.Nature. 1998; 395: 604-608Crossref PubMed Scopus (598) Google Scholar C-terminal binding protein 1,17Brannon M. Brown J.D. Bates R. Kimelman D. Moon R.T. XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.Development. 1999; 126: 3159-3170Crossref PubMed Google Scholar dCBP,18Waltzer L. Bienz M. Drosophila CBP represses the transcription factor TCF to antagonize Wingless signalling.Nature. 1998; 395: 521-525Crossref PubMed Scopus (326) Google Scholar Smads,19Labbe E. Letamendia A. Attisano L. Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-β and wnt pathways.Proc Natl Acad Sci U S A. 2000; 97: 8358-8363Crossref PubMed Scopus (379) Google Scholar and Chibby,20Takemaru K. Yamaguchi S. Lee Y.S. Zhang Y. Carthew R.W. Moon R.T. Chibby, a nuclear β-catenin-associated antagonist of the Wnt/Wingless pathway.Nature. 2003; 422: 905-909Crossref PubMed Scopus (243) Google Scholar have been identified as interacting directly with the β-catenin and TCF/LEF complexes and modulating transcriptional activity. Except for Smads, however, all of these molecules are suppressors of TCF/LEF-mediated gene transactivation, and their biological significance in intestinal carcinogenesis has not been explored. In this study, we took a proteomics approach21Gavin A.C. Bosche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Hofert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Functional organization of the yeast proteome by systematic analysis of protein complexes.Nature. 2002; 415: 141-147Crossref PubMed Scopus (3993) Google Scholar to identifying molecules associated with TCF-4. In consequence, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a member of the TCF complexes. PARP-1 is a nuclear DNA-binding protein that catalyzes the transfer of adenosine diphosphate ribose from oxidized nicotinamide adenine dinucleotide (NAD+) to acceptor proteins.22D’Amours D. Desnoyers S. D’Silva I. Poirier G.G. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.Biochem J. 1999; 342: 249-268Crossref PubMed Scopus (1572) Google Scholar, 23de Murcia G. Menissier de Murcia J. Poly(ADP-ribose) polymerase a molecular nick-sensor.Trends Biochem Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (761) Google Scholar PARP-1 is activated by DNA damage and plays an important role in the process of DNA repair and genomic stability.24Herceg Z. Wang Z.Q. Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death.Mutat Res. 2001; 477: 97-110Crossref PubMed Scopus (408) Google Scholar No mutations or loss of heterozygosity of the PARP-1 gene have been reported in clinical cancers, but the overexpression of PARP-1 has been reported in various human malignancies. PARP-1 was found to be overexpressed in 57% (20/35) of primary breast carcinomas, and the locus of the PARP-1 gene (1q41-44) was amplified in 70% (7/10) of the tumors in the PARP-1-overexpressing group.25Bieche I. de Murcia G. Lidereau R. Poly(ADP-ribose) polymerase gene expression status and genomic instability in human breast cancer.Clin Cancer Res. 1996; 2: 1163-1167PubMed Google Scholar Amplification of this locus has also been reported in other cancers, such as hepatocellular carcinoma26Crawley J.J. Furge K.A. Identification of frequent cytogenetic aberrations in hepatocellular carcinoma using gene-expression microarray data.Genome Biol. 2002; 3 (RESEARCH0075.)Crossref PubMed Google Scholar and brain tumors.27Pleschke J.M. Kleczkowska H.E. Strohm M. Althaus F.R. Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins.J Biol Chem. 2000; 275: 40974-40980Crossref PubMed Scopus (450) Google Scholar PARP-1 is also overexpressed in Ewing’s sarcoma28Soldatenkov V.A. Albor A. Patel B.K. Dreszer R. Dritschilo A. Notario V. Regulation of the human poly(ADP-ribose) polymerase promoter by the ETS transcription factor.Oncogene. 1999; 18: 3954-3962Crossref PubMed Scopus (75) Google Scholar and malignant lymphoma.29Tomoda T. Kurashige T. Moriki T. Yamamoto H. Fujimoto S. Taniguchi T. Enhanced expression of poly(ADP-ribose) synthetase gene in malignant lymphoma.Am J Hematol. 1991; 37: 223-227Crossref PubMed Scopus (71) Google Scholar The PARP-1 protein was significantly increased in hepatocellular carcinoma compared with levels in biopsy specimens of nonneoplastic liver.30Shimizu S. Nomura F. Tomonaga T. Sunaga M. Noda M. Ebara M. Saisho H. Expression of poly(ADP-ribose) polymerase in human hepatocellular carcinoma and analysis of biopsy specimens obtained under sonographic guidance.Oncol Rep. 2004; 12: 821-825PubMed Google Scholar A recent report31Ghabreau L. Roux J.P. Frappart P.O. Mathevet P. Patricot L.M. Mokni M. Korbi S. Wang Z.Q. Tong W.M. Frappart L. Poly(ADP-ribose) polymerase-1, a novel partner of progesterone receptors in endometrial cancer and its precursors.Int J Cancer. 2004; 109: 317-321Crossref PubMed Scopus (49) Google Scholar indicated that PARP-1 expression increased progressively from nonatypical to atypical endometrial hyperplasia, reaching the highest level in grade I endometrial carcinoma, and then decreasing as the disease further progressed, suggesting the involvement of PARP-1 in the early carcinogenesis of the uterine endometrium. Here, we report that PARP-1 is a novel coactivator of TCF-4/β-catenin-evoked gene transactivation and may be involved in the regulation of intestinal epithelial cell differentiation/proliferation and carcinogenesis. The human embryonal kidney cell line HEK293 was obtained from the Riken Cell Bank (Tsukuba, Japan). Colon cancer cell lines SW480 and HCT116 were purchased from American Type Culture Collection (Manassas, VA). Bleomycin was obtained from Sigma Chemical Co (St Louis, MO). Human TCF-4 (splice form E) complementary DNA (cDNA) and its truncated forms (Figure 3A–C) were subcloned into pFLAG-CMV4 (Sigma Chemical Co). Human PARP-1 cDNA (kindly provided by Dr. Miwa, Tsukuba University, Tsukuba, Japan) and its truncated forms (Figure 3D) were subcloned into pcDNA3.1/myc-His (Invitrogen, Carlsbad, CA). Human β-catenin cDNA was subcloned into pFLAG and pCR3.1 (Invitrogen). pCR3.1-β-cateninΔN134 lacks a 134-amino acid sequence in its NH2 terminus. Human ETS1 and ETS2 cDNAs were subcloned into pcDNA3.1/myc-His. pcDNA3.1-ETS2-DN-myc lacks a 328-amino acid sequence in its NH2-terminus and has a dominant negative effect.32Langer S.J. Bortner D.M. Roussel M.F. Sherr C.J. Ostrowski M.C. Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.Mol Cell Biol. 1992; 12: 5355-5362Crossref PubMed Scopus (136) Google ScholarFigure 3Identification of the regions required for interaction between TCF-4 and PARP-1. (A–C) Full-length or truncated forms of FLAG-TCF-4 purified on anti-FLAG affinity gel were incubated with the biotinylated PARP-1 protein prepared by in vitro translation. The complexes were thoroughly washed and analyzed by blotting with anti-FLAG antibody (FLAG) and avidin/horseradish peroxidase (Avidin). Full-length and truncated forms of TCF-4 are schematically represented at the bottom. Asterisks indicate the TCF-4 constructs that bound to the PARP-1 protein. (D) Full-length and truncated forms of biotinylated PARP-1 were prepared by in vitro translation, and their expression was confirmed by blotting with avidin/horseradish peroxidase (Avidin, input). The full-length FLAG-TCF-4 protein was purified with anti-FLAG affinity gel and incubated with each form of PARP-1 for 12 hours at 4°C. The complexes were thoroughly washed, eluted from the gels, and analyzed by blotting with avidin-HRP (Avidin, output) or anti-FLAG antibody (FLAG). The full-length and truncated forms of PARP-1 are illustrated at the bottom (WT, wild-type). Asterisks indicate the PARP-1 constructs that bound to TCF-4.View Large Image Figure ViewerDownload (PPT) The region containing 1.2 kilobases upstream from the transcription start site of the PARP-1 gene (−1200/+152) was amplified from genomic HEK293 DNA by polymerase chain reaction (PCR) and subcloned into pGL3-basic (Promega, Madison, WI) (pGL3-basic-PARP-1-prom). The composition of all of the constructs in this study was confirmed by restriction endonuclease digestion and sequencing. Details of the procedures used for plasmid construction are available on request. Cells were extracted with lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton X-100) containing a protease inhibitor cocktail (Sigma Chemical Co). Nuclear extracts were prepared with the CelLytic nuclear extraction kit (Sigma Chemical Co). Immunoprecipitation was performed with 50 μL of anti-FLAG M2 affinity gel (Sigma Chemical Co) or anti-PARP-1 monoclonal antibody (BD PharMingen, San Diego, CA), anti-β-catenin monoclonal antibody (BD Transduction, Lexington, KY), and anti-TCF-4 monoclonal antibody (Upstate Biotechnology, Waltham, MA) along with 10 μL of Dynabeads Protein G (Dynal, Oslo, Norway). After being washed with wash buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl), immobilized immunocomplexes were eluted from anti-FLAG M2 affinity gel for 1 hour by incubating at 4°C with 150 ng/μL of 3xFLAG Peptide (Sigma Chemical Co) or from Dynabeads by boiling in sodium dodecyl sulfate loading buffer for 5 minutes. Proteins were fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and detected with the negative gel stain MS kit (Wako, Osaka, Japan) or by Western blotting. SDS-PAGE gels were cut into ∼1 mm3 sections, and the protein in the gel was reduced with NH4HCO3 and alkylated with iodoacetamide. The gel sections were washed with acetonitrile, and the protein was hydrolyzed with modified trypsin (Promega). Peptides eluted from the gel were spotted onto a steel target plate with 2,5-dihydroxybenzoic acid (gentisic acid) (Sigma Chemical Co) as a matrix. Mass spectra were obtained in the refractor mode by using a Q-star Pulsar-i (Applied Biosystems, Foster City, CA) as described previously33Seike M. Kondo T. Mori Y. Gemma A. Kudoh S. Sakamoto M. Yamada T. Hirohashi S. Proteomic analysis of intestinal epithelial cells expressing stabilized β-catenin.Cancer Res. 2003; 63: 4641-4647PubMed Google Scholar and were analyzed with Mascot software (Matrix Sciences, London, England). Anti-FLAG M2 monoclonal antibody was purchased from Sigma Chemical Co, anti-PARP-1 polyclonal antibody from Cell Signaling Technology (Beverly, MA), anti-β-catenin monoclonal antibody from BD Transduction, anti-TCF3/4 monoclonal antibody from Upstate Biotechnology, anti-c-myc polyclonal antibody from Roche (Indianapolis, IN), anti-polyADP-ribose polyclonal antibody from Alexis (Lausanne, Switzerland), and avidin/horseradish peroxidase from Amersham (Amersham, Buckinghamshire, England). Total cell lysates were extracted at 4°C with RIPA buffer (150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris-HCl, pH 8.0) containing a protease inhibitor cocktail (Sigma Chemical Co). Samples were fractionated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Billerica, MA),15Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google Scholar and blots were detected by an enhanced chemiluminescence method (Amersham). FLAG-tagged protein was purified from HEK293 cells by using anti-FLAG M2 affinity gel. Full-length or truncated forms of biotinylated PARP-1 protein were prepared by in vitro translation with the TNT quick-coupled transcription/translation system (Promega). Recombinant PARP-1 protein was purchased from Alexis. The anti-FLAG M2 affinity gels combined with FLAG-tagged protein were incubated for 12 hours at 4°C with in vitro translated or recombinant PARP-1 in wash buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl). The gels were washed 3 times with the wash buffer and then eluted by incubation for 1 hour at 4°C with 150 ng/μL of 3xFLAG Peptide (Sigma Chemical Co). A pair of luciferase reporter constructs, TOP-FLASH and FOP-FLASH (Upstate Biotechnology), were used to evaluate TCF/LEF transcriptional activity. Cells were transiently transfected in triplicate with one of the luciferase reporters and phRL-TK (Promega) by using Lipofectamine 2000 reagent; 24 hours after transfection, luciferase activity was measured with the dual-luciferase reporter assay system (Promega) and Renilla luciferase activity as an internal control.15Naishiro Y. Yamada T. Takaoka A.S. Hayashi R. Hasegawa F. Imai K. Hirohashi S. Restoration of epithelial cell polarity in a colorectal cancer cell line by suppression of β-catenin/T-cell factor 4-mediated gene transactivation.Cancer Res. 2001; 61: 2751-2758PubMed Google Scholar Two short hairpin RNA sequences targeting PARP-1 messenger RNA (mRNA) were designed by B-Bridge (Sunnyvale, CA). Synthesized double-stranded oligonucleotides were cloned into pSUPER RNAi vector (OligoEngine, Seattle, WA) carrying the H1 promoter and a neomycin resistance gene. Two targeting constructs, named pSUPER-PARP-1 (A) and (B), or control (empty pSUPER) were transfected into the colorectal cancer cell line HCT116 by using Lipofectamine 2000 reagent. Twenty-four hours later, the transfection medium was replaced with RPMI 1640 containing 1 mg/mL G418 (Geneticin; Invitrogen). Cells were stained with Giemsa staining solution (Wako, Tokyo, Japan) after selection with G418 for 7 days. Cells were labeled with anti-bromodeoxyuridine and propidium iodide following the manufacturer’s protocol (BD Biosciences, San Jose, CA) and analyzed using BD LSR and CellQuest software (BD Biosciences). Formalin-fixed and paraffin-embedded intestinal tissues containing adenoma and carcinoma were stained by the avidin-biotin complex method.33Seike M. Kondo T. Mori Y. Gemma A. Kudoh S. Sakamoto M. Yamada T. Hirohashi S. Proteomic analysis of intestinal epithelial cells expressing stabilized β-catenin.Cancer Res. 2003; 63: 4641-4647PubMed Google Scholar Ten patients with FAP and 28 sporadic colorectal cancer cases were selected from the surgical pathology panel of the National Cancer Center Central Hospital. Male multiple intestinal polyposis (Min) mice (C57BL/6J-ApcMin/+) were obtained from the Jackson Laboratory (Bar Harbor, ME). Animal experiments were reviewed and approved by the ethical committee of the National Cancer Center Research Institute (Tokyo, Japan). The purified protein of FLAG-TCF-4 or FLAG-β-catenin was incubated with 1 μL of the PARP-1 enzyme (Trevigen, Gaithersburg, MD) and 250 ng of histone (Trevigen) in 50 μL of reaction mixture (50 mmol/L Tris-HCl, pH 8.0, 250 mmol/L MgCl2, 400 μmol/L nicotinamide adenine dinucleotide [NAD+], 25 μmol/L biotinylated NAD+) for 30 minutes at room temperature. PolyADP-ribosylation was detected by Western blotting with avidin/horseradish peroxidase. Total RNA was prepared from the normal small intestine and polyp tissues of Min mice with TRIzol reagent (Invitrogen) and from cell lines with the RNeasy Mini Kit (Qiagen, Valencia, CA). One-microgram samples of deoxyribonuclease I-treated total RNA were reverse transcribed. cDNA samples from human sporadic colorectal cancer tissue and the corresponding normal tissue were obtained from Clontech (Palo Alto, CA). The following PCR primers were used: for human c-myc, 5′-GGTCTTCCCCTACCCTCTCAA-3′ and 5′-CGTTGTGTGTTCGCCTCTTG-3′; for human cyclin D1, 5′-CCCGCTGGCCATGAACTA-3′ and 5′-CGGAGGCAGTCTGGGTCA-3′; for human matrilysin (MMP7), 5′-GAGTGCCAGATGTTGCAGAATACT-3′ and 5′-GAATGCCTTTAATATCATCCTGGG-3′; for mouse Parp-1, 5′-CTCCAAAGAGGACGCTGTTGA-3′ and 5′-CCTCGATGTCCAGGAGGTTGT-3′; for mouse Ets1, 5′-CGACTCTCACCATCATCAAGACA-3′ and 5′-GAGAACTCTGAGGGAGGAACACA-3′; for mouse Ets2, 5′-CGATGAATGACTTTGGAATCAAGA-3′ and 5′-GATCATCTGCTCTAGATGTTCCCA-3′; for mouse Gapdh (glyceraldehyde-3-phosphate dehydrogenase), rodent Gapdh forward and reverse primers (Applied Biosystems); for human PARP-1, 5′-GTGTGGGTACGGTGATCGGTA-3′ and 5′- GCCTGCACACTGTCTGCATT-3′; and for human GAPDH, 5′-GAAGGTGAAGGTCGGAGTC-3′ and 5′-GAAGATGGTGATGGGATTTC-3′. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. Quantification analysis was performed using the LAS-3000 scanner and the Science Lab 2003 software (Fujifilm, Tokyo, Japan). To identify the proteins associated with TCF-4, HEK293 cells were transiently transfected with FLAG-tagged TCF-4 (FLAG-TCF-4) or control (FLAG-MOCK). Immunoprecipitation with anti-FLAG antibody and SDS-PAGE revealed that several proteins were selectively coprecipitated with FLAG-TCF-4 (open arrowheads, Figure 1A) but not with the control. From both total cell lysates and nuclear extracts, a protein of approximately 112 kilodaltons (closed arrowheads, Figure 1A) was consistently coimmunoprecipitated with FLAG-TCF-4 and was subjected to protein identification by mass spectrometry. Peptide mass fingerprinting (Figure 1B) and database search revealed that the protein was PARP-1 (Figure 1C).Figure 1Identification of an interaction between PARP-1 and TCF-4. (A) SDS-PAGE analysis of the immunoprecipitates from HEK293 cells transfected with FLAG-TCF-4 or control FLAG-MOCK. Twenty-four hours after transfection, total cell lysates (left) or nuclear extracts (right) were immunoprecipitated with anti-FLAG affinity gel and analyzed by SDS-PAGE. The open arrowhead is pointing to FLAG-TCF-4 and the closed arrowhead to the 112-kilodalton protein. (B) Mass spectrum of the 112-kilodalton protein digested with modified trypsin. (C) Amino acid sequence of PARP-1. Underlining indicates peptides that correspond to peaks identified by mass spectrometry. (D) Western blot analysis of the immunoprecipitates of HEK293 cells transfected with FLAG-TCF-4 (+) or control FLAG-MOCK (−). The input cell lysates (total) and immunoprecipitates with anti-FLAG antibody (IP: FLAG) were blotted with anti-FLAG and anti-PARP-1 antibodies. (E) (Top) Lysates of HEK293 cells transfected with FLAG-TCF-4 or untransfected HEK293 cells (parent) were blotted with anti-FLAG or anti-PARP-1 antibody. (Bottom) Lysates of HEK293 cells transfected with FLAG-TCF-4 were immunoprecipitated with anti-PARP-1 antibody or normal mouse IgG and analyzed by blotting with anti-FLAG and anti-PARP-1 antibodies.View Large Image Figure ViewerDownload (PPT) Western blotting with anti-PARP-1 antibody confirmed the protein identification (Figure 1D). Correspondingly, FLAG-TCF-4 protein was detected in the immunoprecipitate with anti-PARP-1 antibody but not with normal mouse immunoglobulin (Ig) G (Figure 1E). The β-catenin protein accumulates in colorectal cancer cells as a result of mutations in the APC or β-catenin (CTNNB1) genes, resulting in constitutive formation of the β-catenin/TCF-4 complex.12Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Constitutive transcriptional activation by a β-catenin-Tcf complex in APC-/- colon carcinoma.Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2926) Google Scholar The 78-kilodalton TCF-4 (Figure 2A) and β-catenin (Figure 2B) proteins were coimmunoprecipitated with anti-PARP-1 antibody but not with normal mouse IgG from lysates of a colorectal cancer cell line SW480 carrying a mutated APC gene. Conversely, PARP-1 was immunoprecipitated with anti-TCF-4 and β-catenin antibodies (Figure 2C). These results revealed the inclusion of PARP-1 in the native TCF-4/β-catenin complex.Figure 2Inclusion of PARP-1 in the native TCF-4/β-catenin complex. (A) Nuclear extracts of SW480 (Total) were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with anti-TCF-3/4 (TCF-4) and anti-PARP-1 antibodies. (B) SW480 cell lysates were immunoprecipitated with anti-PARP-1 antibody (IP: PARP-1) or normal mouse IgG (IP: IgG) and blotted with ant
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