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 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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