Role of p73 in regulation of cell death specific role in mitotic cell death and potential regulation of caspase 2s

ROLE OF P73 IN REGULATION OF CELL DEATH: SPECIFIC ROLE IN MITOTIC CELL DEATH AND POTENTIAL REGULATION OF CASPASE-2s TOH WEN HONG (BSc Hons, University of Queensland, Australia) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 ROLE OF P73 IN REGULATION OF CELL DEATH: SPECIFIC ROLE IN MITOTIC CELL DEATH AND POTENTIAL REGULATION OF CASPASE-2s TOH WEN HONG NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGMENT I am extremely grateful to the members of my lab for their generosity, creative discussions and mostly acceptance of me, as a weird introverted character Most of all, I would like to thank my supervisor, Associate Professor Kanaga Sabapathy, for his wonderful guidance and advice throughout the period of my candidature Lastly, I would like to say thank you to my dearest wife and cutest daughter, for their understanding and support, which makes the countless experimental failures more bearable i TABLE OF CONTENTS Acknowledgements Table of Contents Summary List of Tables List of Figures List of illustrations Abbreviations List of publications Chapter Introduction i ii vi vii viii xii xii xiv 1.1 p53 tumor suppressor family 1.1.1 p53 1.1.2 p63 1.1.3 p73 1.1.3.1 p73 and cancers 1.1.3.2 p73 regulation 1.1.3.3 p73 protein modifications 1.1.4 p53 family in apoptosis and as tumor suppressors 1.2 Control of genomic integrity 1.2.1 G1 checkpoint 1.2.2 G2 checkpoint 1.2.3 Spindle checkpoint 1.3 Mitotic catastrophe 1.2.1 How mitotic arrest results in apoptosis 1.4 Apoptosis 1.4.1 Caspase-9 activation pathway 1.4.2 Caspase-8 activation pathway 1.4.3 Caspase-2 activation pathway 1.5 Mitochondria membrane permeabilization (MMP) 1.6 BH3-only proteins 1.6.1 Bim 1.7 Aims 10 11 12 12 13 15 16 18 18 20 20 21 23 23 25 Chapter Material and Methods 27 2.1 Materials 2.2 General methods 2.2.1 Tissue culture 2.2.1.1 Cell lines 2.2.1.2 Culture conditions of cell lines 28 39 39 39 39 ii 2.2.1.3 Subculturing of cells 2.2.1.4 Cryopreservation of cells 2.2.1.5 DNA transfections 2.2.1.6 siRNA transfections 2.2.1.7 Synchronization and released of cells from the G1/S phase 2.2.1.8 Drugs treatment 2.2.1.9 γ-irradiation 2.2.2 Molecular Biology procedures 2.2.2.1 Bacterial transformation 2.2.2.2 Site directed mutagenesis 2.2.2.3 DNA sequencing 2.2.2.4 Cloning of p73α, p73β and p53 into pGEX-4T1 2.2.2.5 Cloning of Del4 truncations 2.2.2.6 Miniprep 2.2.2.7 RNA extraction 2.2.2.8 Quantification of nucleic acid concentration 2.2.2.9 1st strand cDNA synthesis 2.2.3 FACS procedures 2.2.3.1 Cell death assays 2.2.3.2 Cell cycle and phospho-histone-H3 analysis 2.2.4 Immunofluorescence and confocal microscopy 2.2.5 Protein isolation and analysis 2.2.5.1 Total cell lysate 2.2.5.2 Cell fractionation 2.2.5.3 Determining protein concentration 2.2.5.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 2.2.5.5 Western hybridization 2.2.6 Semi-quantitative PCR 2.2.7 Chromatin immunoprecipitation 2.2.8 GST-protein purification 2.2.9 In vitro DNA Protein Binding assay 2.2.10 Luciferase assay 2.2.11 Statistical analysis Chapter p73 role in mitotic cell death 3.1 Background 3.1.1 Mitosis 3.1.2 Chemotherapeutic drugs that target mitosis 3.1.2.1 Taxol 3.1.3 Aims 3.2 Taxol-induced mitotic cell death 3.2.1 G2/M arrest induced by taxol treatment 3.2.2 Mitotic arrest in taxol treated cells 3.2.3 Initiation of apoptosis in mitotic arrested taxol treated cells 40 40 40 41 42 42 43 43 43 43 44 44 45 46 47 47 48 48 48 49 50 51 51 51 52 52 53 53 56 59 59 60 61 62 63 63 64 65 66 67 67 68 69 iii 3.2.4 Taxol induced cell death during mitosis 3.2.5 Blockage of Taxol treated cells from entering mitosis prevent apoptosis 3.3 p73 is required for mitotic cell death 3.3.1 Silencing of p73 block taxol induced cell death 3.3.2 Up-regulation of p73 early during taxol treatment 3.3.3 Silencing of p53 does not block taxol induced cell death 3.3.4 p73 null mouse fibroblasts are resistant to taxol induced cell death 3.3.5 p73-/- fibroblasts maintain in G2/M block 60 hrs after taxol treatment 3.3.6 No decrease in number of adherent p73-/- cells after taxol treatment 3.4 p73 play a role in mitotic cell death induced by other means 3.4.1 MG132 induced mitotic cell death 3.4.1.1 p73 is required for MG132 mitotic cell death 3.4.2 Mitotic cell induced by plk1 silencing require p73 3.4.3 p73 is required for γ-irradiation induced cell death 3.5 p73 does not regulate bubr1 and mad2 3.5.1 Mechanism of p73 on mitotic cell death differs from that of bubr1 and mad2 3.6 Caspase-9 is the apical caspase activated during mitotic cell death 3.7 p73 regulate cytochrome c release during mitotic cell death 3.8 p73 does not regulate Bax translocation during mitotic cell death 3.9 Bim is not upregulated in p73-/- fibroblasts upon taxol treatment 3.10 p73 transcriptionally activated Bim 3.10.1 p73 binds to response elements in bim intron region 3.10.2 Silencing of Bim protect cells from mitotic cell death induced by plk1 depletion and taxol treatment Chapter Regulation of Caspase-2S by p73β β 4.1 Background 4.1.1 Caspases 4.1.2 Caspase-2 4.1.3 Transcriptional regulation of Caspase-2S 4.1.4 Aims 4.2 Caspase-2S but not caspase-2L, caspase-8 and caspase-9 is up-regulated by p73β 4.3 p73β can induce caspase-2S promoter reporter construct 4.4 p73β act through a 22 bp sequence in caspase-2S promoter 4.5 p73β bind directly to caspase-2S promoter 4.5.1 p73β does not act through Sp1 to induce caspase-2S promoter 4.5.2 DNA binding domain of p73β is required for inducing caspase-2S activity 72 74 77 77 79 81 83 87 88 89 89 93 95 99 102 105 109 114 115 119 122 124 127 130 131 131 131 132 134 135 137 140 142 142 144 iv 4.5.3 p73β and DNp73β bind to endogenous caspase-2S promoter 4.5.4 p73β bind directly to the identified 22 bp RE site on caspase-2S promoter 4.6 Potential biological role of p73 in regulation of caspase-2S 4.6.1 Increase survival of Sh5y expressing DNp73β during serum deprivation 4.6.2 Caspase-2 silencing has no effect on p73β mediated cell death 146 Chapter 153 Discussion 149 150 150 151 5.1 Overview 5.2 Regulation of mitotic cell death by p73 5.2.1 p73-dependent mitotic cell death require spindle checkpoint activation and mitotic arrest 5.2.2 p73, not p53 has a role in mitotic cell death 5.2.3 p73 regulate mitotic cell death induced by other factors 5.2.4 Prolonged mitosis results in apoptosis 5.2.5 Same phenotype but different mechanism 5.2.6 p73 act upstream of apical caspase-9 5.2.7 p73 regulate cytochrome c release independent of Bax 5.2.8 p73 regulate Bim to prime cell for mitotic catastrophe 5.3 Caspase-2S is a target of p73β 5.3.1 Induction of caspase-2S is specific to p73β isoform only 5.3.2 p73β bind directly to the 22 bp site on Caspase-2S promoter 5.3.3 Role of caspase-2S up-regulation by p73 5.3.4 Over-expression of p73 in cancers 5.4 Conclusion 154 155 Bibliography 173 155 156 158 159 160 161 162 163 166 166 168 168 170 171 v SUMMARY p73 is a member of the p53 family of transcription factors It shares similar properties to p53 such as the induction of genes required for regulating cell arrest and apoptosis Genetic evidences have established an important role of p73 as a tumor suppressor In addition, p73 has been also demonstrated to play vital proapoptotic role in regulating chemotherapeutic drug sensitivity However studies of p73 function is complicated by the expression of numerous N- and C-terminal variants DNp73, an alternate promoter expressed N-terminal p73 variant blocks both p53 and p73 pro-apoptotic functions In human cancers, p73 is rarely found mutated but often over-expressed Other studies have also found that both fulllength p73 and DNp73 have the ability to regulate growth promoting factors Hence suggesting that p73 have unique roles in regulating cell death as well as growth and survival We show in this research proposal that p73 is capable of both playing a pro-apoptotic role in mitotic cell death as well as a survival role in up-regulation of anti-apoptotic caspase-2S gene Mitotic cell death is a form of cell death that is triggered upon prolonged mitotic cell arrest Although many chemotherapeutic drugs trigger mitotic cell death, the mechanism is unknown In this PhD project, we found that p73 but not p53, is important for inducing mitotic cell death Cells lacking p73 or cells in which p73 expression is silenced are resistant to mitotic cell death The phenomenon is not due to the potential p73 regulation on spindle checkpoint proteins bubr1 and mad2, but instead, is due to the inability of p73 knockout cells to trigger mitochondria permeabilization Cytochrome c release from the mitochondria and activation of caspases are defective in p73-silenced cells This is found to be due to the requirement of p73 to trans-activate bim Bim is a BH3-only protein, which interact with other Bcl-2 family members to permeabilize the mitochondria membrane upon apoptotic signal p73 knockout cells have reduced levels of bim Bim silenced cells are also resistant to mitotic cell death Lastly, p73 can bind to bim promoter and induce bim expression Together, our data define a critical regulatory role for p73 in inducing mitotic death, which occurs independently of p53 vi In addition, we have discovered caspase-2S to be a novel target gene of p73 Caspase-2S is a smaller variant of caspase-2 and is known for promoting survival instead of cell death Ectopic expression of p73 not affect the transcript level of any other caspases tested except caspase-2S The induction of caspase-2S is unique to p73β and DNp73β but not p73α, p53 or p63 This induction of caspase2S by p73β depends on a 22 bp unique p73β recognition sequence Additionally, we are able to demonstrate direct binding of p73 to this recognition sequence on caspase-2S promoter Lastly, Sh5y neuroblastoma cells stably expressing DNp73β have higher caspase-2S levels and are resistant to cell death induced by serum starvation Overall, we have demonstrated a dual role of p73 in regulation of cell death: first as a pro-apoptotic protein involved in mitotic cell death, and secondly as an antiapoptotic protein by up-regulating caspase-2S vii LIST OF TABLES Table 1: p73 recognition sequence on Bim intron 124 Table 2: DNA sequence of the caspase-2 gene contain in Del reporter construct 140 viii Jacobs,W.B., Walsh,G.S., and Miller,F.D (2004) Neuronal survival and p73/p63/p53: a family affair Neuroscientist 10, 443-455 Jacobs,W.B., Kaplan,D.R., and Miller,F.D (2006) The p53 family in nervous system development and disease J Neurochem 97, 1571-1584 Jones,S.N., Roe,A.E., Donehower,L.A., and Bradley,A (1995) Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53 Nature 378, 206208 Jost,C.A., Marin,M.C., and Kaelin,W.G., Jr (1997) p73 is a simian [correction of human] p53-related protein that can induce apoptosis Nature 389, 191-194 Kartasheva,N.N., Lenz-Bauer,C., Hartmann,O., Schafer,H., Eilers,M., and Dobbelstein,M (2003) DeltaNp73 can modulate the expression of various genes in a p53-independent fashion Oncogene 22, 8246-8254 Kadonaga,J.T., Carner,K.R., Masiarz,F.R., and Tjian,R (1987) Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain Cell 51, 1079-1090 Kaghad,M., Bonnet,H., Yang,A., Creancier,L., Biscan,J.C., Valent,A., Minty,A., Chalon,P., Lelias,J.M., Dumont,X., Ferrara,P., McKeon,F., and Caput,D (1997) Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers Cell 90, 809-819 181 Kisurina-Evgen'eva,O.P., Briantseva,S.A., Stil',A.A., and Onishchenko,G.E (2006) [Antimicrotubule agents can activate different apoptotic pathways] Biofizika 51, 875-879 Kovalev,S., Marchenko,N., Swendeman,S., LaQuaglia,M., and Moll,U.M (1998) Expression level, allelic origin, and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines Cell Growth Differ 9, 897-903 Korsmeyer,S.J., Wei,M.C., Saito,M., Weiler,S., Oh,K.J., and Schlesinger,P.H (2000) Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c Cell Death Differ 7, 11661173 Koster,M.I., Kim,S., Mills,A.A., DeMayo,F.J., and Roop,D.R (2004) p63 is the molecular switch for initiation of an epithelial stratification program Genes Dev 18, 126-131 Koster,M.I and Roop,D.R (2004) The role of p63 in development and differentiation of the epidermis J Dermatol Sci 34, 3-9 Kroemer,G (2002) Introduction: mitochondrial control of apoptosis Biochimie 84, 103-104 Kumar,S., Kinoshita,M., Noda,M., Copeland,N.G., and Jenkins,N.A (1994) Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3 and the mammalian IL-1 beta-converting enzyme Genes Dev 8, 1613-1626 182 Lane,D.P and Crawford,L.V (1979) T antigen is bound to a host protein in SV40-transformed cells Nature 278, 261-263 Lassus,P., Opitz-Araya,X., and Lazebnik,Y (2002) Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization Science 297, 1352-1354 Lanni,J.S and Jacks,T (1998) Characterization of the p53-dependent postmitotic checkpoint following spindle disruption Mol Cell Biol 18, 1055-1064 Lee,H and Kimelman,D (2002) A dominant-negative form of p63 is required for epidermal proliferation in zebrafish Dev Cell 2, 607-616 Lee,A.F., Ho,D.K., Zanassi,P., Walsh,G.S., Kaplan,D.R., and Miller,F.D (2004) Evidence that DeltaNp73 promotes neuronal survival by p53-dependent and p53independent mechanisms J Neurosci 24, 9174-9184 Lei,K and Davis,R.J (2003) JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis Proc Natl Acad Sci U S A 100, 2432-2437 Leu,J.I., Dumont,P., Hafey,M., Murphy,M.E., and George,D.L (2004) Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex Nat Cell Biol 6, 443-450 Lin,K.W., Nam,S.Y., Toh,W.H., Dulloo,I., and Sabapathy,K (2004) Multiple stress signals induce p73beta accumulation Neoplasia 6, 546-557 183 Li,W., Lan,Z., Wu,H., Wu,S., Meadows,J., Chen,J., Zhu,V., and Dai,W (1999) BUBR1 phosphorylation is regulated during mitotic checkpoint activation Cell Growth Differ 10, 769-775 Linzer,D.I., Maltzman,W., and Levine,A.J (1979) The SV40 A gene product is required for the production of a 54,000 MW cellular tumor antigen Virology 98, 308-318 Liu,X and Erikson,R.L (2003) Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells Proc Natl Acad Sci U S A 100, 5789-5794 Liu,X., Lei,M., and Erikson,R.L (2006) Normal cells, but not cancer cells, survive severe Plk1 depletion Mol Cell Biol 26, 2093-2108 Liu,G and Chen,X (2005) The C-terminal sterile alpha motif and the extreme C terminus regulate the transcriptional activity of the alpha isoform of p73 J Biol Chem 280, 20111-20119 Li,W., Lan,Z., Wu,H., Wu,S., Meadows,J., Chen,J., Zhu,V., and Dai,W (1999) BUBR1 phosphorylation is regulated during mitotic checkpoint activation Cell Growth Differ 10, 769-775 Logette,E., Wotawa,A., Solier,S., Desoche,L., Solary,E., and Corcos,L (2003) The human caspase-2 gene: alternative promoters, pre-mRNA splicing and AUG usage direct isoform-specific expression Oncogene 22, 935-946 184 May,K.M and Hardwick,K.G (2006) The spindle checkpoint J Cell Sci 119, 4139-4142 Mantovani,F., Piazza,S., Gostissa,M., Strano,S., Zacchi,P., Mantovani,R., Blandino,G., and Del,S.G (2004) Pin1 links the activities of c-Abl and p300 in regulating p73 function Mol Cell 14, 625-636 Margottin-Goguet,F., Hsu,J.Y., Loktev,A., Hsieh,H.M., Reimann,J.D., and Jackson,P.K (2003) Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase Dev Cell 4, 813-826 Melino,G., Bernassola,F., Ranalli,M., Yee,K., Zong,W.X., Corazzari,M., Knight,R.A., Green,D.R., Thompson,C., and Vousden,K.H (2004) p73 Induces apoptosis via PUMA transactivation and Bax mitochondrial translocation J Biol Chem 279, 8076-8083 Melino,G., Lu,X., Gasco,M., Crook,T., and Knight,R.A (2003) Functional regulation of p73 and p63: development and cancer Trends Biochem Sci 28, 663-670 Mihara,M., Erster,S., Zaika,A., Petrenko,O., Chittenden,T., Pancoska,P., and Moll,U.M (2003) p53 has a direct apoptogenic role at the mitochondria Mol Cell 11, 577-590 185 Minn,A.J., Boise,L.H., and Thompson,C.B (1996) Expression of Bcl-xL and loss of p53 can cooperate to overcome a cell cycle checkpoint induced by mitotic spindle damage Genes Dev 10, 2621-2631 Miller,L.K (1999) An exegesis of IAPs: salvation and surprises from BIR motifs Trends Cell Biol 9, 323-328 Mills,A.A., Zheng,B., Wang,X.J., Vogel,H., Roop,D.R., and Bradley,A (1999) p63 is a p53 homologue required for limb and epidermal morphogenesis Nature 398, 708-713 Moll,U.M and Slade,N (2004b) p63 and p73: roles in development and tumor formation Mol Cancer Res 2, 371-386 Nigg,E.A (2001) Mitotic kinases as regulators of cell division and its checkpoints Nat Rev Mol Cell Biol 2, 21-32 Nomura,M., Shimizu,S., Sugiyama,T., Narita,M., Ito,T., Matsuda,H., and Tsujimoto,Y (2003) 14-3-3 Interacts directly with and negatively regulates proapoptotic Bax J Biol Chem 278, 2058-2065 Ozaki,T and Nakagawara,A (2005) p73, a sophisticated p53 family member in the cancer world Cancer Sci 96, 729-737 Parant,J.M., Reinke,V., Mims,B., and Lozano,G (2001) Organization, expression, and localization of the murine mdmx gene and pseudogene Gene 270, 277-283 186 Park,S.J., Wu,C.H., Gordon,J.D., Zhong,X., Emami,A., and Safa,A.R (2004) Taxol induces caspase-10-dependent apoptosis J Biol Chem 279, 51057-51067 Pozniak,C.D., Radinovic,S., Yang,A., McKeon,F., Kaplan,D.R., and Miller,F.D (2000) An anti-apoptotic role for the p53 family member, p73, during developmental neuron death Science 289, 304-306 Puthalakath,H., Huang,D.C., O'Reilly,L.A., King,S.M., and Strasser,A (1999) The proapoptotic activity of the Bcl-2 family member Bim is regulated by interaction with the dynein motor complex Mol Cell 3, 287-296 Puthalakath,H., Villunger,A., O'Reilly,L.A., Beaumont,J.G., Coultas,L., Cheney,R.E., Huang,D.C., and Strasser,A (2001) Bmf: a proapoptotic BH3-only protein regulated by interaction with the myosin V actin motor complex, activated by anoikis Science 293, 1829-1832 Ramadan,S., Terrinoni,A., Catani,M.V., Sayan,A.E., Knight,R.A., Mueller,M., Krammer,P.H., Melino,G., and Candi,E (2005) p73 induces apoptosis by different mechanisms Biochem Biophys Res Commun 331, 713-717 Ren,J., Datta,R., Shioya,H., Li,Y., Oki,E., Biedermann,V., Bharti,A., and Kufe,D (2002) p73beta is regulated by protein kinase Cdelta catalytic fragment generated in the apoptotic response to DNA damage J Biol Chem 277, 33758-33765 Roninson,I.B., Broude,E.V., and Chang,B.D (2001) If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells Drug Resist Updat 4, 303-313 187 Rossi,M., De,L., V, Munarriz,E., Green,D.R., Liu,Y.C., Vousden,K.H., Cesareni,G., and Melino,G (2005) The ubiquitin-protein ligase Itch regulates p73 stability EMBO J 24, 836-848 Rupinder,S.K., Gurpreet,A.K., and Manjeet,S (2007) Cell suicide and caspases Vascul Pharmacol 46, 383-393 Sambrook J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: a laboratory manual (New York, Cold Spring Harbour Lab Press) Sbisa,E., Catalano,D., Grillo,G., Licciulli,F., Turi,A., Liuni,S., Pesole,G., De,G.A., Caratozzolo,M.F., D'Erchia,A.M., Navarro,B., Tullo,A., Saccone,C., and Gisel,A (2007) p53FamTaG: a database resource of human p53, p63 and p73 direct target genes combining in silico prediction and microarray data BMC Bioinformatics Suppl 1, S20 Secombe,J., Pierce,S.B., and Eisenman,R.N (2004) Myc: a weapon of mass destruction Cell 117, 153-156 Sedlak,T.W., Oltvai,Z.N., Yang,E., Wang,K., Boise,L.H., Thompson,C.B., and Korsmeyer,S.J (1995) Multiple Bcl-2 family members demonstrate selective dimerizations with Bax Proc Natl Acad Sci U S A 92, 7834-7838 Seong,Y.S., Kamijo,K., Lee,J.S., Fernandez,E., Kuriyama,R., Miki,T., and Lee,K.S (2002) A spindle checkpoint arrest and a cytokinesis failure by the dominant-negative polo-box domain of Plk1 in U-2 OS cells J Biol Chem 277, 32282-32293 188 Shibue,T and Taniguchi,T (2006) BH3-only proteins: integrated control point of apoptosis Int J Cancer 119, 2036-2043 Shearwin-Whyatt,L.M., Harvey,N.L., and Kumar,S (2000) Subcellular localization and CARD-dependent oligomerization of the death adaptor RAIDD Cell Death Differ 7, 155-165 Shi,Y (2002) Mechanisms of caspase activation and inhibition during apoptosis Mol Cell 9, 459-470 Shin,S., Lee,Y., Kim,W., Ko,H., Choi,H., and Kim,K (2005) Caspase-2 primes cancer cells for TRAIL-mediated apoptosis by processing procaspase-8 EMBO J 24, 3532-3542 Shiozaki,E.N., Chai,J., Rigotti,D.J., Riedl,S.J., Li,P., Srinivasula,S.M., Alnemri,E.S., Fairman,R., and Shi,Y (2003) Mechanism of XIAP-mediated inhibition of caspase-9 Mol Cell 11, 519-527 Spankuch-Schmitt,B., Bereiter-Hahn,J., Kaufmann,M., and Strebhardt,K (2002) Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells J Natl Cancer Inst 94, 1863-1877 Srinivasula,S.M., Hegde,R., Saleh,A., Datta,P., Shiozaki,E., Chai,J., Lee,R.A., Robbins,P.D., Fernandes-Alnemri,T., Shi,Y., and Alnemri,E.S (2001) A conserved XIAP-interaction motif in caspase-9 and Smac/DIABLO regulates caspase activity and apoptosis Nature 410, 112-116 189 Stiewe,T and Putzer,B.M (2000) Role of the p53-homologue p73 in E2F1induced apoptosis Nat Genet 26, 464-469 Stott,F.J., Bates,S., James,M.C., McConnell,B.B., Starborg,M., Brookes,S., Palmero,I., Ryan,K., Hara,E., Vousden,K.H., and Peters,G (1998) The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2 EMBO J 17, 5001-5014 Sudo,T., Nitta,M., Saya,H., and Ueno,N.T (2004) Dependence of paclitaxel sensitivity on a functional spindle assembly checkpoint Cancer Res 64, 25022508 Suh,E.K., Yang,A., Kettenbach,A., Bamberger,C., Michaelis,A.H., Zhu,Z., Elvin,J.A., Bronson,R.T., Crum,C.P., and McKeon,F (2006) p63 protects the female germ line during meiotic arrest Nature 444, 624-628 Sunters,A., Fernandez de,M.S., Stahl,M., Brosens,J.J., Zoumpoulidou,G., Saunders,C.A., Coffer,P.J., Medema,R.H., Coombes,R.C., and Lam,E.W (2003) FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines J Biol Chem 278, 49795-49805 Taylor,W.R and Stark,G.R (2001) Regulation of the G2/M transition by p53 Oncogene 20, 1803-1815 Tan,T.T., Degenhardt,K., Nelson,D.A., Beaudoin,B., Nieves-Neira,W., Bouillet,P., Villunger,A., Adams,J.M., and White,E (2005) Key roles of BIM- 190 driven apoptosis in epithelial tumors and rational chemotherapy Cancer Cell 7, 227-238 Teodoro,J.G., Heilman,D.W., Parker,A.E., and Green,M.R (2004) The viral protein Apoptin associates with the anaphase-promoting complex to induce G2/M arrest and apoptosis in the absence of p53 Genes Dev 18, 1952-1957 Thornton,B.R., Ng,T.M., Matyskiela,M.E., Carroll,C.W., Morgan,D.O., and Toczyski,D.P (2006) An architectural map of the anaphase-promoting complex Genes Dev 20, 449-460 Tinel,A and Tschopp,J (2004) The PIDDosome, a protein complex implicated in activation of caspase-2 in response to genotoxic stress Science 304, 843-846 Tomkova,K., El-Rifai,W., Vilgelm,A., Kelly,M.C., Wang,T.C., and Zaika,A.I (2006) The gastrin gene promoter is regulated by p73 isoforms in tumor cells Oncogene 25, 6032-6036 Toh,W.H., Siddique,M.M., Boominathan,L., Lin,K.W., and Sabapathy,K (2004) c-Jun regulates the stability and activity of the p53 homologue, p73 J Biol Chem 279, 44713-44722 Toh,W.H., Kyo,S., and Sabapathy,K (2005) Relief of p53-mediated telomerase suppression by p73 J Biol Chem 280, 17329-17338 Urist,M and Prives,C (2004) The linchpin? Pin1 meets p73 Cancer Cell 5, 515517 191 Urist,M., Tanaka,T., Poyurovsky,M.V., and Prives,C (2004) p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2 Genes Dev 18, 3041-3054 Vikhanskaya,F., Toh,W.H., Dulloo,I., Wu,Q., Boominathan,L., Ng,H.H., Vousden,K.H., and Sabapathy,K (2007) p73 supports cellular growth through cJun-dependent AP-1 transactivation Nat Cell Biol 9, 698-706 Vikhanskaya,F., Vignati,S., Beccaglia,P., Ottoboni,C., Russo,P., D'Incalci,M., and Broggini,M (1998) Inactivation of p53 in a human ovarian cancer cell line increases the sensitivity to paclitaxel by inducing G2/M arrest and apoptosis Exp Cell Res 241, 96-101 Vogelstein,B., Lane,D., and Levine,A.J (2000) Surfing the p53 network Nature 408, 307-310 von,H.C., Wieder,T., Essmann,F., Schulze-Osthoff,K., Dorken,B., and Daniel,P.T (2003) Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspases-3/-8-driven mitochondrial amplification loop Oncogene 22, 2236-2247 Wahl,A.F., Donaldson,K.L., Fairchild,C., Lee,F.Y., Foster,S.A., Demers,G.W., and Galloway,D.A (1996) Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis Nat Med 2, 72-79 192 Wang,L., Miura,M., Bergeron,L., Zhu,H., and Yuan,J (1994) Ich-1, an Ice/ced3-related gene, encodes both positive and negative regulators of programmed cell death Cell 78, 739-750 Wang,X., Arooz,T., Siu,W.Y., Chiu,C.H., Lau,A., Yamashita,K., and Poon,R.Y (2001) MDM2 and MDMX can interact differently with ARF and members of the p53 family FEBS Lett 490, 202-208 Wang,W.K., Bycroft,M., Foster,N.W., Buckle,A.M., Fersht,A.R., and Chen,Y.W (2001) Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha Acta Crystallogr D Biol Crystallogr 57, 545-551 Watson,I.R., Blanch,A., Lin,D.C., Ohh,M., and Irwin,M.S (2006) Mdm2mediated NEDD8 modification of TAp73 regulates its transactivation function J Biol Chem 281, 34096-34103 Weaver,B.A and Cleveland,D.W (2005) Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death Cancer Cell 8, 7-12 Willis,S.N and Adams,J.M (2005) Life in the balance: how BH3-only proteins induce apoptosis Curr Opin Cell Biol 17, 617-625 Yang,A., Kaghad,M., Caput,D., and McKeon,F (2002) On the shoulders of giants: p63, p73 and the rise of p53 Trends Genet 18, 90-95 Yang,A., Walker,N., Bronson,R., Kaghad,M., Oosterwegel,M., Bonnin,J., Vagner,C., Bonnet,H., Dikkes,P., Sharpe,A., McKeon,F., and Caput,D (2000) 193 p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours Nature 404, 99-103 Yuan,Z.M., Shioya,H., Ishiko,T., Sun,X., Gu,J., Huang,Y.Y., Lu,H., Kharbanda,S., Weichselbaum,R., and Kufe,D (1999) p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage Nature 399, 814-817 Zaika,A., Irwin,M., Sansome,C., and Moll,U.M (2001) Oncogenes induce and activate endogenous p73 protein J Biol Chem 276, 11310-11316 Zaika,A.I., Kovalev,S., Marchenko,N.D., and Moll,U.M (1999) Overexpression of the wild type p73 gene in breast cancer tissues and cell lines Cancer Res 59, 3257-3263 Zaika,A.I., Slade,N., Erster,S.H., Sansome,C., Joseph,T.W., Pearl,M., Chalas,E., and Moll,U.M (2002) DeltaNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors J Exp Med 196, 765-780 Zamzami,N., Marzo,I., Susin,S.A., Brenner,C., Larochette,N., Marchetti,P., Reed,J., Kofler,R., and Kroemer,G (1998) The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition Oncogene 16, 1055-1063 Zeng,X., Chen,L., Jost,C.A., Maya,R., Keller,D., Wang,X., Kaelin,W.G., Jr., Oren,M., Chen,J., and Lu,H (1999) MDM2 suppresses p73 function without promoting p73 degradation Mol Cell Biol 19, 3257-3266 194 Zeng,X., Li,X., Miller,A., Yuan,Z., Yuan,W., Kwok,R.P., Goodman,R., and Lu,H (2000) The N-terminal domain of p73 interacts with the CH1 domain of p300/CREB binding protein and mediates transcriptional activation and apoptosis Mol Cell Biol 20, 1299-1310 195 .. .ROLE OF P73 IN REGULATION OF CELL DEATH: SPECIFIC ROLE IN MITOTIC CELL DEATH AND POTENTIAL REGULATION OF CASPASE- 2s TOH WEN HONG NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGMENT... Up -regulation of caspase- 2S in Saos2 p73? ? inducible cell line and Sh5y stably expressing DNp73β 146 Fig 4.10: ChIP of p73 on 22 bp p73 binding site identified on the caspase- 2S using Soas2 p73? ?... dual role of p73 in regulation of cell death: first as a pro-apoptotic protein involved in mitotic cell death, and secondly as an antiapoptotic protein by up-regulating caspase- 2S vii LIST OF TABLES