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Genetic and proteomic analysis of the P33ING1b tumour suppressor in melanoma Campos, Eric I. 2006

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GENETIC A N D PROTEOMIC ANALYSIS OF THE P33ING1B T U M O U R SUPPRESSOR IN M E L A N O M A by ERIC I. CAMPOS B.Sc. with Honours, University of Ottawa, 2000 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Experimental Medicine Program THE UNIVERSITY OF BRITISH C O L U M B I A April 2006 © Eric I. Campos, 2006 11 ABSTRACT The ING1 (Inhibitor of Growth) gene is the founding member of at least five related human genes associated with tumour suppressive properties. ING genes are evolutionarily conserved and express cofactors of histone acetyltransferases (HAT) and histone deacetylases (HDAC). The ING1 locus encodes at least three detectable protein isoforms, including the well-studied pSS^*3"3 protein. Through associated HAT and H D A C activity, pSS^ 0 1 1" is capable of regulating the transcription of various genes, including the p21 w a f l and cyclin BI cell cycle regulators and the pro-apoptotic Bcl-2 family member Bax, leading to inhibition of cell cycle progression and sensitization of cells to apoptosis. P 3 3 I N G l b also enhances the nucleotide excision repair of ultraviolet-damaged D N A . This work describes 1) the generation and use of rabbit polyclonal antiserum that can specifically recognize the p 3 3 I N G l b isoform; 2) the status of the ING1 gene in malignant human melanoma; and 3) the regulation of the p 3 3 I N G l b protein through protein phosphorylation and degradation. Mutational alterations were found within the ING1 gene of nearly a fifth of the melanoma biopsies examined. Two common alterations within the ING1 gene at codons 102 and 260 were found to be detrimental to p33 I N G l b-mediated enhancement of nucleotide excision repair. Mutations within the ING1 gene may also be indicative of a poorer 5-year patient survival. The ING1 gene was also found to be over-expressed in melanoma cells and biopsies compared to normal melanocytes. There was, however, widespread loss of nuclear expression of p 3 3 r N G I b in the melanoma tumours. Ill This thesis further describes the regulation of the pSS™ 0 1 1 1 protein through phosphorylation of serine 126 by the cyclin-dependent kinase 1 in the absence of D N A damage and by the checkpoint kinase 1 upon genotoxic stress. Although serine 126 is near the nuclear-localization sequence, it was not found to affect the sub-cellular localization of the p 3 3 I N G l b protein. Phosphorylation did however alter the half-life of the protein. Serine to alanine site-directed mutagenesis of codon 126 or the inhibition of the Cdkl kinase both resulted in a higher turnover rate of the p 3 3 I N G l b protein. Although p 3 3 I N G l b did incorporate ubiquitin moieties, it was found not to undergo proteolysis through the classical ubiquitin-proteasome pathway. iv T A B L E OF CONTENTS Abstract ii Table of Contents iv List of Tables viii List of Figures ix List of Symbols and Abbreviations xi Preface xiii Acknowledgments xvi Co-authorship Statement xvii 1 Introductory Chapter 1 1.1 The p 3 3 I N G l b Protein: A Literature Review 1 1.1.1 Structure of the p33mGlb Protein 2 1.1.2 Biological Relevance of the p 3 3 I N G , b Protein 4 1.1.2.1 Transcriptional Regulation 5 1.1.2.2 Cell Cycle Checkpoints 7 1.1.2.3 Apoptosis 8 1.1.2.4 D N A Repair 9 1.1.3 Status oflNGl in Human Malignancies 11 1.2 Protein Phosphorylation 12 1.3 Protein Degradation 14 1.4 Thesis Theme 16 1.5 Hypotheses 17 1.6 Figures & Tables 18 1.7 References 21 2 Materials and Methods 31 2.1 Cell Culture and Transfection 31 2.2 Generation of Polyclonal Antibodies 32 2.3 Drug Treatments and Ultraviolet Irradiation 33 2.4 Single-strand Conformation Polymorphism 34 2.5 D N A Sequencing 35 2.6 Western Analysis 36 2.7 Semi-quantitative Reverse Transcriptase Polymerase Chain Reaction .. 37 2.8 Immunohistochemistry 38 2.9 Immunofluorescence 39 2.10 Site-directed Mutagenesis 39 2.11 D N A Repair Assays 40 2.12 Immunoprecipitation and Phosphatase Treatment 41 2.13 Enzyme-linked Immunosorbent Assay 42 2.14 Purification of Recombinant p 3 3 I N G l b 42 2.15 Mass Spectrometry 43 2.16/?7 vitro Kinase Assay 44 2.17 References 46 3 Generation of Rabbit Polyclonal Antisera Directed Against pSS™ 0 1 1 1 47 3.1 Rationale 47 3.2 Results & Discussion 48 vi 3.2.1 Immunization and Affinity Purification 48 3.2.2 Specificity of the Affinity-purified Ant i -p33 I N G , b Antiserum 49 3.3 Figures 51 3.4 References 56 4 Status of the ING1 Gene in Human Melanoma 57 4.1 Rationale 57 4.2 Results 58 4.2.1 ING1 Gene Expression in Human Melanoma 58 4.2.2 ING1 Gene Mutation in Human Melanoma 60 4.2.3 Correlations between ING1 Mutations and Clinical Parameters 62 4.2.4 Effect ofJNGl mutation on D N A repair 63 4.3 Discussion 65 4.4 Figures & Tables 70 4.5 References 83 5 Phosphorylation of the pSS 1* 0 1 1 5 Protein 86 5.1 Rationale 86 5.2 Results 87 5.2.1 pss™ 0 1 1 3 is a Phospho-protein 87 5.2.2 Serine 126 is Phosphorylated upon Genotoxic Stress 89 5.2.3 Serine 126 Phosphorylation Does not Alter p 3 3 I N G l b Sub-cellular Localization 91 V l l 5.2.4 Phosphorylation of Serine 126 Increases the Half-life o f p 3 3 I N G l b 92 5.2.5 Serine 126 is Phosphorylated by both Chkl and Cdkl .... 93 5.2.6 P 3 3 1 N G l b is not Degraded through the Classical Ubiquitin-proteasome Pathway 96 5.3 Discussion 97 5.4 Tables & Figures 103 5.5 References 116 6 Concluding Remarks 118 viii LIST OF T A B L E S Table 1-1 List of p33m G l b-regulated genes 19 Table 1 -2 Summary of reported ING1 expression levels and gene alterations in human malignancies 20 Table 4-1 Summary of ING1 gene mutations found by SSCP-D N A sequencing in melanoma cell lines 80 Table 4-2 Summary of ING1 gene mutations found by SSCP-D N A sequencing in melanoma biopsies 81 Table 4-3 Correlation between ING1 gene mutations in human cutaneous malignant melanoma and clinical parameters 82 Table 5-1 NetPhosK 1.0 kinase prediction for p 3 3 I N G l b serine 126 115 ix LIST OF FIGURES Figure 1-1 Structural features of the p 3 3 I N G l b protein 18 Figure 3-1 Peptide used for the generation of a pSS^^-specific polyclonal antibody 51 Figure 3-2 Purity of the peptide used to raise a p33 I N G l b-specific polyclonal antiserum 52 Figure 3-3 ELISA of serum from rabbits inoculated with a synthetic peptide corresponding to the p 3 3 I N G l b protein 53 Figure 3-4 Western analysis of p 3 3 I N G l b using the rabbit polyclonal p 3 3 i N G i b _ s p e c i f l c a n t i b o d y 54 Figure 3-5 Epifluorescent images of pINGl b-FLAG transfected cells immunolabeled with anti-p33 I N G l b rabbit polyclonal antibody .... 55 Figure 4-1 Western analysis of p33 1 N G l b protein levels in fourteen melanoma cell lines 70 Figure 4-2 Immunohistochemical analysis of p 3 3 I N G l b expression in human cutaneous malignant melanoma 71 Figure 4-3 SSCP analysis of the ING1 gene in human melanoma cell lines and cutaneous malignant melanoma biopsies 73 Figure 4-4 Representative sequencing analysis of samples with aberrant SSCP migration 74 Figure 4-5 Location and relative frequency of mutations affecting the p 3 3 1 N G l b protein 75 Figure 4-6 Correlative analysis of pSS^ 0 1 1 1 immunohistochemical parameters and ING1 gene mutations in melanoma biopsies 76 Figure 4-7 Kaplan-Meier curves illustrating a potential correlation between ING1 mutation and 5-year patient survival 77 Figure 4-8 Site-directed mutagenesis of the pCIneo-JA^G/ft-FLAG plasmid ... 78 Figure 4-9 ING1 mutations found in human cutaneous malignant melanoma abrogate p33 ,NGlb-dependent repair of UV-damaged D N A 79 Figure 5-1 Posttranslational modification of the p3 3 I N G l b protein 103 Figure 5-2 P 3 3 I N G l b is subject to phosphorylation in vivo and in vitro 104 Figure 5-3 Mass spectrometry analysis of phosphorylated AspN-digested recombinant GST-pSS™ 0 1" 105 Figure 5-4 Generation of phospho-serine 126 antisera and D N A damage-induced phosphorylation of endogenous p 3 3 I N G I b 106 Figure 5-5 Site-directed mutagenesis of serine 126 107 Figure 5-6 Phosphorylation of serine 126 does not affect protein sub-cellular localization 108 Figure 5-7 Abolishment of serine 126 phosphorylation shortens the half-life of thep33 I N G l b protein 109 Figure 5-8 Serine 126 is phosphorylated by Chkl upon genotoxic stress but by Cdkl under normal conditions 110 Figure 5-9 Proposed model of p 3 3 I N G l b regulation through serine 126 phosphorylation 112 Figure 5-10 Proteasome-independent degradation o f p 3 3 I N G l b 113 XI LIST OF S Y M B O L S A N D ABBREVIATIONS Abbreviation Full Name 6-4PP pyrimidine (6-4) pyrimidone photoproducts AFP alpha fetoprotein A T M ataxia-telangiectasia mutated ATP adenosine triphosphate ATR ATM-related protein Bax Bcl2-associated X protein CAT chloramphenicol acetyltransferase Cdk cyclin-dependent kinase ChIP chromatin immunoprecipitation Chkl/2 checkpoint kinase 1/2 CIP calf intestinal alkaline phosphatase CKII casein kinase II CPD cyclobutane pyrimidine dimers D N A deoxyribonucleic acid DNA-PK DNA-dependent protein kinase ELISA enzyme-linked immunosorbent assay E M S A electrophoretic mobility shift assay GSK3a glycogen synthase kinase 3 a GST glutathione S-transferase HAT histone acetyltransferase HDAC histone deacetylase HRP horseradish peroxidase h-SMG-1 human homologue of the C. elegans protein CeSMG-1 ING inhibitor of growth K L H keyhole limpet hemocyanin MALDI-TOF matrix assisted laser desorption ionization time-of-flight M A P K mitogen-activated protein kinase M D M 2 mouse double minute NER nucleotide excision repair NLS nuclear localization sequence NTS nucleolus-targeting sequence NuA3 nucleosome acetylating H3 NuA4 nucleosome acetylating H4 PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PBS-T phosphate-buffered saline tween-20 PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PCR domain potential chromatin regulatory domain PCR-SOEing polymerase chain reaction splicing overlap extension PDEVI phosphorylation-dependent interacting motif xii PHD plant homeodomain PI3K phosphatidylinositol-3 kinase PIKK phosphatidylinositol-3 kinase-related kinase PPM peptide-interacting motif PIP PCNA-interacting protein domain Ptdlns phosphatidylinositol PTEN phosphatase and tensin homolog NiNTA nickel-nitrilotriacetic acid Rb retinoblastoma RIA radioimmunoassay RNA ribonucleic acid R N A i R N A Interference ROS reactive oxygen species RT-PCR reverse transcriptase polymerase chain reaction sec squamous cell carcinoma SDS Sodium dodecyl sulphate siRNA small interfering R N A SSCP single strand conformational polymorphism Ub ubiquitin U V ultraviolet Wafl wild-type p53-activated factor XP xeroderma pigmentosum Xlll PREFACE Melanoma results from the malignant transformation and uncontrolled proliferation of melanocytes, the melanin-producing cells normally found in the basal layer of the skin epidermis. Melanoma is one of the most deadly cutaneous neoplasms. With an incidence increase of 4.1 percent per year in the US in the past few years (according to the American Cancer Society, 2005), there is great interest in finding the underlying genetic causes leading to its formation. There are numerous risk factors for melanoma, including sun sensitivity, light skin tone (little pigmentation), family predisposition, immunosuppression and the number of typical and large congenital nevi (MacKie et al, 1989). However there is a great deal to be learned on the genetic factors that contribute to the cellular transformation of melanocytes and on the regulation of tumour suppressors that may be involved. Although the exact mechanism of melanoma development remains to be elucidated, ultraviolet (UV) radiation is thought to be an important environmental factor linked to the transformation of cutaneous melanocytes (reviewed by Jhappan et al., 2003). There are two mutually inclusive cellular pathways thought to contribute to melanomagenesis: 1) oncogenic activation of the Ras - mitogen-activated protein kinase (MAPK) cascade; and 2) inactivation of pathways that promote hypo-phosphorylation of the retinoblastoma (Rb) tumour suppressor. For the first, various studies have observed RAS (notably NRAS) gene mutations in approximately 5-25% of melanomas, or in some cases RAS gene amplification (Gorden et al., 2003; Demunter et al., 2001). Most xiv mutations were found to constitutively activate Ras which in turn activates the M A P K (RAP - M E K - ERK), or the PI3K - A K T - N F - K B pathway known to cross-talk with the former (Ackermann et al, 2005; Chudnovsky et al, 2005; Wu et al, 2003), both of which promote cellular growth. Alternatively melanoma cells may also activate the M A P K or PI3K pathways without constitutive Ras activation. Loss of heterozygosity within the PTEN locus was observed in 30-50% of melanomas (Wu et al., 2003); however, patients with Cowden disease (inherited PTEN mutation) are not reported to be susceptible to melanoma. Although it has also been found that up to 70% of melanomas may express mutant B-Raf (Davies et ah, 2002, Uribe et al., 2003), a similar proportion of normal melanocytic nevi were found to carry the same mutation (Uribe et al., 2003) suggesting that other genetic factors may be necessary for melanocytic transformation. There are several pathways leading to the regulation of the Rb protein. Once thought to be the 'melanoma gene' the CDKN2A locus encodes two tumour suppressors within different reading frames (Quelle et al., 1995) that can influence Rb activity. P16 I N K 4 a sequesters CDKs to prevent Rb phosphorylation (Rb inhibits cell cycle progression when hypo-phosphorylated) and p l 4 A R F prevents mdm2-mediated p53 degradation, thus also Rb phosphorylation (see introductory chapter). Germline mutations of the CDKN2A gene are observed in a high proportion of familial melanomas (Hussussian et al., 1994), however somatic mutations of this gene in sporadic primary melanoma are rare (Chachia et al, 2000). Unlike other skin cancers there is a low (less than 15%) incidence of TP53 mutations in melanoma (Gwosdz et al., 2006). Both Rb inactivation and the activation of the Ras-MAPK/PI3K pathway are increasingly thought X V to contribute to melanomagenesis (Ackermann et al, 2005; Chudnovsky et al., 2005). Since p53 activation does lead to the hypo-phosphorylation and activation of Rb; and unlike other cancers p53, p\6mK4a, p l 4 A R F and Rb are infrequent targets in sporadic primary melanoma, there is a great interest to find other melanoma susceptibility genes that may lead to the inactivation of the retinoblastoma protein. A novel family of tumour suppressors, the Inhibitor of Growth (ING) family, has recently been identified and found to directly cooperate with the p53 protein. The p 3 3 I N G l b protein is also capable of up-regulating the expression of the p21 W a f l C D K inhibitor thereby contributing to Rb activation. Much of the present thesis concentrates on the p 3 3 I N G l b protein. In order to determine whether p 3 3 I N G l b inactivation may contribute to the susceptibility of sporadic melanoma, we sought to examine the status the ING1 gene in malignant melanoma. Since p 3 3 I N G l b impedes cellular growth and enhances the repair of damaged DNA, the regulation of the p 3 3 , N G l b protein is also explored in the later part of the thesis. The observations made throughout this work were at times surprising but nonetheless interesting and will further contribute to the understanding of cutaneous melanoma development and of the p 3 3 I N G l b tumour suppressor protein. xvi A C K N O W L E D G M E N T S I would like to acknowledge the Michael Smith Foundation for Health Research, the UBC & Vancouver Hospital Foundation, the Natural Science and Engineering Research Council, the National Cancer Institute of Canada (NCIC), the U B C Faculty of Graduate Studies and the U B C Department of Medicine, Division of Dermatology for their support. I also sincerely thank my family and friends, as well as past and present members of Dr. Li 's laboratory (particularly Yvonne, Jason, Mei, Marco and William for all their help). I also thank my supervisor Dr. Gang L i as well as my supervisory committee: Drs Vince Duronio, Bi l l Salh, Chris Ong and Vincent Ho as well as Dr. Hao Xiao (ImmuneChem Pharmaceuticals Inc.) and Dr. Magda Martinka (Vancouver Hospital) for guidance and help. XVII CO-AUTHORSHIP STATEMENT Certain experiments and reagents were performed or obtained in collaboration with others, or under industrial contract, as listed below: Synthetic peptides were generated by the Nucleic Acid and Protein Service Unit (NAPS) unit at the University of British Columbia. The radioimmunoassay of D N A samples was performed by Dr. David L. Mitchell (University of Texas M.D. Anderson Cancer Center). The KinaseProfiler™ kinase selectivity screening is a service offered by Upstate Inc. Mass spectrometry analysis of p 3 3 I N G l b was done in the laboratory of Dr. Jeffrey Smith (The Burnham Institute). Certain kinase assays were performed with the help of Dr. Marco Garate (University of British Columbia) as well as the analysis for half-life estimations; triplicates for the half-life time point experiments were also obtained with the help of Wei-Hung Kuo (University of British Columbia). Permission to reproduce copyright material was granted for the following published articles in written or as part of the publishing agreement: 1- Campos E.I., Chin M . Y . , Kuo W.H. and L i G. Biological functions of the ING family tumour suppressors. 2004. Cell Mol Life Sci 61:2597-613. 2- Campos E.I., Martinka M . , Mitchell D.L., Dai D.L. and L i G. Mutations of the ING1 tumour suppressor gene detected in human melanoma abrogate nucleotide excision repair. 2004. Int J Oncol 25:73-80. XV111 3- Campos E.I., Xiao H . and L i G. Generation of a polyclonal antibody specifically against the p33(INGlb) tumour suppressor. 2004. J Immunoassay Immunochem 25:71-80. 4- Campos E.I., Cheung K.J . Jr., Murray A. , L i S. and L i G. The novel tumour suppressor gene ING1 is overexpressed in human melanoma cell lines. 2002. Br J Dermatol 146:574-80. 1 1. INTRODUCTORY CHAPTER 1.1 The p 3 3 I N G l b protein: A Literature Review The ING1 gene was originally identified using a strategy developed by Gudkov et al. (1994), for the recovery genetic elements that can promote neoplastic growth deregulation when lost or inactivated. Specifically, through subtractive hybridization between normal human mammary epithelial cells and seven breast cancer cell lines, genetic suppressor elements that were lost in the cancer cell lines were isolated. These genetic fragments were then cloned in an inverse orientation and the resulting antisense constructs retained i f found to possess oncogenic characteristics in vivo. The sequence of these oncogenic fragments were subsequently used to retrieve the full gene from a normal human fibroblast cDNA library; hence the cloning of the ING1 gene (Garkatsev et al., 1996). ING1 has been cytogenetically mapped to 13q34 at 5Mb of the telomeric region of the chromosome (Zeremski et al., 1997). The ING1 gene is also the founding member of at least five additional related human genes, namely ING2-5 and INGX (INGX encodes a truncated ING-like PHD domain and expression has yet to be detected) (He et al, 2005; Shimada et al, 1998; Nagashima et al, 2003; Shiseki et al, 2003). It is interesting to note that ING genes have been shown to flank important functional genes. For example, ING1 is flanked by the RAB20 gene which encodes a Ras family member and the Rho guanidine nucleotide exchange factor gene ARHGEF7 (He et al, 2005). However, a A version of this chapter has been published in: Campos E.I., Chin M . Y . , Kuo W.H. and L i G. Biological functions of the ING family tumour suppressors. 2004. Cell Mol Life Sci 61:2597-613. 2 coordinated regulation of proximal genes within 7M?-containing clusters has yet to be explored. Most importantly, ING proteins are found to be highly conserved through evolution and have so far been observed among members of all four eukaryotic kingdoms (animal, plant, fungi and protista) (He et al, 2005; Mayanagi et al., 2005). The ING1 gene comprises three exons that encode three detectable alternatively spliced protein isoforms of 47 (p47 [ N G l a), 33 (p33 1 N G l b) and 24 (p24 I N G l c ) kDa (Gunduz et al, 2000; Saito et al, 2000), of which p 3 3 I N G l b is the most abundant and ubiquitously expressed in human tissues (Saito et al, 2000; Shimada et al, 1998). The following sections review studies on the structure of the p 3 3 I N G l b protein, its implication in the cell cycle, in apoptosis and in D N A repair, as well as the status of the ING1 gene in human cancers. 1.1.1 Structure of the p 3 3 I N G l b Protein ING family proteins are defined by the presence of a carboxyl C4-H-C3 zinc finger known as the Plant Homeodomain (PHD), as well as by the presence of an uncharacterized KIQI /KVQL motif named the 'Potential Chromatin Regulatory (PCR) domain' (initially termed the MDS00105 motif) (Kawaji et al, 2002; He et al, 2005). Most ING proteins also comprise an amino terminal leucine zipper. The p 3 3 I N G l b protein is one of the few family members that lack the leucine zipper. Conversely, the p 3 3 I N G l b protein has some unique structural features. This ING protein is the only human isoform to possess an amino terminal proliferating-cell nuclear antigen (PCNA)-interacting 3 protein (PIP) domain. Although all human ING proteins have a Nuclear Localization Sequence (NLS), the p33mGlh isoform is one of the few to possess two functional nucleolus-targeting motifs within its NLS (Scott et al., 2001a). Based on sequence alignment analysis, p 3 3 I N G l b is also predicted to contain at least two additional domains: a 'N-REASP-C' N-terminal sequence termed the phosphorylation-dependent interacting motif (PDIM) due to its resemblance to the RSXpSXP binding motif found on 14-3-3 proteins (He et al., 2005; Yaffe et al., 1997); and a C-terminal peptide-interacting motif (PEVl) (He et al., 2005) thought to1 mediate protein-protein interactions due to the presence of various basic residues (He et al., 2005) (Figure 1-1). Unfortunately little effort has so far been done to characterize most of the p33 1 N G l b domains. However, some data has been obtained on the role of the PHD domain. The PHD finger belongs to the Treble class of zinc-binding motifs and is most often found in chromatin-interacting proteins. Like the closely related F Y V E zinc finger, the p33 1 N G l b and p33 I N G 2 PHD domains can serve as nuclear phosphoinositide (Ptdlns)-binding modules with a strong preference for PtdIns(5)P (Gozani et al., 2003). Phosphoinositide binding may help regulate ING protein functions in at least two distinct manners based on observations made on the p33 I N G 2 PHD domain. First, overexpression of the lipid PIKIip kinase (which converts PtdIns(5)P into PtdIns(4,5)P2) caused a sharp reduction of chromatin bound r)33mG2 and increase of free p33 I N G 2 , while overexpression of the IpgD phosphatase had the opposite effect (Gozani et al., 2003). Secondly, PIKIip overexpression inhibited pSS^^-dependent acetylation of the p53 protein and enhancement of apoptosis without affecting overall p33 1 N G 2 protein levels (Gozani et al., 4 2003). These observations imply a regulatory role for the ING PHD zinc fingers as Ptdlns receptors and modulators of ING protein localization and activity. 1.1.2 Biological Relevance of the pSS™ 0 1 6 Protein The p 3 3 I N G l b protein is a type II tumour suppressor. It is involved in a wide range of vital biological functions including cell cycle regulation, gene regulation, D N A repair and apoptosis. ING proteins lack enzymatic activity but serve as co-factors of histone acetyltransferases (HATs) and histone deacetylases (HDAC). H A T enzymes catalyze the transfer of an acetyl group from a given substrate onto the s-NH3 + groups of histone amino terminal lysine residues. This transfer alters local charges and increases histone hydrophobicity and is thought to affect chromatin dynamics. The p 3 3 r N G l b protein has been co-purified with the p300 HAT and the Sin3 complex which includes HDAC1 and HDAC2 (Vieyra et al, 2002; Kuzmichev et al, 2002; Skowyra et al, 2001). The role of ING proteins in histone acetylation was first clarified in yeast. The Saccharomyces cerevisae ING-family member Yng l has been described as a core component of the NuA3 HAT complex (Howe et al, 2002). Yng l was found non-essential for NuA3 HAT activity or for the structural integrity of the complex, but did however regulate the substrate specificity of the complex. In the absence of Y n g l , NuA3 was found capable of acetylating free histones but lacked enzymatic activity on nucleosomes; whereas Y n g l -proficient cells exhibited specific NuA3 activity on nucleosomal histones 3 and 4 but not on free histones (Howe et al, 2002). The same observation has now been extended to other ING family members (Campos et al, 2004; Doyon et al, 2006) as it is believed that 5 they may help recruit H A T and H D A C complexes onto chromatin; therefore underlying ING protein implication in transcriptional regulation, cell cycle checkpoints, apoptosis and D N A repair. 1.1.2.1 Transcriptional Regulation Much of the anti-proliferative effect of the p53 tumour suppressor is attributed to the up-regulation of the cyclin-dependent kinase (CDK) inhibitor p21 W a f I (el-Deiry et al., 1993). P33 INGlb-dependent suppression of growth is only evident in the presence of wild-type p53 (Garkatsev et al, 1998). In fact, expression of anti-sense p 3 3 i N G i b h a g b e e n s h o w n t Q alleviate p53-dependent inhibition of growth (Garkatsev et al., 1998). The activation of various p53-inducible promoters, including Wafl , M D M 2 and Bax and the repression of AFP is now known to require both p53 and p 3 3 I N G l b (Takahashi et ah, 2002; Kataoka et al., 2003). Both proteins have been reported to physically associate (Garkatsev et al., 1998; Leung et al., 2002), and the expression of either antisense ING1 or antisense p53 has been shown to result in a three to five-fold repression of the Wafl promoter (Garkatsev et al, 1998). Transcriptional expression of p21 W a f l is also known to correlate with the amount of p 3 3 r N G l b in cells over-expressing p53 (Shimada et al., 2002). While the exact mechanism of p33 I N G l b-mediated transcriptional regulation remains to be elucidated, it is evident that associated H A T and H D A C activities are likely involved. Like p53, pSS™ 0 1 1 3 is believed to mediate contacts with chromatin. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChlP) experiments have shown that p 3 3 I N G l b is capable of binding D N A either directly or indirectly (Kataoka et al, 2003; Vieyra et al, 2002). The PCR domain of pSS™0 1 1 5 is hypothesized to help associate this protein to various H A T and H D A C complexes, and is thought necessary for interactions with Sap30 (He et al., 2005; Skowyra et al., 2001), a component of the Sin3/HDAC complex involved in transcriptional repression. P 3 3 I N G l b is also known to associate with the p300 transcriptional up-regulator H A T (Vieyra et al., 2002) and the Sir2 transcriptional repressor H D A C (Kuzmichev et al., 2002). There are additional clues suggesting that p 3 3 I N G I b is a transcriptional regulator. First, p 3 3 I N G I b accumulates within the nucleolus where it may up-regulate various ribosomal components (Takahashi et al., 2002; Scott et al., 2001a). Most importantly, microarray analysis of cells expressing antisense INGlb significantly altered the expression of various genes in murine epithelial cells (Takahashi et al., 2002) (known p 3 3 r N G I b transcriptional targets are listed in Table 1-1). In a study aimed to investigate pSS11"10111 repression of AFP, it was found that p 3 3 I N G l b can directly act through specific promoter domains such as the AFP A T motif (Kataoka et al., 2003). Although p 3 3 I N G l b repression of AFP was stronger in p53-proficient HepG2 cells, it could also be observed in a p53-null isogenic cell line invoking the possibility of p53-dependent and p53-independent regulatory mechanisms. Although it remains to be tested, the authors of this study proposed that p 3 3 I N G l b may repress AFP transcription by binding to the A T motif, thus excluding HNF1 (the primary AFP positive transcriptional regulatory factor) binding, and by increasing p53 acetylation through binding and inhibition of Sir2. 7 1.1.2.2 Cell Cycle Checkpoints P 3 3 I N G l b is reported to enhance both Gi and G2 cell cycle checkpoints (Campos et al, 2004). P 2 1 W a f l (also know as Cipl) is a potent mediator of the Gi cellular checkpoint (Harper et al, 1993). The amino-terminus of p21 W a f l avidly binds and disables cyclin dependent kinases (Cdks) by forming ternary complexes with the Cdks and associated cyclins (Chen et al, 1995). This inhibition results in the hypo-phosphorylation of the retinoblastoma (Rb) tumour suppressor protein, which in turn leads to Rb binding and sequestration of E2F, a critical transcription factor for the Gi/S transition (reviewed by Harbour and Dean 2000). PSS™0"3 has been shown to enhance p53-dependent up-regulation of p21 W a f l (Garkatsev et al, 1998). Initiation of the G2 arrest requires phosphorylation of Cdkl and is p53 and ING-independent. However like p53, pSS™0"3 is believed to help prolong the G2 cell cycle checkpoint upon genotoxic stress. Several studies demonstrate that p53 can promote the maintenance of a G2 cell cycle arrest by suppressing both Cdkl and cyclin B expression (Imbriano et al, 2005; Clifford et al, 2003; Lakin and Jackson, 1999; Manni et al, 2001; Innocente et al, 1999). Cyclin B is the regulatory subunit of Cdkl and is required for mitotic onset (reviewed by Porter and Donoghue, 2003). Microarray analysis of p331 N G l b-regulated genes identified CCNB1 (cyclin BI gene) as a repressed target of p 3 3 I N G l b (Takahashi et al, 2002), suggesting a similar involvement of p 3 3 I N G l b in the cellular G2/M checkpoint. This is substantiated by a few observations: First, overexpression of p 3 3 I N G l b was shown to prolong adriamycin-induced G2 arrest in the 8 p53-null H1299 non-small cell lung carcinoma cell line (Tsang et al, 2003). Most importantly, reverse transcriptase polymerase chain reaction (RT-PCR) analysis of p53-deficient SAOS2 cells infected with INGlb also resulted in a decrease in cyclin B l mRNA levels after 72 hours (Takahashi et al, 2002). However, infection of both p53 and INGlb constructs caused a marked reduction of cyclin B l mRNA 24 h post-infection, suggesting that p 3 3 I N G l b repression of cyclin B l is enhanced in the presence of p53. Although p33 I N G 2 but not p 3 3 I N G l b has been found to promote p53 K382 acetylation (Nagashima et al, 2001) it remains to be determined whether p 3 3 I N G l b may still promote acetylation of other p53 lysine residues to enhance p53 transcriptional regulation. This may be important since p53 protein lacking C-terminal lysine residues has minimal effects on cyclin B l regulation and can promote Gi but not G2 cell cycle arrest (Nakamura et al, 2002). 1.1.2.3 Apoptosis Observations regarding developmental remodeling of regressing tails of Xenopus laevis tadpoles initially linked p 3 3 I N G l b activity to programmed cellular death, or apoptosis. It was noted that the Xenopus homolog of p 3 3 I N G l b was widely present in the receding tail of the tadpoles but was absent in the growing hind limbs (Wagner et al, 2001). Antisense expression of INGlb, was subsequently shown to promote anchorage-independent growth and neoplastic transformation of mammalian cells as well as tumour formation in nude mice (Garkatsev et al, 1996). Like in transcriptional regulation, p33 ,NGlb-dependent sensitization to apoptosis is largely influenced by p53. 9 Overexpression of pSS^0113 was shown to have little effect on the survival of human and murine fibroblasts in the absence of p53; while expression of both p33mG]b and p53 were required for efficient suppression of colony formation (Garkatsev et al, 1998). In agreement, ectopic expression of both p33 r N G l b and p53 was shown to synergistically induce apoptosis in various cultured cell lines, inhibit anchorage-dependent cell growth and reduce cell viability more efficiently than with p53 alone (Shinoura et al, 1997; Shimada et al, 2002). It is believed that pSS™0"5 is involved in the p53-mediated intrinsic apoptotic pathway since it can enhance the transcriptional up-regulation of the pro-apoptotic Bcl-2 family member Bax at the promoter level and consequently promotes changes in mitochondrial membrane potential favorable to cytochrome C release (Cheung and Li, 2002; Nagashima et al, 2001). 1.1.2.4 DNA Repair Various observations imply a role of the pSS^0115 protein in nucleotide excision repair (NER). NER is one of the five major mammalian DNA repair pathways (the other four being 1- base excision repair for removal of deaminated bases; 2- mismatch repair in DNA replication; 3- non-homologous end-joining and 4- homologous recombination for the repair of double strand breaks). NER is the pathway responsible for the removal of bulky DNA lesions caused by intra-strand cross-linking such as those caused by UV irradiation (Eveno 1995). The most prominent UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) (reviewed by Cadet et al, 2005). PSS™0113 was observed to be transcriptionally up-10 regulated upon U V exposure in the M M R U melanoma cell line in a time and dose dependent manner (Cheung et al, 2001). Most importantly, overexpression of p 3 3 r N G l b was shown to enhance the repair of a reporter plasmid irradiated with ultraviolet light ex vivo and help reduce the amount of UV-induced genomic D N A lesions in vivo while antisense expression of INGlb had the opposite effect (Cheung et al, 2001). P33 I N G l b-mediated enhancement of D N A repair is again dependent on wt p53. However unlike p53, p 3 3 1 N G l b is incapable of interacting with the core NER factors xeroderma pigmentosum complementation group A and B (XPA and XPB) (Cheung et al, 2001). The exact mechanism of p33 I N G l b-mediated enhancement of NER is currently under investigation and likely involves chromatin remodeling capabilities coupled to p33 I N G l b-associated HATs (unpublished data). p 3 3 I N G l b j s a i s o believed to enhance the repair of damaged D N A through interactions with PCNA. P C N A is a highly conserved protein which forms a homo-trimeric complex that can encircle D N A as part of larger multi-protein complexes involved in D N A replication and D N A repair (Maga and Hubscher, 2003). In NER, P C N A promotes the re-synthesis of damaged D N A strands by promoting D N A polymerase activity (Maga and Hubscher, 2003). The p21 W a f l protein is known to bind and regulate P C N A activity thereby inhibiting D N A replication (Maga and Hubscher, 2003; Waga et al, 1994). Through its PIP domain, p 3 3 I N G l b is believed to competitively bind P C N A and displace p21 W a f l to favor D N A repair over D N A replication (Scott et al, 2001b). 11 1.1.3 Status of ING1 in Human Malignancies The status of the ING1 gene is reported to vary in different human neoplasms (see Table 1-2). In most cancers observed to date, there are infrequent gene alterations but rather persistent loss of ING1 gene expression (reviewed by Campos et al, 2004). In a recent study, ING1 expression was found to inversely correlate with the progression of astrocytomas (Vieyra et al, 2003). ING1 mRNA expression levels in pilocytic astrocytomas (WHO grade I) and in diffuse astrocytomas (WHO grade II) were significantly higher than in anaplastic astrocytomas and glioblastomas (WHO grades III and IV). Although expression of ING1 was found to be independent of neuroblastoma progression, low ING1 mRNA levels were associated with poor patient prognosis (Takahashi et al, 2004). It is therefore not surprising that INGl-vmW mice are hypersensitive to D N A damaging events and are prone to lymphoma development (Kichina et al, 2005). Loss of ING1 expression was also found to correlate with the invasiveness of bladder tumours as reduced levels of p 3 3 I N G l b are found in most advanced cancers relative to early-stage transitional cell carcinoma of the bladder (Sanchez-Carbayo et al, 2003). Intriguingly, p 3 3 I N G l b expression was reported in this latter study to correlate with the survival of bladder cancer patients. Although the role of p 3 3 I N G l b in bladder cancer needs further elucidation, this observation may be at least partly explained by the fact that much of this correlation is attributed to invasive cancers and also by the use of platinum-based chemotherapy in the treatment of this type of cancer (a combination of 12 methotrexate, vinblastine, doxorubicin, and cisplatin ( M V A C ) or of cisplatin, methotrexate, and vinblastine (CMV) (reviewed by Hussain and James, 2003). Platinum-based compounds act by creating intra-strand D N A cross-links (Bernges and Holler, 1991), which like UV-induced D N A photoproducts are also repaired through NER (Reardon et al, 1999). Since NER can be enhanced by p 3 3 I N G I b overexpression (Cheung et al., 2001), the observed high levels of pSS 0" 1 0" 3 in this type of cancer may contribute to the repair of platinum-induced damage. 1.2 Protein Phosphorylation Posttranslational modifications or the proteolytic cleavage or covalent addition of a modifying group to one or more amino acids, is a versatile mechanism to regulate protein function in space and time. There are over two hundred forms of posttranslational modifications known to exist (Banks et al., 2000). Protein phosphorylation, once a mere phenomenon observed in glycogen metabolism (Rail and Sutherland, 1958), is now recognized as a ubiquitous and reversible transient mechanism capable of regulating protein activity. The added phosphoryl group is dianionic under most physiological conditions. The negative charges and capability to form extensive hydrogen bonds with the four phosphoryl oxygen atoms can have significant effects on protein conformation, interactions and functions. Up to two percent of the human genome is though to encode various protein kinases (Lander et al., 2001). It is not surprising that protein phosphorylation is implicated in nearly all aspects of cellular function. 13 Various phosphorylation events are known to regulate the cellular response to D N A damage. For example, a number of protein kinases, notably the phosphatidylinositol-3 kinase-related kinase (PIKK) family members including ataxia-telangiectasia mutated (ATM), ATM-related protein (ATR), DNA-dependent protein kinase (DNA-PK), and the human homologue of the C. elegans protein CeSMG-1 (hSMG-1) are mobilized upon genomic injury and play critical roles initiating the G2 cell cycle arrest (Bakkenist et al, 2004; Brumbaugh et al., 2004). A T M mainly responds to D N A strand breaks through altered chromatin topology which leads to auto-activation and subsequent phosphorylation of downstream modulators (Bakkenist et al., 2004). Chemical agents such as the type II topoisomerase inhibitor doxorubicin are also reported to cause A T M auto-phosphorylation and activation through the generation of reactive oxygen species (ROS) (Kurz et al, 2004). ATR is mainly activated through stalled replication forks, such as those resulting from ultraviolet-induced bulky D N A photo-lesions (Ward et al, 2004; Unsal-Kacmaz et al, 2002; Guo et al, 2000). Upon activation A T M phosphorylates p53 and the damage checkpoint effector kinases Chkl and Chk2, while A T R selectively phosphorylates p53 and Chkl but not Chk2 (Helt et al, 2005). Chkl plays a critical role in inducing a G2 cell cycle arrest, which allows genetic repair prior to mitotic onset. Chkl phosphorylates and inhibits the protein phosphatase Cdc25 (Sanchez et al, 1997), thus inhibiting the cyclin dependent kinase 1 (Cdkl) by preventing de-phosphorylation (Furnari et al, 1997). Both Chkl and Chk2 can also phosphorylate p53 on serine 20 (Shieh et al, 2000; Hirao et al, 2000). Mdm2 is an E3 recognin enzyme that can recognize the N-terminus of the p53 tumour suppressor and promote p53 degradation (Honda et al, 1997; Kubbutat et al, 1997). Phosphorylation of 14 p53 on serine 20 can prevent mdm2 binding and therefore p53 degradation (Unger et al., 1999). There are no reports on phosphorylated p 3 3 I N G l b residues prior to this thesis. 1.3 Protein Degradation Protein degradation composes an important regulatory system necessary for proper cellular function. There are various pathways that regulate protein turnover. The most extensively studied proteolytic path involves ubiquitination of the target protein and subsequent degradation within a proteic complex known as the proteasome. In this model, ubiquitin (Ub) is transferred unto proteins through a series of well-orchestrated events to mark them for destruction. Typically, Ub is activated by proteins termed E l enzymes, which hydrolyze ATP to form a complex with adenylated Ub. E l proteins then transfer activated Ub unto E2 or Ub ligases and then onto the target protein through an E3 recognin enzyme which can recognize specific proteic targets (reviewed by Welchman et al., 2005). Successive ligation of Ub onto an ubiquitinated residue (polyubiquitination) then marks proteins for degradation, unlike monoubiquitination which serves other regulatory functions (Welchman et al, 2005). The proteasome is composed of a multicatalytic cylinder-like protease termed the 20S core particle which is flanked on both ends by a 19S regulatory particle that serve as 'lids' regulating the entry into the proteasome (Groll et al., 2005). Several Ub-binding components then help 'thread' proteins into the core particle for proteolysis (Groll et al., 2005). 15 The lysosome is another important proteolytic cellular component responsible for protein degradation. This organelle contains the highest concentration of proteases in the cell (de Duve, 1983) and is involved in both targeted and non-specific degradation of proteins. There are various digestive processes mediated by the lysosome such as receptor-mediated endocytosis, pinocytosis and phagocytosis of extracellular material, but also the micro- and macro-autophagy and chaperone-assisted transport and degradation of intracellular material. Micro- and macro-autophagy results from lysosomal internalization of the cytosol and can be selective or non-selective of its targets (Regiorri et al., 2005). While selective-targeting of proteins through autophagy is poorly understood, non-selective autophagy is influenced by extracellular factors, such as nutrients and cytokines, including IFN-y and the IL-3 growth factor (Gutierrez et al., 2004; Lum et al., 2005). Chaperone-mediated lysosomal degradation differs from autophagy since it does not require vesicular trafficking of the target proteins. Instead proteins are targeted to a receptor within the lysosomal membrane, the lysosome-associated membrane protein type 2a (lamp2a) receptor (Majeski and Dice, 2004). Proteins containing a " K F E R Q " or related recognition sequences are bound by the Hsc70 chaperone and an associated protein complex, which then target the substrate to the lamp2a receptor where it is denatured and imported into the lysosome (reviewed by Majeski and Dice, 2004). 16 1.4 Thesis theme The main objectives of this thesis are to: 1) better understand the status of the ING1 gene in human melanoma; and 2) to determine i f the p 3 3 I N G l b tumour suppressor protein is subject to posttranslational regulation through protein phosphorylation and if so, when and how. The first objective was met by raising polyclonal antisera to specifically study the p 3 3 I N G I b protein and by observing the expression pattern of this protein in human melanoma. Since p 3 3 I N G l b closely associates with the p53 tumour suppressor and the TP53 gene is rarely mutated in human melanoma, the ING1 gene was also scanned for mutations in primary melanoma tumours and the ING1 mutations studied for loss of function. The latter objective was met by demonstrating that the p 3 3 I N G l b is a phospho-protein and identifying serine 126 as a phospho-residue. This work also identifies the kinases that phosphorylate serine 126 and addresses the effect of phosphorylation on the turnover rate of the p 3 3 1 N G l b through lysosomal degradation. 17 1.5 Hypotheses The hypothesis for chapter 4 is: 1. The ING1 gene is silenced or mutated in human melanoma. The hypotheses for chapter 5 are: 2. The p 3 3 [ N G l b protein is phosphorylated upon genotoxic stress. 3. The D N A damage-responsive PIKK-related kinases A T M , ATR, D N A -P K or hSMG-1 phosphorylate pSS™0 1 1 3. 4. Protein phosphorylation regulates the pSS™ 0 1 1 5 protein. 18 1.6 Figures & Tables Figure 1-1 Structural features of the p33 1 N G l b protein. A l l ING proteins are defined by the presence of a C-terminal plant homeodomain (PHD) zinc finger motif which interacts with rare nuclear phosphoinositides populations and enhances binding to chromatin (Gozani 2003) and by a yet to be defined but highly conserved potential chromatin regulatory (PCR) domain (He 2005). The p 3 3 I N G l b protein is also the only human ING protein to contain a functional PCNA-interacting (PIP) motif through which p33 I N G ! b may help mediate D N A repair (Scott 2001b) and a truncated bromodomain still not known to mediate binding to acetylated residues. Like all mammalian ING proteins (except INGx), p 3 3 1 N G l b also contains a nuclear localization sequence (NLS); however p33 1 N G l b and p33 I N G 2 are the only PNG proteins to further contain two functional nucleolar translocation sequences within the NLS sequence (Scott 2001a). The p 3 3 I N G l b protein is also predicted to contain a N-terminal sequence that resembles the RSXpSXP binding motif found on 14-3-3 proteins and a C-terminal lysine-rich sequence hypothesized to mediate protein-protein interactions (He 2005) (yet to be tested and therefore not shown). Numbers denote amino acid position. PIP 16 45 74 PCR NLS 194 211 PHD PARTIAL BROMODOMAIN NTS-2 NTS-1 Table 1-1 List of p33 I N G , b-regulated genes. Target Regulation Reference y-actin i Takahashi 2002 ^-proteasome i Takahashi 2002 AFP i Takahashi 2002 Aldehyde dehydrogenase II I Takahashi 2002 Cyclin BI i Takahashi 2002 D E K i Takahashi 2002 IGF-II receptor i Takahashi 2002 Myosin light chain i Takahashi 2002 Osomotic stress protein 1 Takahashi 2002 Osteopontin I Takahashi 2002 SDR i Takahashi 2002 Serum albumin i Takahashi 2002 TDE 1 i Takahashi 2002 TIS11 i Takahashi 2002 Bax T Cheung 2002 Ef-2 (elongation factor-2) T Takahashi 2002 Int-6 T Takahashi 2002 Wafl T Garkavtsev 1998 RPL12 T Takahashi 2002 RPS7 T Takahashi 2002 RPS11 T Takahashi 2002 RPS29 T Takahashi 2002 TPT1 T Takahashi 2002 20 Table 1-2 Summary of reported ING1 expression levels and gene alterations in human malignancies. Tumour type Expression l o r t Rate (%) Mutations References _.(%) Bladder cancer i ND N D Sanchez-Carbayo et al. 2003 Brain tumour i 100 3.4 Vieyra et al. 2003 Brain tumour i ND 0.0 Tallen et al. 2004 Breast cancer i 43.8 0.3 Toyamae/a/. 1999 Breast cancer i 70.8 N D Tokunaga et al. 2000 Breast cancer i 80.2 ND Nouman et al. 2003 Breast cancer cell lines i 100 0.0 Toyamae^a/. 1999 Esophageal SCC i 54.8 12.9 Chen etal. 2001 Esophagogastric junction adenocarcinoma i 63.2 5.3 Hara et al. 2003 Gastrointestinal cancers I 75.0 ND Okxet al. 1999 Hepatocarcinoma t 54.7 ND Ohgi et al. 2002 Lymphoblastic leukaemia i 76.5 ND Nouman et al. 2002b Lymphoid cancer cell lines i 56.3 • 0.0 Ohmori etal. 1999 lung cancer i 42.0 0.0 Kameyama et al. 2003 Basal cell carcinoma 1 25.0 1.9 Chen et al. 2003 Brain tumour cell lines f 80.0 ND Tallen et al. 2003 Malignant melanoma ] 80.0 ND Nouman et al. 2002a Malignant melanoma ] 96.3 19.6 Campos et al. 2004 Melanoma cell lines t 100 7.1 Campos et al. 2002 Oral SCC T 93.0 N D Hoque et al. 2002 Colorectal carcinoma N D ND 0.0 Sarelae/a/. 1999 Gastrointestinal carcinoma cell lines N D ND 8.3 Okie? a/. 1999 Head & neck SCC N D N D 13.0 Gunduz et al. 2000 Head & neck SCC N D ND 0.0 Sanchez-Cespedes et al. 2000 Various haematological malignancies N D N D 0.0 Bromidge and Lynas 2002 Myeloid leukemia N D ND 0.0 Ito et al. 2002 Non-small cell Oral SSC N D ND 0.0 Krishnamurthy et al. 2001 Ovarian cancer N D ND 0.0 Toyama et al. 1999 ND: Not Determined 21 1.7 References Ackermann J., Frutschi M . , Kaloulis K. , McKee T., Trumpp A. and Beermann F. 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The M M A N , M M R U , R P E P and P M W K cell lines were kind gifts from Dr R. Byers, Boston University School. The Sk-mel-2 and Sk-mel-5 cell lines were obtained from the Tissue Bank at the National Institutes of Health, U . S . A . The M E W O , Sk-mel-3, Sk-mel-24, Sk-mel-93, Sk-mel-110, K Z - 2 , KZ-13 and KZ-28 cell lines were kind gifts from Dr A . P . Albino (Memorial Sloan-Kettering Cancer Center, New York, U .S .A . ) . Normal human epithelial melanocytes and fibroblasts were purchased from Clonetics. A l l melanoma cell lines and human fibroblasts were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Canadian Life Technologies), 100 units/ml penicillin and 100 ug/ml streptomycin in a 5% C 0 2 atmosphere at 37°C. Normal melanocytes were maintained in melanocyte growth medium (Clonetics) at 37°C in a 5% C O 2 atmosphere. Cells were grown to 50-60% confluency prior to transfection. Transient transfections were done using Effectene reagent (Qiagen) as directed by the manufacturer at a ratio of 1 mg D N A to 25 m l Effectene. A T M s i R N A (Ambion) was introduced into cells using Lipofectine (Invitrogen) reagent according to the manufacturer's protocol. The pCl-INGlb plasmid was a kind gift from Dr. K . Riabowol (University of Calgary). The pINGlb-FLAG plasmid was constructed by inserting B g l l l and K p n l restriction sites up- and down-stream the INGlb c D N A by P C R (forward primer: 5'-32 G A A G A T C T A C C A T G C T G A G T C C T G C C A A C - 3 ' ; reverse primer: 5'-CGGGGTACCCCTGTTGTAAGCCCTCTC-3 ' ) and re-ligating in the p C M V - F L A G vector. BamHI and EcoRI restriction sites were also introduced upstream and downstream of pCl-INGlb by PCR (forward primer: 5' - C T C G A G G A T C C C T G C A G C -3'; reverse primer: 5 ' -CGAATTCCTACCTGTTGTAAGCCC-3 ' ) to sub-clone into the pGEX-2T glutathione-S-transferase (GST) vector (Amersham Bioscience). 2.2 Generation of Polyclonal Antibodies Antibodies were generated as previously described (Campos et al., 2004). Briefly, the synthetic peptides N - L S P A N E Q L H L V N C - C and N - E L G D T A G N p S G K A G A D R P - C (where p denotes phosphorylation) corresponding to the N-terminus of the p33 1 N G l b protein and a p 3 3 I N G l b peptide containing phosphorylated serine 126 were conjugated to keyhole limpet hemocyanin (KLH). Twenty milligrams of K L H were initially denatured by boiling in the presence of 5% (v/v) sodium dodecyl sulfate (SDS) and 10 m M sodium carbonate. The solution was then activated with N-hydroxysuccinimide-iodoacetate ester (NHS-Iac) at room temperature for 10 min. Activated K L H was then purified by passing though a G15 sephadex bead column. Five milligram of the INGlb peptide were dissolved in 400 ml of 10 m M sodium phosphate [pH 8.5] and conjugated to the iodoacetate-activated K L H by incubating overnight at room temperature with gentle rotation. The concentration of the K L H conjugated INGlb protein solution was adjusted to 1 mg/ml in PBS. Two New Zealand rabbits were immunized with 1 mg of the 33 conjugated ING lb peptide in Freund's complete adjuvant, and every 2 weeks subsequently in Freund's incomplete adjuvant. Ten milliliters of sepharose C L beads were washed with 100 ml of 10 m M sodium carbonate and then incubated for 20 min with 100 mg of sodium m-periodate at room temperature with gentle rotation. The beads were then washed with 0.1 M citrate buffer and conjugated with 10 mg of the synthetic p 3 3 I N G l b peptides (N-terminal for anti-p 3 3 I N G l b antisera and phosphorylated peptide for anti-p-S126 antisera) by rotating at room temperature for 30 min. Ten milligrams of sodium cyanoborohydride was then added to the beads left to rotate at room temperature overnight. The peptide-conjugated beads were extensively washed with PBS prior to use. Approximately 80 ml of the antiserum was passed through the affinity column. The column was then washed with 100 mL of PBS followed by 50 ml of 1 M NaCl. The specific anti-p33 I N G l b antibodies retained in the column were eluted with 3% acetic acid. Antibodies were then precipitated with 30% of ammonium sulphate and reconstituted with PBS. Anti-p-S126 serum was further affinity purified after incubating with glutathione agarose beads-bound recombinant GST-p33 I N G l b at 4°C overnight with gentle rocking and the non-bound fraction kept for use. 2.3 Drug Treatments and Ultraviolet Irradiation The topoisomerase inhibitors doxorubicin and etoposide were dissolved directly into culture media at 0.1-1 (J-g/ml and 0.25 ug/ml respectively. The okadaic acid phosphatase 34 inhibitor was used at a working concentration of 100 uM in culture media. The kinase inhibitors wortmannin, staurosporine, roscovitine, kenpaullone and H-89 were used at 100 mM, 50 uM, 50 uM, 10 uM and 25 uM working concentrations respectively. The 80S cytosolic ribosome inhibitor cycloheximide was diluted to 20 ug/ml directly into the culture media. The proteasome inhibitor MG132 was also used at 50 uM in culture media. A l l drugs were obtained from Sigma. Ultraviolet irradiation was performed by removing media and exposing cells to 20 mJ/cm2 U V B (290-320 nm) light using a bank of four unfiltered FS40 sunlamps (Westinghouse). 2.4 Single-strand Conformation Polymorphism SSCP was initially developed to identify genetic polymorphisms (Orita et al, 1989) and was adapted as previously described (Chen et al, 2003). D N A was isolated from hematoxylin- and eosin-stained 12 um sections of paraffin-embedded tumour blocks. Normal and tumour tissues were dissected under a microscope, and the paraffin removed through a series of xylene washes prior to D N A extraction. D N A from both fixed tissues and cultured cells was extracted using the DNeasy Tissue Kit (Qiagen) according to the manufacturer's protocol. A l l three exons of the ING1 gene were then amplified by PCR using the following primers: forward 5 ' -AGCAGCTCCACCTGGTGAAC-3 ' and reverse 5 ' -ACTGAAGCGCTCGT A G C ACT-3' (exon la); forward 5'-TGGCTGTGATGTCCTTCGTG-3 ' and reverse 5 ' - A G G C C A G G A G G A G A A C C A A C - 3 ' (exon lb set 1); forward 5'-GTGTGGTTGGTTCTCCTC-3' and reverse 5'-GGATC ACTGCT A C T G C T A-3' (exon lb set 2); forward 5'-35 C G A G A A G A T C C A G A T C G T G A - 3 ' and reverse 5 ' -GGCTTGTCAGACTGCGCTAC-3 ' (exon 2 set 1); and forward 5 ' - G A C C T C C A A G A A G A A G A A G C - 3 ' and reverse 5'-CCATGGTCTTCTCGTTCTCC-3 ' (exon 2 set 2). D N A samples were denatured at 94°C for '3 min and amplified by 40 cycles of 30 sec denaturing at 94°C, 1 min primer annealing at 60°C (55°C for set 2 of both exon lb and exon 2) and 1 min extension at 72°C with a final 5 min extension following the last cycle. PCR samples were then diluted 1:4 in SSCP loading buffer (95% formamide, 0.05% bromo-phenol blue, 20 mM NaOH), denatured at 80°C for 20 min and quickly chilled on ice prior loading onto a 6% non-denaturing polyacrylamide gel. "Electrophoresis was carried out at 80 V at 4°C in a water-cooled apparatus overnight and the D N A visualized by staining with ethidium bromide. 2.5 D N A Sequencing Genomic D N A from samples exhibiting band-shifts in the SSCP analysis were re-amplified for direct sequencing. Products were sequenced using the Big Dye Terminator Kit on a ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Sequences were aligned and compared to the ING1 gene under the GeneBank accession nos. AB024401, AB024402 and AF078835 (differing by known SNPs). 36 2.6 Western Analysis Whole cell extracts were obtained by scraping cells in cold PBS and centrifuging cells at 500 x g for 2 min at 4°C. Cell pellets were solubilized by adding 3 volumes of lysis buffer (50 m M Tris-Cl [pH 8.0], 150 m M NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40) in the presence of a premixed protease inhibitors (Roche) and a Ser/Thr phosphatase inhibitor cocktail mix (Sigma) where applicable. Samples were left on ice for 15 min and sonicated twice for 10 s using a Microson sonicator (Heat Systems -Ultrasonics) at setting 8. Lysates were then centrifuged at 12,000 x g for 30 min at 4°C and supernatants kept for analysis. Nuclear extracts were obtained by washing cells with cold PBS and scraping cells off plates. Cells were then centrifuged at 500 x g at 4°C for 2 min. The volume of the packed pellet was estimated and four volumes of NP-40 lysis buffer (50 m M Tris-HCl, 10 mM NaCl, 5 m M M g C l 2 , 0.5% Nonidet P-40) containing the abovementioned inhibitors. Samples were left on ice for 5 min, and centrifuged at 4°C, 500 x g for 2 min. The supernatant was discarded, and the pellet was washed once in four volumes of NP-40 lysis buffer. The supernatant was discarded, and the packed nuclei solubilized with approximately three volumes of nuclear extraction buffer (20 m M Hepes [pH 7.9], 0.5 M NaCl, 1 m M EDTA, 20% glycerol, 1 m M dithiothreitol) containing protease inhibitors, left on ice for 30 min and sonicated as previously described. Extract and then centrifuged at 12,000 x g for 30 min at 4°C and supernatant kept for analysis. 37 Concentration of proteins was determined using the DC Protein Assay (Bio-Rad) system. Fifty ug/lane of proteins were separated by 15% SDS-PAGE and electrotransferred onto polyvinylidene difluoride filters (Bio-Rad). Low-bis band shift assays were performed using gels containing a 118.5:1 acrylamide:6w crosslinker ratio instead of the usual 37.5:1. Filters were incubated with primary antiserum at room temperature for 1 h or at 4°C overnight followed by three washes in 0.04% Tween-20 PBS for 5 min each and subsequent incubation with horseradish peroxidase-conjugated secondary antiserum for 1 h at room temperature. Blots were washed and signals detected with SuperSignal enhanced chemiluminescence (Pierce). The following antibodies were used in this study: anti-ATM, anti-Chkl, anti-Cdkl antibodies (Santa Cruz Biotechnology), anti-FLAG (Sigma), anti-INGl (PharMingen), anti-P-actin (Sigma). Densitometry analysis was carried using the Quantity One software (Bio-Rad). Intensity of the signal of interest was corrected for the different amounts of cellular protein loaded on the gel by using P-actin as the input control. 2.7 Semi-quantitative Reverse Transcriptase Polymerase Chain Reaction Total RNA was extracted with TriZol reagent (Canadian Life Technologies) and the concentrations were determined by U V spectrophotometry. Five micrograms of total R N A were reverse transcribed into cDNA using Superscript II RNase H reverse transcriptase (Canadian Life Technologies) in a 20 ul reaction using oligo d(T) primers as 38 prescribed by the manufacturer. ING1 mRNA was amplified from 2 p.1 of the RT-PCR reaction using the following primers: forward 5 ' - G A T C C T G A A G G A G C T A G A C G - 3 \ reverse 5' - A G A A G T G G A A C C A C T C G A T G - 3 ' using Taq D N A polymerase (Qiagen) as prescribed. Amplification was carried at an annealing temperature of 50°C as previously described. 2.8 Immunohistochemistry Six-micron slides were cut from paraffin-embedded blocks of melanoma biopsies. Tissues were de-waxed by heating at 55°C for 30 min followed by three 5 min washes in xylene. Samples were re-hydrated by washing for 5 min each in 100%, 90% and 70% ethanol and 30 min in PBS. Antigen retrieval was performed by microwaving the samples for 5 min at full power in a 10 m M sodium citrate solution (pH 6.0). Endogenous peroxidase activity was quenched with a 0.3% H2O2 solution for 10 min and non-specific antibody binding blocked by incubating with 10% pre-immune goat serum in PBS for 20 min. Proteins were immunolabeled with primary antisera and subsequently HRP-conjugated secondary antisera diluted 1:500 in blocking serum. Labeling was finalized by developing with 3,3'-diaminobenzidine (DAB) substrate (Vector Laboratories). 39 2.9 Immune-fluorescence M M R U cells were grown on coverslips at a density of 2 x 105 cells/well in a 6-well plate. Twenty four hours following transfection cells were simultaneously fixed and extracted in 2% paraformaldehyde, 0.5% Triton X-100 in PBS for 30 min at 4°C. Cells were incubated in 10% pre-immune goat serum diluted in PBS for 1 h at room temperature to minimize non-specific signal. Indirect immunolabeling was performed by incubating fixed cells on primary antibody diluted 1:50 in PBS at room temperature for 1 h, washing 3 times with PBS and incubating for 1 h at room temperature with Cy3 or Cy5-conjugated secondary antiserum (Jackson ImmunoResearch) diluted 1:500 in PBS. Cells were counterstained with 650 ng/ml Hoechst 33258 diluted in PBS to visualize DNA. Slides were visualized under a Zeiss Axioplan 2 microscope. 2.10 Site-directed mutagenesis Site-directed mutagenesis was performed by splicing overlap extension (PCR-SOEing) (Ho et al., 1989) using the high-fidelity Pfx D N A polymerase (Invitrogen) using 2* Pfx buffer in the final recommended reaction. Mutagenesis of the ING lb coding sequence was performed on the pINGlb-VL AG or the pCmeo-/A7G7£>-FLAG vectors using the following primer pairs: 5 ' -GAGAACCTCACGCGGCA-3 ' (forward) and 5'-TGCCGCGTGAGGTTCTCC-3 ' (reverse) for R102L; 5'-A G A G C G A G A A G A C C A T G G A - 3 ' (forward) and 3 ' -CCATGGTCTTCTCGCTCTC-3' (reverse) for N260S; and 5 '-GGC A A C G C A G G C A A G G C - 3 ' (forward) and 5'-40 CCTTGCCTGCGTTGCCC-3 ' (reverse) for S126A. A l l constructs were verified by D N A sequencing. 2.11 D N A Repair Assays Use of the host-cell reactivation assay to assay for cloned D N A repair genes was first described by Henderson et al. (1989). We followed this protocol using the chloramphenicol acetyltransferase (CAT) gene as a reporter in the plasmid pCMVcat (kind gift from Dr. Lawrence Grossman, Johns Hopkins University). The reporter plasmid was irradiated ex vivo at 40 mJ/cm2 using a ultraviolet crosslinker. The damaged reporter was then co-transfected (as abovementioned) with empty pCIneo or pCIneo plasmids expressing wild-type or mutant p33 I N G l b . Cells were collected 40 h after transfection, resuspended in 30 pi of a 250 m M Tris-Cl [pH 7.8] and 5 m M EDTA solution and cell-free extracts obtained by repeatedly freezing the samples in liquid nitrogen and centrifuging the lysates at 12,000 x g for 15 min. Supernatants were then individually mixed with 7.5 ul of 5 m M chloramphenicol, 1 pi of 2.5 mM [ 3H ]-acetyl-CoA and 16.5 pi distilled water. The chloramphenicol acetyltransferase reaction was allowed to proceed for 90 min at 37°C. The organic phase was then isolated, dried and the amount of radioactivity that was transferred onto the chloramphenicol determined using a scintillation counter. A l l reactions were performed in triplicates. Measurement of damaged D N A using the radioimmunoassay (RIA) technique was first described by Hsu et al. (1981). RIA was performed using rabbit antibodies 41 capable of recognizing UV-induced pyrimidine (6-4) pyrimidinone D N A photoproducts (6-4PPs). D N A used for RIA was obtained from M M R U cells expressing wild-type or mutant p 3 3 I N G l b and irradiated with 20 mJ/cm2 U V B or ambient light for an equivalent amount of time. D N A was isolated 4 h after U V irradiation by standard phenol:chloroform extractions. Two milligrams of heat-denatured D N A samples were then incubated with 5 pg of poly(deoxyadenylated TMP; labeled over 5 x 108 cpm/pg by nick translation with [32P] dideoxythymidine 5'-triphosphate) in a total volume of 1 ml of 10 m M Tris [pH 7.8], 150 m M NaCl, 1 m M EDTA, and 0.15% gelatin. 6-4PP antibodies were then added at a dilution that yielded 30-60% binding to labeled ligand, and then immunoprecipitated using with anti-rabbit secondary antisera. After centrifugation the pellet was dissolved in tissue solubilizer (Amersham) and radioactivity quantified by scintillation. The experiment was then repeated in the presence of increasing amounts of an unlabeled competitor that inhibits antibody binding to the radiolabeled ligand. Sample inhibition was then extrapolated through a dose-response curve to determine the number of photoproducts in 106 D N A bases (6-4PPs/Mb DNA). Salmon sperm D N A (Sigma) irradiated with increasing doses of U V was used as an internal standard control. 2.12 Immunoprecipitation and Phosphatase Treatment Immunoprecipitations were performed from nuclear extracts with 2 ug/ml of either anti-Cdkl or anti-Chkl antibodies (Santa-Cruz Biotech.) or anti-FLAG M2 antibodies (Sigma) with end-to-end rotation at 4°C overnight. Samples were further incubated with 20 jul (50%o slurry) protein A or protein G-agarose beads (Pharmacia) with rotation for 1 h 42 at 4°C and beads washed 3 times with 500 ul PBS. Alkaline calf intestinal phosphatase (CIP) (New England Biolabs) treatment of immunoprecipitated p 3 3 I N G l b was done on-beads in NEBuffer-3 and 10 U CIP per immunoprecipitation. Reaction was carried at 37°C for 2 h and stopped upon addition of sample buffer. 2.13 Enzyme-linked Immunosorbent Assay A 96-well ELISA plate was incubated with 100 ul of phosphorylated or non-phosphorylated synthetic peptide corresponding to amino acids 118-134 of p 3 3 ! N G l b diluted at 50 p.g/ml in 50 m M Na2CC>3 at room temperature overnight, and then blocked with 5% milk PBS-T at room temperature for 20 min. Plates were then incubated with 100 ul of the purified anti-p-S126 antibody at different dilutions at room temperature for 60 min. The plates were then washed with PBS-T, and incubated with a HRP-conjugated goat anti-rabbit antibody (Santa-Cruz Biotech). 100 ul of the HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) (Moss Inc.) was added to develop for 15 min and the reaction stopped with 50 ul of 0.1 N HC1. O.D. was measure at 450 nm on a Titertek plate reader. 2.14 Purification of Recombinant p 3 3 ! N G l b Recombinant p 3 3 [ N G l b was expressed from the pGEX-INGlb vector in E. Coli BL-21 DE3. Bacteria was grown to an optical density of 0.6 and induced with isopropyl-P-D-thiogalactopyranoside (Sigma) for 4 h at room temperature. Bacteria was then pelleted 43 by centrifugation and incubated in resuspension buffer (25 m M Tris [pH 7.5], 150 mM NaCl, 5 m M P-mercaptoethanol and protease inhibitors) and lysed for 30 min on ice in the presence of 50 ng/ml lysozyme prior to freeze-thaw cycles. Lysate was further incubated on ice for 30 min in the presence of 50 ng/ml DNase I (Sigma) and 5 mM M g C l 2 and for another 30 min with 1% Nonidet P-40 at 4°C with gentle rotation. Lysates were then centrifuged at 12,000 x g for 30 min at 4°C. Supernatant was recovered and GST-p33 I N G l b affinity purified using glutathione agarose beads (Sigma). Beads were extensively washed with PBS and GST cleaved from the fusion protein by incubating the INGlb-bound beads with 50 U thrombin (Sigma) at room temperature for 12 h. 2.15 Mass Spectrometry F33™G]h protein samples were resolved by SDS-PAGE and excised from the gel. Proteins were then digested with AspN using an Abimed Digest Pro robot, and the resultant peptides dried. A newly described methodology (Xu et al, 2005) was used to reduce the ionization of the positively charged ions, therefore enabling easier detection of negatively (i.e. phosphorylated) ions. After the digested protein was dried, it was resuspended in methanolic hydrochloric acid and incubated for 3 h at room temperature. The solution was dried and the peptides were next resuspended in 0.1% trifluoroacetic acid, 30%) acetonitrile. The matrix used for this preparation was 50 m M 2,6-dihydroxyacetophenone in 90%) methanol and 100 m M diammonium citrate in Mil l i -Q water. These two solutions were mixed in equal volumes and then this mixture was mixed at a 1:1 ratio with the sample. The mass spectra were acquired on an Applied 44 Biosystems Voyager DE-Pro in both positive ion, reflector detector mode and negative ion, reflector detector mode. The spectra were calibrated using the autolytic trypsin peaks at m/z 842.5099 and 2211.0968. The results of the mass spectroscopic analysis were compared to an in silico digest using the PAWS program. This method adds +14 to D, E and C resides and can add +15 to N and Q residues. The phosphorylated peptides were also subjected to on-target phosphatase treatment. Approximately 1 pi of 0.05 U/ul CIP was spotted onto each sample on the M A L D I target. The target was placed in a humidified chamber at 37°C for 2 h. The CIP was blotted and allowed to dry. The samples were then run on the mass spectrometer and a loss of -80 (HPO3) or -98 (H3PO4) was expected of the phosphorylated samples. 2.16 In vitro Kinase Assay Initial kinase predictions were performed with NetPhosK 1.0 (Blom et al., 2004). The peptide ELGDT AGNS G K A G ADRP corresponding to p 3 3 I N G l b amino acids 118-134 was sent for KinaseProfiler™ (Upstate) kinase selectivity screening service and assayed as substrate for rabbit CaMKII, human Cdkl-Cyclin B, human Chk l , human CK2 and human GSK3a. Chkl , Cdkl and A T M were immunoprecipitated as aforementioned. After washing with PBS, beads were washed twice more with 50 ul kinase buffer (20 m M MOPS [pH 7.2], 25 m M B-glycerol phosphate, 1 mM dithiothreitol, 1 m M CaCl 2) the beads were resuspended in 20 u,l kinase buffer. Kinase assays were performed by mixing 45 0.5 mM of substrate peptide (full length p 3 3 I N G , b or the L G D T A G N S G K A G A D R P K peptide corresponding to amino acids 119-135 of p 3 3 I N G l b diluted in kinase buffer to above mentioned immunoprecipitates and 5 ul of 1:10 diluted [gamma-32P]ATP mixture (Perkin-Elmer) in the presence of phosphatase inhibitors. The peptide used in the kinase assays differs slightly from the one used in the KinaseProfiler™ screen because of a change in pi (4.56 to 8.59) and therefore better binding to P81 paper. Reactions were incubated at 30°C for 30, 90 or 120 min and stopped by transferring a 25 ul aliquot onto the center of phosphocellulose P81 paper. P81 squares were washed with 0.75% phosphoric acid three times and once with acetone prior to scintillation counting. Radioactive labeling of full-length p 3 3 I N G l b was performed in the same manner but using 5 jig of M M R U nuclear cell extracts as the kinase source and incubating the reactions for 30 min. Reactions were stopped upon the addition of loading buffer and resolved by SDS-PAGE prior to autoradiography. P K C kinase assays were performed using a PKC assay kit (Upstate) comparing INGlb peptide to control peptide as indicated by the manufacturer. 46 2.17 References Campos E.I., Xiao H. and L i G. Generation of a polyclonal antibody specifically against the p33(INGlb) tumour suppressor. 2004. J Immunoassay Immunochem 25:71-80. Chen B., Campos E. I., Crawford R., Martinka M . and L i G. Analyses of the tumour suppressor ING1 expression and gene mutation in human basal cell carcinoma. 2003. Int J Oncol 22:927-31. Blom N.T., Sicheritz-Ponten T., Gupta R., Gammeltoft S., and Brunak S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. 2004 Proteomics 4:1633-49. Henderson E.E., Valerie K. , Green A.P., and de Riel J.K. Host cell reactivation of CAT-expression vectors as a method to assay for cloned DNA-repair genes. 1989 Mutat Res 220:151-60. Ho S.N., Hunt H.D., Horton R .M. , Pullen J.K., and Pease L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. 1989 Gene 77:51-9. Hsu I.C., Poirier M.C. , Yuspa S.H., Grunberger D., Weinstein I.B., Yolken R.H., and Harris C C . Measurement of benzo(a)pyrene-DNA adducts by enzyme immunoassays and radioimmunoassay. 1981 Cancer Res 41:1091-5. Orita M . , Iwahana H. , Kanazawa H. , Hayashi K. , and Sekiya T. Detection of polymorphisms of human D N A by gel electrophoresis as single-strand conformation polymorphisms. 1989 Proc Natl Acad Sci USA 86:2766-70. Xu C.F., Lu Y . , Ma J., Mohammadi M . , and Neubert T A . Identification of phosphopeptides by M A L D I Q-TOF MS in positive and negative ion modes after methyl esterification. 2005 Mol Cell Proteomics 4:809-18. 47 3 GENERATION OF RABBIT P O L Y C L O N A L ANTISERA DIRECTED AGAINST THE p s s ™ 0 1 8 PROTEIN 3.1 Rationale In order to fully study the cellular function of a protein, it is crucial to have the right tools that enable its detection and purification. At the onset of this thesis there were two commercially available antibodies capable of recognizing p33 I N G l b . The first, a rabbit monoclonal was raised against full length recombinant p 3 3 I N G l b and was not affinity purified (Garkatsev et al., 1996). Since the ING1 gene expresses at least three protein variants with identical carboxyl-termini and the carboxyl-terminus further shares a high degree of homology with other ENG proteins, it is not surprising that this antiserum lacked specificity. This antibody has since been discontinued by the manufacturer. The second antibody is a mouse monoclonal (Garkatsev et al., 1997), which is specific for P 3 3 I N G l b but that lacks avidity in various basic molecular assays. For this reason we envisaged to raise a rabbit polyclonal antisera that could specifically recognize the p 3 3 I N G l b protein. A version of this Chapter has been published in: Campos E.I., Xiao H. and L i G. Generation of a polyclonal antibody specifically against the p33(LNGlb) tumour suppressor. 2004. J Immunoassay Immunochem 25:71-80. 48 3.2 Results & Discussion 3.2.1 Immunization and Affinity Purification The carboxyl-terminus of all three ING1 proteins is encoded by exon 2 of ING1. The peptides largely differ at their amino-terminus, encoded by exon lb for p47 I N G l a , exon l a for p 3 3 I N G l b and exon 2 for p24 I N G l c (Figure 3-1). A 12-amino acid peptide corresponding to the amino-terminus of p33 1 N G l b was therefore used to raise rabbit antisera (Figure 3-1). A glycine residue found within this part of the protein was not included in the peptide to avoid the formation of a cyclic structure in the synthetic peptide upon synthesis. A cysteine residue was also added at the carboxyl terminus to allow conjugation to the highly immunogenic keyhole limpet hemocyanin (KLH) carrier protein to serve as an immune stimulant and carrier for the synthetic p 3 3 I N G l b hapten. SELDI mass spectrometry confirmed the purity of the synthesized peptide. The observed molecular weight of the peptide corresponds to the predicted molecular weight of 1437.56 (Figure 3-2) and minimal impurity was detected. New Zealand rabbits were immunized with the conjugated N-terminal p 3 3 I N G l b peptide and after 6 weeks of immunization the serum was tested for an immune response by ELISA. The serum was capable of detecting a bacterially produced recombinant GST-p33 I N G l b , but not the GST tag alone (Figure 3-3). Since the serum demonstrated strong immunogenic properties against pSS™0"3 peptides in the ELISA, further serum was collected and affinity purified. 3.2.2 Specificity of the Affinity-purified anti-p33 I N G l b Antiserum 49 The affinity-purified polyclonal antibody was found to be highly specific for the p 3 3 I N G l b protein in biochemical and immunocytochemical assays. The specificity of this antibody was first assessed by western analysis. Protein extracts were obtained from untransfected M M R U cells or M M R U cells transfected with vectors encoding a FLAG-tagged or an untagged p 3 3 I N G l b protein. The extracts were resolved by SDS-PAGE, transferred to PVDF membranes and probed with the affinity purified antibody (Figure 3-4). The antibody demonstrated high specificity and avidity. As expected, the F L A G fusion protein was detected at a slightly higher molecular weight compared to untagged p33 I N G l b . Importantly, endogenous p 3 3 I N G l b was also detected with this antibody in untransfected cells. The F L A G epitope was only detected in extract from cells expressing FLAG-tagged p 3 3 I N G l b when the membrane was stripped and re-probed using an anti-FLAG antibody. Endogenous and exogenous pSS1™0115 protein could also be detected through indirect immunofluorescence using this N-terminal specific polyclonal antibody (Figure 3-5). Consistant with previous reports, the p 3 3 I N G l b protein was largely nuclear and the FLAG-tagged exogenous protein co-localized with the F L A G epitope. Antibodies are essential tools that help dissect the biochemical functions and properties of proteins. Designing an antibody that specifically recognized the p 3 3 I N G l b protein was especially important to this study, since all proteins encoded by the ING1 gene share an identical carboxy-terminus that is also highly conserved in other ING family members. Furthermore, isoforms encoded by the ING1 gene do have certain 50 antagonistic effects, notably in H A T / H D A C associations and in the transcriptional regulation of certain genes (Campos et al, 2004). For these reasons the generation of this antibody has evident advantages over the use of an antibody that recognizes all ING1 protein variants; especially in immunocytochemical studies where multiple proteins that are recognized by the same antibody cannot be differentiated. 51 3.3 Figures Figure 3-1 Peptide used for the generation of a p33 I N G l b-specific polyclonal antibody. Sequences of the p47 I N G l a , p33 I N G l b , and p24 1 N G l c proteins (GeneBank accession numbers BAA82887, BAA82886 and BAA83496, respectively) are compared. The shaded carboxyl-terminal protein sequence encoded by exon 2 of the ING1 gene is common to all three protein isoforms. The boxed sequence in the amino-terminus of the p33 protein highlights the sequence of the synthetic peptide that was used to immunize rabbits to raise a p33 I N G l b-specific polyclonal antibody. The glycine residue was omitted to avoid bending of the peptide during synthesis. A cysteine residue was also added to the carboxyl-terminus of the synthetic peptide to facilitate its conjugation to keyhole limpet hemocyanin. P 4 7 I N G 1 a : M S F V E C P Y H S P A E R L V A E A D E G G P S A I T G M G L C F R C X L F S F S G R S G V E G G R V D L N V F G S L G L Q P W I G S S R C W G G P C S S A L RCGWFSSWPPreKSAIPIGGGSRGAGRVSRWPPPHWLEAWRVSPRPLSPLSPXXFGRGFIAVAVIPGLWARGRGCSSDRL PRPAGPARRQFQAASLLTRGWGRAWPWKQILKELDeCYERFSRETDGAQKRRMLHCVQRALIRSQELGDEKIQIVSQWIVEL V E N R T R Q V D S H V E L F E A Q Q E L G D T V G N S G K V G A D R P N G D A V A Q S D K P N S K R S R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y C L C N Q V S Y G E M I G C D N D E C P I E W F H F S C V G L N H K P K G K W Y C P K C R G E N E K T M D K A L E K S K K E R A Y N R p 3 3 I N G 1 b : ^^§^JjG (^^I^3YVEDYLDSIESLPFDLQRNVSLMRElDAKYOEILKELDECYERFSRETOGAQKRRMLI- tCVQRALIRSQE L G D E K I O I V S Q M V E L V E N R T R Q V D S H V E L F E A Q Q E L G D T V G N S G K V G A D R P N G D A V A Q S D K P N S K R S R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y C L C N Q V S Y G E M I G C D N D E C P I E W F H F S C V G L N H K R K G K W Y C P K C R G E N E K T M D K A I : E K S K K E R A Y N R p 2 4 I N G 1 c -M L H C V Q R A U R S Q E L G D E K I Q I V S Q M V E L V E N R T R Q V O S H V E L F E A Q Q E L G D T I V G N S G K V G A D R P N ' G D A V A Q S D K P N S K R S R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y C L C N Q V S Y G E M I G C D N D E C P I E W F H F S C V G L N H K P K G K W Y C P K C R G E N E K T M D K A L E K S K K E R A Y N R " 52 Figure 3-2 Purity of the peptide used to raise a p33 r N G l b-specific polyclonal antiserum. Purity was assessed by SELDI mass-spectrometry. The predicted molecular weight of the synthetic ' L S P A N E Q L H L V N C ' peptide is of 1437.56. The observed weight of the synthetic peptide is of 1437.3 and contains little impurities. 15 >> <D I 5 0 I 1437.3+H | | 1296.5+H J . 1200 1400 1600 Mass (m/z) 53 Figure 3-3 Enzyme-linked immunosorbent assay of serum from rabbits inoculated with a synthetic peptide corresponding to a sequence of the p 3 3 I N G l b protein. The ELISA plate was pre-coated with serial dilutions of bacteria-expressed recombinant GST tag (left column) or GST-p33 I N G l b (right column), and incubated with 0.2 ug/ml of total serum and a HRP-tagged goat anti-rabbit antibody subsequently. The numbers on the right of the plate correspond to the dilution of the GST and GST-p33 I N G l b proteins on each row. The last row ' C ' contained buffer only. Bound HRP was detected by adding a o-phenylenediamine dihydrochloride substrate. Immunized rabbits did develop p33 I N G l b -specific antibodies and was therefore further purified by affinity chromatography. 54 Figure 3-4 Western analysis of p33 I N G l b-expressing M M R U cells using the rabbit polyclonal p33 1 N G l b-specific antibody. Lanes were loaded with 50 pg of protein extracts from untransfected cells (control) or cells transfected with pINGl b-FLAG or pCl-INGlb plasmids. Membranes were probed with the rabbit anti-p33 I N G l b antibody diluted to 0.4 ng/ml and then striped and re-probed with an anti-FLAG antibody. FLAG-tagged p33 1 N G , b appears slightly higher that the untagged counterpart. The p 3 3 I N G l b polyclonal antibody can also detect endogenous protein. kDa ^0 7 5 -54 -3 5 -2 4 -• 16-10-anti-p33 I N G 1 b anti-FLAG 55 Figure 3-5 Epifluorescent images of M M R U cells transiently transfected with the pINGlb-FLAG plasmid and immunolabeled using the anti-p33 I N G l b rabbit polyclonal and anti-FLAG antibodies. The p 3 3 I N G l b protein was found to be mainly nuclear using both antibodies. Both endogenous and exogenous p 3 3 I N G l b was detected with the p33 I N G l b -specific rabbit polyclonal antibody while exogenous protein was detected using both pSS™0"5- and FLAG-specific antibodies. Cells were counterstained with Hoechst 33258 and further photographed under Nomarski's differential interference contrast (DIC). Scale bar = 50 p.m. f % anti-FLAG / m Hoechst merged / | | | 56 3.4 References Campos E.I., Chin M . Y . , Kuo W.H. and L i G. Biological functions of the ING family tumour suppressors. 2004. Cell M o l Life Sci 61:2597-613. Campos E.I., Xiao H. and L i G. Generation of a polyclonal antibody specifically against the p33(INGlb) tumour suppressor. 2004. J Immunoassay Immunochem 25:71-80. Garkavtsev I., Boland D., Mai J., Wilson H. , Veillette C. and Riabowol K. Specific monoclonal antibody raised against the p33INGl tumour suppressor. 1997. Hybridoma 16:537-40. Garkatsev I., Kazarov A . , Gudkov A . and Riabowol K . Suppression of the novel growth inhibitor p33ING 1 promotes neoplastic transformation. 1996. Nat Genet 14:415-20. 57 4 STATUS OF THE INGl GENE IN H U M A N M E L A N O M A 4.1 Rationale Cutaneous malignant melanoma is a skin cancer that results from the transformation and uncontrolled proliferation of melanocytes. Of all types of cancer, melanoma has seen the highest increase in incidence in recent years. According to the American National Cancer Institute (NCI), the lifetime risk of developing melanoma jumped from 1 in 1,500 in 1935 to roughly 1 in 74 in 1999 within its Caucasian population (Brochez and Naeyaert, 2000; Ries et al., 2002). Melanoma is also a highly heterogeneous disease (Freitas et al., 2004) and displays a high degree of chemoresistance (Serrone and Hersey, 1999). Exposure to sunlight is widely thought to be an important risk factor in the genesis of cutaneous melanoma (Gilchrest et al, 1999). This is further supported by a study that suggests a significantly higher melanoma development in sun-exposed body parts compared to non-exposed parts (Brochez and Naeyaert, 2000). Indeed biological evidence strongly suggests a role of U V light in D N A damage and pathogenesis of melanoma (Brochez and Naeyaert, 2000; Gilchrest et al., 1999). Recent evidence suggests that p 3 3 1 N G l b plays an important role in the removal of UV-induced D N A damage and is transcriptionally up-regulated after U V irradiation (Cheung et al., 2001). A version of this chapter has been published in: Campos E.I., Cheung K.J . Jr., Murray A. , L i S. and L i G. The novel tumour suppressor gene INGl is overexpressed in human melanoma cell lines. 2002. Br J Dermatol 146:574-80. and in: Campos E.I., Martinka M . , Mitchell D.L., Dai D.L. and L i G. Mutations of the I N G l tumour suppressor gene detected in human melanoma abrogate nucleotide excision repair. 2004. Int J Oncol 25:73-80. 58 Furthermore, p 3 3 1 N G l b closely cooperates with the p53 tumour suppressor (reviewed by Campos et al. 2004) also known to enhance the repair of UV-induced D N A lesions. Since p53 is rarely mutated in human melanoma (Gwosdz et al., 2006) and INGl, a gene that cooperates with p53 in apoptosis and D N A repair is also reported to be lost in various cancers (see Table 1-2), we therefore hypothesized that p 3 3 I N G l b might be down-regulated or mutated in human melanoma. 4.2 Results 4.2.1 INGl Gene Expression in Human Melanoma To determine i f INGl expression is altered in human melanoma the p 3 3 1 N G l b protein levels were first assessed in fourteen human melanoma cell lines. Results from western analysis demonstrated that p 3 3 I N G l b was clearly overexpressed in all 14 melanoma cell lines compared to normal cultured human epithelial melanocytes (Figure 4-1). Densitometric readings corrected for total protein loading confirmed that the levels of p 3 3 I N G l b protein in the melanoma cell lines ranged from a twofold to up to a 15-fold increase compared to normal human melanocytes (Figure 4-1). To investigate i f the accumulation of p 3 3 I N G l b results from aberrant INGl gene transcription mRNA levels were also assessed by semi-quantitative RT-PCR. A l l cell lines were found to overexpress the INGl gene at the transcriptional level, while INGl mRNA levels were nearly undetectable in normal melanocytes (Dr. K.J . Cheung, personal communication). In order to determine i f p 3 3 1 N G l b also accumulates in cutaneous malignant melanoma, 59 twenty seven biopsies were collected from the 1995-2001 archives of the Pathology Department of the Vancouver General Hospital. Levels of p 3 3 I N G l b were assessed in the 27 malignant melanoma biopsies by immunohistochemistry (Figure 4-2). The intensity of the staining was rated as negative (0), weak (1+), moderate (2+) or strong (3+). Negligible or no nuclear expression of p 3 3 I N G l b protein was detected in normal melanocytes from normal skin biopsies. Among the 27 biopsies, 20 (74.1%) showed high levels of pSS™0 1 1 5, 6 (22.2%) had moderate expression and only one sample (3.7%) had undetectable p 3 3 I N G I b protein. Normal melanocytic nevus, but not normal epidermal melanocytes where also found to contain high levels of the p 3 3 I N G l b protein (Figure 4-2). Although p33 I N L , l b was highly expressed in the melanoma biopsies, all twenty seven biopsies also showed a curious phenomenon. Unlike normal melanocytes and normal melanocytic nevi, all melanoma biopsies contained melanoma cells in which p ^ r N G i b w a g e X p r e s s e c | m a n alternate sub-cellular compartment. Unlike melanocytes, all melanoma cells expressed cytoplasmic pSS™3 1 1 1 and all biopsies contained large cancerous populations that no longer displayed nuclear pSS1 1^0 1 1 3. Curiously, normal epidermal keratinocytes also expressed cytoplasmic pSS™0 1 1 1. Figure 4-2 compares normal nuclear p 3 3 I N G l b in normal nevus melanocytes and cytoplasmic accumulation of p33 I N G I b in a malignant melanoma biopsy. Since all biopsies contained a given proportion of the cell population which there no longer was nuclear p33 1 N G l b , cells with cytoplasmic but no nuclear p 3 3 I N G l b and cells with both nuclear and cytoplasmic p 3 3 I N G l b were counted. In all of the melanoma biopsies only 10-20% of the counted melanoma 60 cells retained some nuclear staining, while all cells expressed cytoplasmic pSS™0"5 (Figure 4-6). 4.2.2 INGl Gene Mutation in Human Melanoma There are reported correlations between p53 tumour suppressor overexpression and TP53 gene mutations in certain types of cancers (Kandioler-Eckersberger et al, 2000; Lukas et al, 2000). For this reason the mutational status of the INGl gene was further assessed in the fourteen melanoma cell lines and in 46 malignant melanoma biopsies. Single strand conformational polymorphism (SSCP) analysis was used to screen for mutations within the INGl gene. SSCP requires PCR amplification of the entire INGl gene in fragments under 300 bp using a high-fidelity D N A polymerase and non-denaturing P A G E of single-stranded PCR products. Since the electrophoresis is performed under non-denaturing conditions, single-stranded D N A is free to fold into a given secondary structure and therefore migrates with a given velocity that differs of the same denatured or double-stranded product (Orita et al., 1989). When there are single base alterations or insertions and deletions, the single-stranded products uptake a slightly different secondary structure detectable as mobility shifts in the P A G E (Orita et al, 1989). A band-shift indicative of D N A base alterations, deletion and /or addition was detected in the PCR product corresponding to exon la of the INGl gene in the Sk-mel-110 cell line. A l l other band-shifts occurred in PCR products corresponding to exon 2 of the INGl gene of the Sk-mel-24 cell line and of 9 of the 46 biopsies (Figure 4-3). 61 Samples demonstrating band-shifts in the SSCP assay were further sequenced to confirm and identify genomic alterations within the ING1 gene (Figure 4-4). A l l samples, but the SK-mel-110 cell line contained at least one missense or nonsense mutation within exon 2 of the ING1 gene. The SK-mel-110 cell line only contained silent mutations. Sequencing results were compared to published ING1 sequence and SNPs (GeneBank accession numbers: AB024401; AB024402 and AF078835). Sequencing of melanoma sample D N A was further compared to D N A from normal human fibroblasts and from adjacent normal epidermal cells (for the melanoma biopsies). No mutations were found in the matched normal skin suggesting that these mutations were of somatic origin. No mutations were found in exon lb of the ING1 gene, which encodes the p 4 7 I N G l a isoform. Table 4-1 recapitulates the sequencing results in all melanoma biopsies and cell lines with aberrant electromobility in the SSCP assay. Interestingly, the mutations were restricted to two areas encoded by exon 2 (within the PCR and the PHD domains of p33 I N G l b ) . Of further interest, two melanoma patients harbored the same mutation at codon 102 (R102L) and two patients and one melanoma cell line harbored the same mutation within codon 260 (N260S) (Figure 4-4). Overlapping sequencing signals in certain melanoma biopsies (exemplified by an overlapping T and G in the bottom-left sequencing sample of the figure) may result from heterozygosity but most likely from the presence of normal tissue found within the tumour biopsy (i.e. blood vessels and cells, etc.) that could not be removed under microscopic dissection using a needle. 62 4.2.3 Correlations between INGl Mutation and Clinical Parameters The biopsies collected from the 46 melanoma patients were from 24 males and 22 females which ranged from 26 to 91 years old, with an average of 55 years. The melanomas were classified into Clark's level of invasiveness (I - V) based on the following parameters: Clark's I, melanoma in situ; Clark's II, the tumour has spread to the upper dermis; Clark's level III, the tumour involves most of the upper dermis; Clark's level IV, the tumour has spread to the lower dermis; and Clark's level V , the tumour has spread beneath the dermis. Only 27 of the 46 biopsies were large enough for both immunohistochemistry and mutational analysis of the INGl gene. No correlations were found between INGl mutation and gender, tumour site or Clark's level of invasion (Table 4-2). Interestingly, the INGl mutation rate was significantly higher in patients aged of 40 years or younger (6/12, 50%) than those older than 40 years of age (3/34, 8.8%) (PO.01, x 2 test). Five-year survival data was also obtained for 34 of the 46 patients. Kaplan-Meier survival curves were plotted for tumour biopsies which contained INGl mutation and for those with wild-type INGl (Figure 4-7). Our data revealed a trend in which patients with INGl mutation have a poorer 5-year survival (P=0.06, log-rank test) in which half of patients harboring INGl mutations in the melanoma cells had died of the disease compared to only 18%> of the patients with no INGl mutation in the melanoma cells. However, due to the relatively small number of cases with INGl mutations, this trend could not be considered statistically significant in 63 our analysis. Finally, there was no correlation between ING1 mutation and levels of p 3 3 I N G l b detected by immunohistochemistry (Figure 4-6). 4.2.4 Effect of ING1 mutation on D N A repair To investigate the effect of ING1 mutation on the biological functions of the tumour suppressor, site-directed mutagenesis was performed to obtain constructs expressing FLAG-tagged pSS™ 0 1 1 1 mutants with point mutations at codon 102 (arginine to leucine) or codon 260 (asparagines to serine) since they occurred more than once and represent two areas prone to mutations (Figure 4-5). Figure 4-8 confirms successful mutagenesis of the two mutant plasmid constructs. A l l constructs further had comparable expression levels when transiently expressed in the M M R U melanoma cell line, as demonstrated by western blotting (Figure 4-8). A host-cell-reactivation assay (Figure 4-9) was used to determine whether the R102L and N260S point mutations have any effect on p33 I N G l b-mediated enhancement of UV-damaged D N A . The vCMVcat plasmid, which encodes the CAT reporter gene, was exposed to 40 mJ/cm2 U V - C ex vivo and then co-transfected into M M R U cells with either empty vector or vectors expressing wt or mutant p33 I N G l b . The C A T activity, which indirectly reflects the repair efficiency of UV-damaged reporter plasmid (Figure 4-9), was then measured after 40 hours. The CAT activity was 10-fold higher in cells overexpressing wild-type p 3 3 1 N G l b compared to cells transfected with empty vector (Figure 4-9), confirming previous reports on p33 I N G l b-mediated enhancement of D N A 64 repair (Cheung et al, 2001). The CAT activity of cells transfected with R102L or N260S mutant pSS™0 1 1 1 was however comparable to that of cells transfected with empty vector suggesting that these two mutations are detrimental to p33 I N G l b-mediated enhancement of D N A repair (Figure 4-9). Co-transfection of wild-type p 3 3 [ N G l b with either one of the two mutants rescued the deficiency in D N A repair further suggesting that these are not dominant negative mutations (Figure 4-9). To confirm that the R102L and N260S alterations of p 3 3 I N G l b can abolish p33 1 N G l b enhancement of UV-damaged D N A repair in vivo, a radioimmunoassay with antibodies that directly recognize UV-induced 6-4PP was performed (Figure 4-9). M M R U cells were transfected with empty vector, wild-type or mutant p 3 3 I N G l b and exposed to ultraviolet radiation (20 mJ/cm2 TJVB) the next day. Genomic D N A was then collected from cells before, immediately after or four hours after exposure to U V light. The RIA assay helped determine the amount of 6-4PPs remaining in each sample. The results demonstrated that cells overexpressing wild-type p 3 3 1 N G l b had 47% less 6-4PPs 4 hours following exposure to U V irradiation compared to cells transfected with empty vector. However, the p33 I N G l b-mediated enhancement of D N A repair was greatly reduced in cells transfected with the R102L or N260S mutant forms with only 18.6% and 18.5% less 6-4PPs compared to the empty vector control (Figure 4-9). 65 4.3 Discussion The p 3 3 I N G l b plays an important role in the cellular stress response to U V radiation. Expression of the INGl gene is responsive to cellular exposure to U V light in cultured melanoma cells (Cheung et al., 2001), but most importantly it induces cell cycle arrest (Garkatsev et al., 1998) an important step to allow cells to repair sub-cellular damage. The key observation that p 3 3 1 N G l b drastically enhances the removal of damaged D N A by nucleotide excision repair (Cheung et al, 2001) confirmed that like p53, p 3 3 I N G l b is a guardian of genomic integrity in the cells. Since p 3 3 I N G l b closely cooperates with p53 to indirectly promote the hypo-phosphorylation of the Rb tumour suppressor (see introductory chapter) and p53 is rarely mutated in human melanoma (Gwosdz et al., 2006), we therefore hypothesized that the INGl gene might be silenced or mutated in human cutaneous melanoma. Although nearly a fifth of the melanoma biopsies observed did contain a missense mutation there were a few surprising results, such as the high levels of p 3 3 I N G l b in melanoma cells, which command a revision of the initial hypothesis. The expression level of p 3 3 [ N G I b in human tumours appears to be dependent on the tissue type. Diminished expression was reported in lymphoid tumour cell lines, gastric carcinomas, breast cancer, esophageal squamous cell cancer, hepatocellular carcinoma and adenocarcinomas of the esophagogastric junction. However, higher p33 I N G l b expression was observed in neuroblastoma and brain cancer cell lines and in basal cell and oral squamous cell carcinomas on top of the here-reported melanoma cell lines and melanoma biopsies (Table 1-2). Although the results in this chapter argue that 66 the expression of the ING I gene is clearly not silenced in cutaneous human melanoma silenced, nearly 85% of all melanoma cells within the melanoma biopsies had little accumulation of p 3 3 I N G l b within the nucleus. This observation was further confirmed by an independent report in which melanoma cells are found to relocate p 3 3 I N G l b in an alternative sub-cellular compartment (Nouman et al, 2002a). Furthermore, observation of the same phenomenon has now been reported in brain tumours (Vieyra et al, 2003), childhood acute lymphoblastic leukemia (Nouman et al, 2002b), seminoma (Nouman et al, 2002c), papillary thyroid carcinoma (Nouman et al, 2002c) and ductal breast carcinoma (Nouman et al, 2002c). The nuclear to cytoplasmic compartment shift occurs independently of ING J gene alterations (Figure 4-6). It is interesting to note that there are three reported missense mutations within the NLS, some observed in cancers reported to have mislocalized p 3 3 I N G l b protein (Hara et al, 2003; Vieyra et al, 2003). However, none of these mutations were observed in melanoma. The cytoplasmic translocation in melanoma may perhaps be due to posttranslational modifications and/or interactions with proteins that can alter pSS1™0113 sub-cellular localization, such as in the case of de-phosphorylation and subsequent mdm2-mediated p53 nuclear to cytoplasmic shuttling (Kubbutat et al, 1997). Although the reason for the nuclear to cytoplasmic compartment shift in melanoma cells, and other types of cancer, has yet to be identified it can certainly contribute to a functional inactivation of p 3 3 I N G l b since most of its functions are attributed to its role in histone acetylation and deacetylation. This is an important observation that certainly merits further investigation. 67 In this study 46 primary melanoma biopsies and 14 melanoma cell lines were further screened for ING1 mutations. SSCP and sequencing analysis revealed that the ING1 gene was mutated in 9 of 46 (19.6%) of melanoma primaries and 2 of 14 (14.3%) melanoma cell lines. This mutation rate is the highest among all human cancers examined so far (Table 1-2) after head and neck SCCs (3/23, 13%) (Gunduz 2000), and esophageal SCCs (4/31, 12.9%) (Chen et al, 2001). In the majority of cancers studied to date ING1 is however rarely mutated. The relatively high ING1 mutation in human melanoma supports the notion that p 3 3 I N G l b plays an important role in cellular stress response to U V radiation and melanomagenesis. There where six G to T transversions among the 10 missense mutations identified through sequencing (Table 4-1). G to T transversions frequently result from oxygen free radical damage in D N A (Reid et al, 1991). The G to T transversions in the ING1 gene of melanoma biopsies may therefore result from oxidative damage associated with solar U V , as G to T mutation has been found in N-Ras and p53 genes in UV-induced skin cancers (Pierceall et al, 1992; Seidl et al, 2001). However, the fact that no ING1 mutations were found in sun-exposed sites (face, ear and neck) (Table 4-2) adds to the subtleness of U V role in melanoma gene mutation. Since the etiology of melanoma suggests that exposure to sunlight in early childhood may be an important factor in adult melanoma formation (Gilchrest et al, 1999), the site of the tumour with ING1 mutation may not correlate with adult exposure but rather with childhood recreational exposures (e.g., sunburn). One interesting finding from this study is that melanomas from younger individuals (40-years or less) had a significantly higher ING1 mutation rate than those over 40-years of age (Table 4-2). Although the cause for this age-related mutation rate and its biological significance have 68 yet to be determined, our findings could indicate an involvement of INGl mutation in melanomagenesis since melanoma affects younger patients unlike non-melanoma skin cancers which are directly related to cumulative sun exposure and mainly diagnosed in older populations. However, the lack of correlation between INGl mutation and Clark's level of progression argues otherwise; or at least that INGl mutation is not a common event leading to melanomagenesis. Cytoplasmic shuttling of p 3 3 I N G l b may therefore be a more critical event contributing to melanoma formation and/or progression since all melanocytic nevus (often considered precursors to melanoma formation) observed clearly contained predominantly nuclear p 3 3 I N G l b (Figure 4-2). However, cancer formation is a multi-step process and involves the activation of growth promoting genes and inactivation of cell cycle regulators and promoters of genomic stability. Since p 3 3 I N G l b plays an important role in the cellular stress response to U V radiation, INGl mutations would certainly contribute to genomic instability and promote cellular transformation. A l l reported INGl mutations to date are located within exon 2 (Figure 4-5). INGl mutations in cutaneous melanoma were found to be clustered within the PHD domain, which can regulate the binding of ING proteins to chromatin (Gozani et al., 2003) and a region reported to interact with components of the sin3 H D A C complex and is believed important in p33 I N G l b-mediated acetylation (Kuzmichev et al., 2002). The unique locations of INGl mutations therefore lead to the hypothesis that they may affect p33 I N G l b-mediated enhancement of D N A repair. Using a host-cell-reactivation assay and a radioimmunoassay specific for UV-induced 6-4PP D N A lesions the two most common mutations, R102L and N260S, were indeed found to abrogate 69 p33 I N G l b enhancernent of NER (Figures 4-9 and 4-10). Since cancer development is a multi-step process, the defects in NER caused by INGl mutation would have severe consequences on melanoma formation, progression and prognosis of melanoma patients. For instance, the reduced capacity of NER caused by INGl mutation will increase the genomic instability by failing to repair UV-induced D N A damage in crucial genes which are important for regulating cell cycles, leading to unnecessary cell divisions and propagation of UV-damaged DNA. Since p 3 3 I N G l b also promotes UV-induced apoptosis (Cheung and L i , 2002), mutation of the INGl gene may reduce the removal of cells containing severely damaged D N A by apoptosis. Increased genomic instability could further lead to tumour progression. This may explain why higher percentage of patients with INGl mutations (50%) died within five years compared to 18% of the patients who retained wild-type INGl. INGl mutation occurs in only 20% of the melanoma biopsies, and therefore cannot be a sole critical contributing factor in melanoma progression. p^^iNGib c i o s e j y cooperates with the p53 protein and resides within in a pathway capable of regulating Rb phosphorylation. It is interesting to note that like p33 1 N G l b , p53 is also overexpressed but rarely mutated in human malignant melanoma (Gwosdz et al, 2006; Sparrow et al, 1995). Exploration of the functionality of this pathway and further experiments exploring the relation between ING1/TP53 expression and mutations and p21 W a f l regulation, Rb phosphorylation and cell cycle progression would certainly prove interesting. 70 4.4 Figures Figure 4-1 Western analysis of p 3 3 I N G l b protein levels in fourteen melanoma cell lines. (A) Fifty micrograms of total cell extracts were resolved by SDS-PAGE and blotted with anti-p33 I N G l b antibody. Normal human epithelial melanocytes (NHEM) were used as control and p-actin as the internal loading control. (B) Densitometric measurement of protein expression levels in (A) corrected for total loading. Compared to N H E M all fourteen melanoma cell lines accumulated high levels of p 3 3 I N G l b protein. 1 <b <o 1^  <8> \^ ^ / / / / / / " » • i • <mmmmm~mmmmmmmmmm0> p-actin <f^# ^ # <^ 4 f 4 ^ 4 # p 3 3 I N G 1 b P-actin B 20n 71 Figure 4-2 Immunohistochemical analysis of p 3 3 I N G l b expression in normal epidermis (A), a normal melanocytic nevus (C), and in a typical cutaneous malignant melanoma tumour (E). Although some cytoplasmic p 3 3 r N G l b was found in normal epithelial keratinocytes no or little cytoplasmic p 3 3 I N G l b was detected in normal melanocytes (panel A indicated by arrows). There was evident nucleolar accumulation of p 3 3 I N G l b in various keratinocytes and basal cells in the normal epidermis but low levels of nuclear p 3 3 I N G l b in normal melanocytes. Benign melanocytic nevi were often found to stain strongly for p33 I N G l b ; however p 3 3 , N G l b was largely confined to the nucleus in these cells (indicated by arrows in panel C). Virtually all melanoma tumours were found to accumulate high amounts of p33 I N G l b , however most melanoma cells displayed a loss of nuclear pSS™0"5 staining. The black arrows in panel E point at melanoma cells expressing high p 3 3 I N G l b levels in both nucleus and cytoplasm while blue arrows point to melanoma cells having lost nuclear expression of p33 I N G l b ; exemplary of typical heterogeneity in this type of cancer. Tissues were indirectly immunolabeled using anti-pSS1™0111 antibodies and subsequent HRP-DAB staining. Tissues were counterstained with hematoxylin to visualize DNA. Primary antibody was omitted in tissues in panels B, D and F (from the same biopsies as in panels A, B and C respectively). Magnification, 250x. 72 B D If**-/ F 73 Figure 4-3 Single-strand conformational polymorphism (SSCP) analysis of the ING1 gene in human melanoma cell lines (A) and cutaneous malignant melanoma biopsies (B). DNA from cultured normal human fibroblasts (NHFB) containing wild-type ING1 was used as a reference to detect mutations in the melanoma cell lines. Sequences from the first and last halves of exon 2 were separately screened for mutations (Exon 2(a) and Exon 2(b) respectively). DNA extracted from normal (N) and tumour (T) tissues from the same patient (represented by a different number) were compared for ING1 alterations. No mutations were detected in exons la and lb of the ING1 gene in genomic DNA from the melanoma cell lines. Arrowheads highlight altered mobility of single-stranded DNA in given samples, indicative of potential ING1 gene alterations. ^ ^ & 4 4 4 <^ 4 4 ^ 4 <f^^ • O O - - V J ? » 4 I 4 ^ ,4 iterf Ifcwd Updi JpMf[ k^wi^  tfcrtP fc*"^ & ^  (h^w Exon 1a Exon 2(a) Exon 2(b) B 2258 15679 19383 26627 32147 T N T N T N T N T N ML .mm mk turn, l i t m BE B H T T • » 3339 5601 15545 19384 T N T N T N T N Exon 2(a) Exon 2(b) 74 Figure 4-4 Representative sequencing analysis of samples with aberrant SSCP migration. A l l samples were found to contain at least one point mutation. (A) Sequencing of the INGl gene in two melanoma cell lines, Sk-mel-24 and Sk-mel-110 (left and right panels respectively). A missence mutation was found to change asparagine of codon 260 of the p 3 3 1 N G l b protein to a serine in the Sk-mel-24 cell line. The Sk-mel-110 cell line was the sole sample sequenced not to contain a missence or nonsense mutation. (B) Missence mutations of codons 102 and 260 of the p 3 3 1 N G l b protein in the melanoma biopsies number 32147 and 5601 respectively. The INGl gene of melanoma cell lines and of melanoma biopsies was compared to that of cultured normal human fibroblasts (NHFB) and normal tissue from the same patients, respectively. A Glu Asn Glu 6 fl G , H H C G fl B I iU ibl i I\1M Asn Arg Thr n n c c G c n c G Asn Lys Thr fl R C C T C H C G Glu Lys Ser l l V ! n W W ]! V \l I NHFB Glu Lys Ser G fl G fl fl G T C C I ! I V Melanoma f \ ii v /\ Glu Asn Glu G ft G fi n c G n G vVA/iww Normal Glu Ser Glu G f l G R G C G H G \ Tumor 75 Figure 4-5 Location and relative frequency of mutations affecting the p 3 3 I N G l b protein. Codons (represented by numbers) in black are indicative of mutations occurring in human cutaneous malignant melanoma and human melanoma cell lines. Codons in grey represent mutations found in breast cancer, gastrointestinal cancers, head and neck squamous cell carcinoma, esophageal squamous cell cancer, basal cell carcinomas, adenocarcinoma of the esophagogastric junction and exocrine pancreatic carcinoma. These alterations do not include reported polymorphisms, but a nonsense mutation (codon 253) is included. Codons 1-56 are encoded by exon la and codons 57-279 are encoded by exon 2 of the INGl gene. 118 102 117 | 111 126 II I 215 260 2591 253 Missense II i " i |i \f li 244 257 247 253 Silent m UJ P C R N L S PHD PIP Bromodomain 76 Figure 4-6 Correlative analysis of immunohistochemical parameters of p 3 3 I N G l b and INGl gene mutations in human cutaneous malignant melanoma biopsies. There was little difference in p 3 3 l N G l b intensity staining between samples with mutant and wild-type INGl (top panel). There also was little difference between the amount of cells expressing nuclear p 3 3 I N G I b in melanoma biopsies with wild-type and mutant INGl (bottom panel) suggesting that p 3 3 I N G l b expression and localization occurs independently of INGl gene alterations. 3 - i c a J: cn c c A A A A A A A Mut WT 77 Figure 4-7 Kaplan-Meier curves illustrating a potential correlation between ING1 mutation and 5-year patient survival. Patients with missense or nonsense ING1 alterations had a poorer 5-year survival than patients with a wild-type ING1 gene. Within five years 50% of patients with ING1 gene mutations died of the disease compared to 18%) of patients with no ING1 gene alterations. Due to the relatively small sample number this result only borders statistical significance (p = 0.06, log-rank test). 100 '£ so -3 ^ 60 -No mutation 50 A Mutation 40 0 10 20 30 40 50 60 Time (months) 78 Figure 4-8 Site-directed mutagenesis of the p C I n e o - T M ^ - F L A G plasmid. Point mutations were introduced into two separate plasmids to mimic the R102L and N260S mutations (panels A and B, respectively). (C) Western analysis of total cell extracts from M M R U cells transiently transfected with an empty vector or the pCI-/A rG76-FLAG, pCI-77VG76-FLAG-R102L or the pCI-/7VG7&-FLAG-N260S plasmids. The F L A G epitope could be detected from all but the vector-transfected cells. P 3 3 I N G l b was expressed in all cases and was clearly overexpressed in cells transfected with either 77VG76-expressing vector. A Asn Arg Thr flflc c GC fl C G B Glu Asn Glu G AG A R C G R G X L A V wt Asn Lys Thr fine C T C BC G wt Glu Ser Glu R102L G B G B G C G B G N260S & ^° <F ^ FLAG p 3 3 I N G 1 b 79 Figure 4-9 INGl mutations found in human cutaneous malignant melanoma can abrogate p33 INGlb-dependent repair of UV-damaged DNA. (A) Effect of INGl gene mutation on the repair of a UV-damaged reporter plasmid D N A by the host-cell-reactivation assay. Undamaged or UV-damaged pCMV-CATreporter plasmids were co-transfected with undamaged empty vector or vectors expressing wild-type or mutant p33 1 N G l b in M M R U cells. Cells were collected after a 40 h period, and the activity of the CAT enzyme assayed in vitro by measuring its capability to transfer a radiolabeled acetyl group into an appropriate substrate. The CAT activity was determined by scintillation counting and expressed as net dpm damage dose/net dpm zero dose. Experiments were performed in triplicates. (B) Effect of p 3 3 I N G l b R102L and N260S mutations on the repair of UV-damaged genomic D N A by RIA. M M R U cells transfected with either empty vector or a vector expressing wild-type or mutant p33 I N G l b . Twenty-four hours later cells were exposed to 20 mJ/cm2 U V B light and genomic D N A collected harvested after 4 h. The percentage of remaining 6-4PPs was then measured using antisera specific for 6-4PPs. Data represents the average of two independent experiments. •WT •WT Table 4-1 Summary of INGl gene mutations found by SSCP-DNA sequencing in the human melanoma cell lines Sk-mel-24 and Sk-mel-110. Cell line Codon Nucleotide Amino acid Type of alteration Sk-mel-24 239 T C G ^ T C C Ser^Ser Silent 244 A A T - > A A C Asn-^Asn Silent 247 C C C ^ C C A Pro^Pro Silent 253 TGT->TGC Cys^Cys Silent 257 CGG->CGT A r g ^ A r g Silent 260 AAC-> A G C Asn -> Ser Missense 270 A A A - > A A G Lys-> Lys Silent 272 A A A ^ A A G Lys -> Lys Silent Sk-mel-110 89 GTG->GTA V a l ^ V a l Silent 101 G A C ^ G A T Asp -> Asp Silent Table 4-2 Summary of INGl gene mutations found by SSCP-DNA sequencing in the human cutaneous malignant melanoma biopsies. Codons represent those of the p 3 3 I N G l b protein and not other I N G l isoforms. Case no. Sex Age Site Clarks level Codon Nucleotide Amino acid Type of Alteration 32147 F 29 Abdomen I 102 C G C ^ C T C Arg->Leu Missense 3339 M 33 Scalp II 257 CGG->CGT A r g ^ A r g Silent 260 AAC-> A G C Asn -> Ser Missense 26627 F 38 Shoulder II 102 C G C ^ C T C Arg->Lys Missense 15545 M 75 Calf III 257 CGG->CGT Arg->Arg Silent 259 G A G ^ G A T G l u ^ A s p Missense 15679 F 26 Arm III 126 AGC-> A A C Ser->Asn Missense 19383 F 35 Calf III 117 C A G ^ C A T G l n ^ H i s Missense 19384 F 40 Abdomen III 244 A A T - > A A C Asn-^Asn Silent 247 C C C ^ C C A Pro->Pro Silent 253 TGT->TGA Cys^Stop Nonsense 5601 M 56 Shoulder IV 253 TGT->TGC C y s ^ C y s Silent 260 AAC-> A G C Asn -> Ser Missense 2258 M 64 Back IV 111 G A G ^ G A T G l u ^ A s p Missense 118 G A G ^ G A T G l u ^ A s p Missense Table 4-3 Correlations between INGl gene mutation in human cutaneous malignant melanoma and clinical parameters. P-value is based on %2 or Fisher's exact test. Sun-exposed sites include face, ear and neck while sun-protected sites indicate other body sites. INGl mutation +(%) -(%) Total P-value Age <40 6(50) 6(50) 12 <0.05 >40 3 (8.8) 31 (91.2) 34 Sex Male 4(16.7) 20 (83.3) 24 Female 5 (22.7) 17(77.3) 22 >0.05 Clarks level 11 (14.3) 6(85.7) 7 >0.05 II 2(18.2) 9(81.8) 11 III 4(33.3) 8 (66.7) 12 IV 2(14.3) 12(85.7) 14 Site Sun-exposed 0(0) 10(100) 10 Sun-protected 9(25) 27 (75) 36 >0.05 83 4.5 References Brochez L. and Naeyaert J .M. Understanding the trends in melanoma incidence and mortality: where do we stand? 2000. Eur J Dermatol 10:71-6. Campos E.I., Cheung K.J . Jr., Murray A. , L i S. and L i G. The novel tumour suppressor gene INGl is overexpressed in human melanoma cell lines. 2002. Br J Dermatol 146:574-80. Campos E.I., Chin M . Y . , Kuo W.H. and L i G. Biological functions of the LNG family tumour suppressors. 2004. Cell Mol Life Sci 61:2597-613. Campos E.I., Martinka M . , Mitchell D.L., Dai D.L. and L i G. 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Ultraviolet exposure as the main initiator of p53 mutations in basal cell carcinomas from psoralen and ultraviolet A-treated patients with psoriasis. 2001. J Invest Dermatol 117:365-70. Serrone L. and Hersey P. The chemoresistance of human malignant melanoma: an update. 1999. Melanoma Res 9:51-8. Sparrow L.E. , Soong R., Dawkins H.J., Iacopetta B.J., Heenan P.J. p53 gene mutation and expression in naevi and melanomas. 1995. Melanoma Res 5:93-100. Vieyra D., Senger D.L., Toyam T., Muzik H. , Brasher P .M. , Johnston R.N., Riabowol K. and Forsyth P.A. Altered subcellular localization and low frequency of mutations of INGl in human brain tumours. 2003. Clin Cancer Res 9:5952-61. 86 5 PHOSPHORYLATION OF THE P 3 3 1 N G 1 B PROTEIN 5.1 Rationale Posttranslational modifications, such as protein phosphorylation and acetylation, are fundamental mechanisms through which intracellular events can be rapidly and effectively regulated. A number of protein kinases, notably various phosphatidylinositol 3 kinase (PBK)-related kinase family members have been identified as transducers which initiate a series of phosphorylation events in response to D N A damage (Bakkenist and Kastan, 2004). For example, p53 activation and stabilization occurs upon activation of the PI3Ks A T M and ATR. Serine 15 of p53 is directly phosphorylated by both A T M and ATR, which in turn is thought to enhance its transactivating activity (Bakkenist and Kastan, 2004). Phosphorylation of p53 serine 20 is also important for p53 stabilization because the phosphate group sterically interferes with binding of the E3 Ub recognin enzyme mdm2 which contributes to p53 degradation (Shieh et al., 2000; Hirao et al., 2000). The Chkl/2 kinases are key regulators of the G 2 / M checkpoint which are activated by A T M and ATR (Bakkenist and Kastan, 2004). The p 3 3 I N G l b protein is transcriptionally up-regulated by UV-induced genotoxic stress in a time- and dose-dependent manner (Cheung et al., 2001). Not only does p 3 3 I N G l b closely cooperate with the p53 protein but it also enhances the nucleotide A version of this chapter will be submitted for publication. 87 excision repair of UV-damaged D N A (Campos et al., 2004; Cheung et al., 2001). However no regulatory mechanisms for the p 3 3 I N G l b protein have yet been described. Since protein phosphorylation is a crucial mechanism for the activity of many tumour suppressors, and forms a major mechanism through which various cellular stress response events are orchestrated upon D N A damage; it is therefore credible to hypothesize that p33 I N G l b is subject to protein phosphorylation upon genotoxic stress and that phosphorylation of p 3 3 I N G l b may represent a key mechanism for the regulation of its activity. 5.2 Results 5.2.1 P 3 3 1 N G l b is a Phospho-protein To test the hypothesis that the p33 1 N G l b protein is subject to posttranslational modifications upon genotoxic stress, we first performed a low-to SDS-PAGE comparing protein extracts of untreated and topoisomerase II inhibitor-treated human M M R U melanoma cells. Since the transiently expressed FLAG-tagged p 3 3 r N G l b appeared as a doublet in untreated cells, it is likely that some posttranslational modification event occured early on after translation (Figure 5-1). The upper F L A G signal would represent posttranslational modified (i.e. phosphorylated) p33 I N G l b , due to altered charged interactions between phosphopeptides and SDS, while the lower signal would represent unmodified p33 1 N G l b . Increasing doses of the DNA-damaging agents etoposide and doxorubicin resulted in altered mobility of p33 1 N G l b . Both topoisomerase II inhibitors 88 lead to a net accumulation of a higher molecular weight protein variant 24 h after exposure, suggestive of a stress-induced posttranslational modification. To further investigate i f the upper F L A G signal was due to protein phosphorylation, we incubated the cells in the presence of the cell permeable phosphatase inhibitor okadaic acid or the kinase inhibitors wortmannin and staurosporine. Okadaic acid, a potent inhibitor of protein phosphatase IA and 2A, caused the disappearance of the bottom (presumably native) proteins (Figure 5-1), indicating that the upper band was likely a result of protein phosphorylation. Furthermore, the addition of the serine-kinase inhibitor staurosporine dramatically reduced the appearance of the upper band (phosphorylated proteins) (Figure 5-1). Wortmannin an inhibitor of phosphoinositide kinase-3 (PI-3K) and PI-3K related kinases also reduced the upper band albeit to a lesser extent. To further confirm that p 3 3 I N G I b is a phospho-protein, we performed western blot analysis using antibodies directed against phosphorylated serine residues. Transiently expressed FLAG-tagged p 3 3 I N G l b was immunoprecipitated from doxorubicin-treated M M R U cells with a mouse anti-FLAG antibody. The immunoprecipitate was divided and half incubated in an alkaline phosphatase reaction, while the other half was incubated in a mock reaction. Western analysis using antibodies directed against phosphorylated serine residues indicated that at least some serine residue(s) within the p33 1 N G l b protein can be phosphorylated (Figure 5-2). This was further supported by the loss of phospho-serine signal, but not of the overall p 3 3 r N G l b signal, that ensued following a phosphatase 89 treatment suggesting that p 3 3 I N G ! b is phosphorylated on at least one serine residue in vivo. Radiolabeling of GST-tagged p 3 3 I N G l b further confirmed that p 3 3 1 N G l b can also incorporate phosphate groups in vitro using cell extracts as the kinase source (Figure 5-2). 5.2.2 Serine 126 is Phosphorylated upon Genotoxic Stress Chemical manipulations that utilize differences in survey modes when doing M A L D I -TOF mass spectrometry (Xu et al., 2005) allowed the identification of serine 126 as a phosphorylatable residue. The modified sample preparation enhances the detection of negative ions, therefore eliminating the need to phospho-enrich samples. It has the advantage of direct mass measurement analysis of any modification that adds a negative charge to a peptide (e.g., phosphorylation) without the background signal of acidic residues. Since the negative charges associated with carboxylate groups of glutamic and aspartic acid residues can be removed through methyl esterification, it is used to suppress the ion intensity of non-phosphorylated acidic peptides in a negative ion mode, thereby enhancing phospho-peptide detection. The analysis was performed on recombinant GST-p33 I N G l b exposed to either untreated or doxorubicin-treated M M R U cell extracts. Minimal phosphorylation of serine 126 was observed when extracts from untreated cells were used to phosphorylate recombinant p 3 3 I N G l b (Figure 5-3 top panels) compared to doxorubicin-treated M M R U cell extracts (Figure 5-3 bottom panels), indicating that this is a stress-responsive phospho-residue. To confirm that serine 126 is a bona fide phosphorylation site, the samples were subjected to an alkaline phosphatase treatment 90 prior to a second round at MALDI-TOF (Figure 5-3 right panels). Such treatment resulted in a reduction of the 1044.8 m/z peak corresponding to the putative phospho-peptide D T A G N p S G K A G A to 964 m/z due to a neutral loss of 80 phosphates (HP0 3) group. (3-casein, a protein containing multiple phospho-residues, was used as CIP positive control (Dr. J. Bush, personal communication). To further confirm that serine 126 is phosphorylated in vivo, rabbit polyclonal antibodies were raised against a phosphorylated peptide and used in western analysis of extracts from cells subjected to genotoxic events. The antibody was sequentially affinity purified using a synthetic phosphorylated serine 126 peptide corresponding to amino acids 118-134 and subsequently using non-phosphorylated recombinant full-length p33 1 N G l b . In order to test the specificity of the antibody, the sera was incubated with a phosphorylated or non-phosphorylated peptide corresponding to amino acids 118-134 of p 3 3 I N G l b and the antibody-peptide binding analyzed by ELISA (Figure 5-4). The binding of the antibody to phosphorylated peptide was much stronger (> 8-fold at 1:2000 dilution) than to non-phosphorylated peptide. To further test the specificity of the antibody, a western analysis was done comparing bacterially-expressed recombinant p33 I N G l b left untreated or phosphorylated using M M R U cell extracts. In eukaryotes, protein phosphorylation occurs mainly as phosphoester in serine, threonine, and tyrosine residues. However, while prokaryotic cells are known to have serine/threonine protein kinases (Kenelly et al, 1998), phosphorylation events largely result from a two-component system in which a phosphate group is transferred from a histidine residue of the kinase protein onto an aspartate residue of the substrate protein (Hoch, 2000). It is 91 therefore not surprising that the polyclonal anti-p-S126 antibody did not recognize bacterially-produced recombinant p 3 3 I N G l b unless it was phosphorylated in vitro using eukaryotic cell extracts. To determine the kinetics of serine 126 phosphorylation within endogenous p 3 3 I N G l b protein upon genotoxic events, we exposed M M R U cells to 20 mJ/cm2 U V B or 1 ug/ml doxorubicin and monitored phosphorylation levels at various time points. Since p 3 3 I N G l b is up-regulated by DNA-damaging events (Cheung et al., 2001), cycloheximide was added to the media to prevent de novo protein synthesis. Using the anti-p-S126 antibody, over two-fold increase in serine 126 phosphorylation in endogenous p33 1 N G l b were observed as quickly 1 minute following U V R , and 60 minutes after exposure to doxorubicin (Figure 5-4). However, a basal level of serine 126 phosphorylation was detected in the untreated asynchronous cells, suggesting that phosphorylation of this residue also occurs under non-stress physiological conditions. Cycloheximide alone had little effect on the phosphorylation of serine 126. 5.2.3 Serine 126 Phosphorylation Does not Alter pSS™0 1 1 3 Sub-cellular Localization Altered expression and sub-cellular localization as well as gene mutation of the p 3 3 I N G l b tumour suppressor have been found in various cancer types (Campos et al., 2004). Since serine 126 is 16 residues from the nuclear localization sequence we considered important to examine the localization of the protein in the context of protein phosphorylation. In order to prevent phosphorylation, serine 126 was mutated to alanine by site-directed 92 mutagenesis (Figure 5-5). Serine to alanine substitutions can be used to prevent phosphorylation since the alanine cannot be phosphorylated and its methyl group side chain is unlikely to participate in most hydrophobic or hydrophilic interactions. The SI26A mutant was then transfected into M M R U melanoma cells and compared to wild-type protein by immunofluorescent staining to examine the protein sub-cellular localization. Both wild-type pSS™0 1 1 1 and the S126A mutant showed nuclear signals, suggesting that phosphorylation of serine 126 does not modulate the cellular localization of the protein (Figure 5-6). 5.2.4 Phosphorylation of Serine 126 Increases the Half-life of p 3 3 1 N G l b The use of the kinase inhibitors staurosporine and wortmannin reduced the level of posttranslationally-modified p 3 3 I N G l b but also overall protein levels (Figure 5-1). These results implicate a role for serine 126 phosphorylation in protein stability. To further explore this possibility the turnover rate of p 3 3 I N G l b was assessed in M M R U cells by overexpressing FLAG-tagged protein and harvesting cycloheximide-treated cells at fixed time intervals (Figure 5-7). Densitometry analysis of SDS-PAGE blots established a half-life at approximately 16.8 h for wild-type p33 1 N G l b compared to 5.7 h for its S126A mutant counterpart in M M R U cells (Figure 5-7). The intensity of p 3 3 1 N G l b bands were corrected for total protein loading and ploted as a natural logarithm of remaining p 3 3 I N G l b in function of time (based on three independent experiments). It therefore reasonable to deduce that phosphorylation at this site enhances protein stability since a proportion of 93 the pSS1™0113 protein pool is phosphorylated in untreated cells as shown by both band-shift assays and western blots on endogenous pSS™0"3 (Figure 5-1). It is noteworthy to examine the effect of p 3 3 I N G l b phosphorylation on p53 protein stability as a previous study has shown that p 3 3 I N G l b promotes p53 acetylation on lysines 373 and/or 382 (Kataoka et al., 2003). Carboxyl-acetylation of p53 has been proposed to affect p53 stability by preventing mdm2-dependent ubiquitination (Ito et al., 2002) although some studies demonstrate contradicting results (Feng et al., 2002; Nakamura et al., 2000). As previously established, the short-lived p53 protein underwent rapid degradation upon cycloheximide treatment (Figure 5-7). However, the p53 turnover rate was not influenced by the expression of mutant p 3 3 I N G ! b compared to the wild-type counterpart. In both cases the half-life of p53 was of approximately 50 min. 5.2.5 Serine 126 is Phosphorylated by both Chkl and Cdkl Our initial attempt to identify the kinase responsible for serine 126 phosphorylation was focused on the protein kinase C (PKC) based on two observations: 1) Staurosporine, a potent PKC inhibitor caused drastic de-phosphorylation of p 3 3 I N G l b (Figure 5-1) The peptide sequence N-S-G-K, where 'S ' corresponds to amino acid 126 of p 3 3 I N G l b matches the consensus P K C substrate sequence (X-S/T-X-R/K). However, an I N G l peptide containing serine 126 could not be phosphorylated in vitro by P K C , while the PKC control peptide Q K R P S Q R S K Y L easily incorporated radiolabeled-phosphate groups (Dr. M . Garate, personal communication). The NetPhosK 1.0 prediction algorithm based on 94 primary protein sequence (Blom et al, 2004) was used to predict candidate kinases which can phosphorylate serine 126 of p 3 3 , N G l b (Table 5-1). NetPhosK 1.0 prediction is restricted to 17 protein kinases, and excludes the DNA-damage responsive ATR and Chkl kinases. Kinases scoring above 0.4 (Cdkl, CaM-II and GSK3) were chosen to be screened by the commercially available KinaseProfiler™ service. Serine 126 is phosphorylated in response to genotoxic stress. Yet the most probable kinase predicted to phosphorylate serine 126 is Cdkl and not any of the PIKKs. We therefore decided to add the ATM/ATR-downstream and Cdkl-upstream Chkl kinase in the KinasePro filer™ screening ( A T M and A T R were unavailable). Unexpectedly, both Chkl and Cdkl were able to phosphorylate serine 126 in vitro (Figure 5-8). To further confirm that both Chkl and Cdkl can phosphorylate serine 126, we immunoprecipitated Chkl and Cdkl proteins from untreated, doxorubicin- or UV-treated M M R U cells. Our data indicated that Cdkl immunoprecipitated from untreated cells was able to phosphorylate p 3 3 I N G l b peptide at serine 126, while doxorubicin and U V suppressed Cdkl phosphorylation of p33 I N G l b . In contrast, Chkl easily phosphorylated serine 126 when immunoprecipitated from U V - or doxorubicin- treated cells but not from untreated cells. This is likely linked to Cdkl and Chkl degradation as little Cdkl was immunoprecipitated from U V - or doxorubicin- treated cells, while the opposite was observed with Chkl (Figure 5-8). While doxorubicin caused an increase in endogenous serine 126 phosphorylation, the kinase inhibitors kenpaullone and staurosporine also caused a decrease in phosphorylation levels (Figure 5-8). The Cdkl inhibitor roscovitin and Chkl inhibitor H-89 also attenuated the phosphorylation of p 3 3 I N G l b peptide 95 (personal communication). Since Chkl is activated by A T M upon doxorubicin treatment we further observed the effect of validated A T M R N A i on the phosphorylated of endogenous pSS™0 1 1 3. As expected, A T M directed short interfering R N A inhibited p ^ i N G i b p j 1 0 S p j l o r y i a t i o n a t s e r i n e 126 in vivo after doxorubicin treatment (Figure 5-8). To confirm the kinase specificity of this result we further immunoprecipitated A T M and found A T M incapable of phosphorylating serine 126 in vitro in kinase assays (Dr. M . Garate, personal communication). Although this experiment implicates the A T M / A T R cascade it remains however preliminary data and much more should be done to examine the role of A T M , but also A T R since A T R is mainly responsive to U V damage. To further confirm that serine 126 phosphorylation is important for protein stability, we treated M M R U cells with the Cdkl inhibitor roscovitin and analyzed the half-life of p 3 3 I N G l b protein. Consistent with the finding that mutation of serine 126 to alanine greatly shortens the half-life of p33 1 N G l b protein (Figure 5-7), the Cdkl inhibitor roscovitin also drastically reduced the half-life of wild-type p 3 3 1 N G l b (Figure 5-8). This is an important observation since a reduction in the half-life of wild-type p 3 3 I N G l b protein through the inhibition of the Cdkl kinase, confirms that the reduction in the half-life of the S126A mutant is linked to serine phosphorylation and not necessarily to missfolding of the S126A p33mGlh protein. 96 5.2.6 P 3 3 I N G l b is not Degraded through the Classical Ubiquitin-proteasome Pathway In order to verify i f p 3 3 I N G I b could be ubiquitinated, M M R U cells were co-transfected with a construct expressing polyhistidine (His)-tagged Ub and a vector expressing either wild-type or S126A FLAG-tagged pSS™0 1". Cells were then treated with the proteasome inhibitor MG132 or with both MG132 and cycloheximide. Histidine has a strong natural affinity for certain metals and His-tagged proteins can therefore easily be purified through binding of a metal moiety of a metal chelator complexes such as the Ni(2+)-nitrilotriacetic acid (Ni-NTA) bound to beads. Proteins incorporating His-tagged ubiquitin were therefore isolated through immobilized metal affinity purification using Ni-NTA beads and the bound fraction analyzed by SDS-PAGE/western blotting. The p53 protein, which is degraded through the classical ubiquitin-proteasome pathway was found bound to the N i -NTA beads; however upon inhibition of the proteasome there was more p53 bound to the Ni -NTA beads since it could still be ubiquitinated but not degraded (Figure 5-10). As expected this p53 accumulation was attenuated if cells were also treated with cycloheximide, which prevents de novo protein synthesis. The amount of isolated p53, which incorporated His-Ub did not change with the expression of wild-type or mutant p33 I N G l b . A n appreciable amount of exogenous p 3 3 I N G l b was found bound to the Ni -NTA beads, suggesting that it can be ubiquitinated. As expected, cycloheximide-treatment translated into a reduced amount of pSS11"10115 that incorporated the His-Ub group. However, upon inhibition of the proteasome, there was no accumulation of N i -NTA bound p33 I N G I b , suggesting that although it may be ubiquitinated it may not be degraded by the proteasome. The same was observed 97 regardless of the mutational status of the p 3 3 I N G l b protein (Figure 5-10). Ubiquitin was also detected on immunoprecipitated endogenous p 3 3 1 N G l b (Dr. M . Garate, personal communication), confirming that it can be ubiquitinated. Our assays cannot however discriminate between mono- and poly-ubiquitination (mono-ubiquitination is not necessarily involved in protein degradation). SDS-PAGE analysis of cycloheximide-treated cells collected at various points in time further support the observation that p 3 3 I N G l b may not be degraded through the proteasome. The S126A p 3 3 I N G l b protein displayed a higher turnover rate than its wild-type counterpart. Little S126A pSS™0 1 1 3 and p53 protein remained twelve hours after cycloheximide treatment (Figure 5-10). As expected the p53 turnover rate was greatly reduced when the cells were co-treated with cycloheximide and the MG132 proteasome inhibitor since p53 is mainly degraded by the proteasome (Figure 5-10). However, the turnover rate of the pSS1™0111 protein remained unchanged even after inhibition of the proteasome, suggesting that it is still degraded independently of the proteasome. 5.3 Discussion The steady state level of any cellular protein is dependent on its synthesis, degradation, and/or secretion. Upon exposure to genotoxic agents, cells must quickly and efficiently regulate biosynthesis and proteolysis of proteins to fulfill the immediate requirements of a stress response. Posttranslational modifications such as protein phosphorylation, are an elegant and transient way of regulating such processes. ING1 greatly enhances 98 genotoxic-stress induced-NER and is transcriptionally up-regulated within 4 h following U V irradiation in melanoma cells (Cheung et al, 2001). Yet kinetic studies demonstrate that NER factors are mobilized seconds after cellular exposure to U V (Mone et al., 2004). Since no regulatory mechanism has been described for any ING protein, we investigated whether the p 3 3 I N G l b protein would be subject to posttranslational modifications upon genotoxic stress. The identification of phospho-residues within a protein has always been a challenging task. Traditionally, studies followed the archetypal steps: 1) Radiolabeling of the protein of interest; 2) Enzymatic or chemical hydrolysis; 3) Separation by HPLC, TLC and/or electrophoresis; and 4) Identification of the phospho-amino acid through Edman sequencing or mass spectrometry. This time-consuming and demanding process is being replaced by more direct and non-radioactive enrichment techniques such IMAC phospho-peptide isolation followed by mass spectrometry. Here we demonstrate the application of a chemical procedure that uses the difference in survey modes when doing MALDI-TOF mass spectrometry, therefore allowing direct analysis of peptides without the need for phospho-peptide enrichment. Using this technique we have identified a phospho-amino acid at position 126 of the pSS^ 0 1 1" tumour suppressor (Figure 5-3). By low-bis acrylamide SDS-PAGE, one of the first observations that can be made is the striking net accumulation of p 3 3 I N G l b protein upon treatment with topoisomerase inhibitors and the overall reduction when cells are treated with kinase inhibitors (Figure 5-1). A T M , A T R and D N A - P K largely influence cellular responses to genotoxic stress 99 rendering p 3 3 I N G l b a likely target of DNA-damage responsive PIKKs. Both Chkl and Cdkl are downstream of PIKKs and can phosphorylate serine 126 of p33 I N G l b . It is therefore not surprising that the reduction of phosphorylated p 3 3 r N G l b caused by wortmannin was limited compared to staurosporine (Figure 5-1). In the absence of genotoxic stress Chkl would not get activated by A T M / A T R and PIKK inhibition would therefore have a limited effect. However, it is possible that other P I K K phospho-acceptor residues exist and could account for the reduction of phospho-p33 I N G l b observed upon wortmannin treatment. In fact, the prediction algorithm NetPhosK 1.0 implies high likelihood of A T M and D N A - P K phosphorylation on multiple serines. Staurosporine is recognized as a highly potent albeit non-selective inhibitor of P K C with reported IC50 values as low as 3 n M in cultured cells (Vegesna et al., 1988). However, it has been recently reported to inhibit both Chkl and Cdkl within a comparable range (Zhao et al., 2002), thus explaining the drastic reduction of phosphorylated pSS1™0113 as well as overall reduction of protein levels that we observed upon treatment with staurosporine (Figure 5-1). Although serine 126 of p 3 3 I N G l b encompassed well the P K C consensus X-S /T-X-R/K recognition motif, immunoprecipitated PKC failed to phosphorylate serine 126 but did radiolabel a validated P K C substrate in vitro. However, P K C is also predicted to phosphorylate serine 190 and cannot yet be ruled out as a potential p 3 3 I N G l b kinase. Although serine 126 does not alter sub-cellular localization of the p 3 3 I N G l b protein it is noteworthy to mention that serine 126 was found mutated in one melanoma patient (see previous chapter) further supporting the importance of this residue. Furthermore serine 126 is highly conserved in human ING proteins and is the last residue of the 1 0 0 undefined but highly conserved 'potential chromatin remodeling' domain. Like the PHD domain, the PCR domain is present and defines all ING proteins of all organisms. It would be interesting to investigate whether this residue can also be phosphorylated and if it can also alter the turnover rate of other ING proteins. Site-directed mutagenesis of serine to alanine prevents phosphorylation. In p33 I N G l b , S126A substitution results in a near 3-fold decrease in the half-life of the protein (Figure 5-7) confirming that the protein level changes after treatment with kinase inhibitors observed on low-bis gels are largely due to protein stability (Figure 5-1). This not likely a result of protein misfolding due to the SI26A substitution since the use of a Cdkl inhibitor also drastically shortens the half-life of wild-type p 3 3 I N G l b (Figure 5-8) and also because the use of topoisomerase inhibitors and kinase inhibitors seem to have opposite effects on overall wilt-type p 3 3 I N G l b protein levels (Figure 5-1). Unlike p53, the degradation of p 3 3 I N G l b does not seem to occur through the proteasome. Although p33 I N G l b can be ubiquitinated our experiments could not discriminate between mono-ubiquitination, a regulatory mechanism independent of degradation, and poly-ubiquitination, which often leads to proteasomal but also lysosomal degradation. There also seems to be a natural equilibrium between non-phosphorylated and phosphorylated p33 I N G l b (regardless of whether it is endogenous or exogenous) in asynchronous cells (Figure 5-1). It would be a great interest to verify whether serine 126 phosphorylation levels correlate with Cdkl-Cyclin B activity in synchronized cells. However, upon genotoxic stress, p 3 3 I N G l b is quickly mobilized to prolong its turnover rate as our phospho-specific antibody showed that p 3 3 I N G l b is phosphorylated on serine 126 within a 1 0 1 minute after U V R (Figure 5-4). The delay in serine 126 phosphorylation upon doxorubicin treatment is likely due to the lag in which the drug incorporates into the cell nucleus and affects cellular functions. Thus, stabilized p 3 3 I N G l b phosphorylated on serine 126 is likely more apt to mediate cellular stress responses such as cell cycle arrest and D N A repair. The initial finding that both Chkl and Cdkl can phosphorylate p 3 3 1 N G l b therefore presented a paradox. On one hand Chkl is a direct PIKK target, thus leading to the phosphorylation of p 3 3 I N G l b but also to the inhibition of Cdc25, the phosphatase that activates Cdkl-cyclin B , required for mitotic entry. However, this very same Cdkl protein which promotes cell division also phosphorylates pSS^ 0 1 1". Closer investigation revealed that while Cdkl mainly phosphorylates serine 126 under normal conditions it is not responsible for the phosphorylation of serine 126 when cells are exposed to genotoxic stress (Figure 5-8). This is likely since Chkl is activated upon D N A damage events and activated Chkl inhibits Cdkl activity in the cells. On the contrary, in the absence of genotoxic stress, inactive Chkl does not phosphorylate pSS™0 1 1 3. However, genotoxic stress triggers the A T M / A T R signaling pathway and activated Chkl can thus phosphorylate p33 I N G l b . Using the anti-p-S126 antibody, we observed attenuated phosphorylation in vivo caused by the Cdkl inhibitor kenpaullone in the absence of D N A damage and by the broad-range Chkl inhibitor staurosporine in the presence of doxorubicin (Figure 5-8), further confirming the duality of the phospho-residue. However again, the use of the Cdkl inhibitor roscovitin greatly reduced the half-life of wild-type pSS™0"3. Our results therefore suggest that p 3 3 I N G I b is a downstream 102 component in the A T M / A T R signaling pathways in response to genotoxic events but that p 3 3 I N G l b is also be regulated under normal conditions through the cell cycle. A past study demonstrated that CCNB1, which expresses cyclin B I , is transcriptionally down-regulated by p 3 3 I N G l b (Takahashi et al, 2002). This report, together with our finding that Cdkl can phosphorylate p33 I N G l b , suggest the existence of a potential negative feedback loop where upon mitotic onset Cdkl-cyclin BI phosphorylate serine 126, leading to p 3 3 I N G l b accumulation and down-regulation of CCNB1 (Figure 5-9). It would therefore be interesting to further investigate this potential negative regulation between Cdkl-Cyclin BI and p 3 3 I N G l b through the cell cycle in synchronous cells. However, although this idea remains to be tested, it may be supported by a few observations. Overexpression of p33 1 N G l b can enhance doxorubicin-induced G 2 arrest in p53-null H1299 cells (Tsang et al, 2003), while CCNB1 is repressed in cells that are arrested in G 2 in response to genotoxic stress (Manni et al, 2001). Furthermore, p ^ i N G i b c o n t a m s a potential cyclin-dependent kinase binding (Cy) motif (a.a. 76-79). This motif can act as a substrate recognition site that directly interacts with cyclin to promote phosphorylation by the Cdk-cyclin complex (Takeda et al, 2001) and may help Cdkl-cyclin BI phosphorylate p33 I N G l b . Taken together, our data suggests that p33 1 N G l b potentially participates in the regulation of mitotic cyclin BI and is itself regulated by the Cdkl-cyclin BI complex. Upon genotoxic stress however, A T M / A T R activate Chkl , which inhibits cdc25 and Cdk l , but also prolongs pSS™ 0 1 1 5 activity necessary for cell cycle arrest and repair of D N A damage (Figure 5-9). 103 5.4 Figures Figure 5-1 Posttranslational modifications of the p 3 3 1 N G l b protein. (A) Low-bis SDS-PAGE analysis of FLAG-tagged pSS™01* in M M R U cells 24 h following exposure to 0.5 ug/ml or 1 ug/ml doxorubicin and 0.25 ug/ml or 0.5 ug/ml etoposide topoisomerase inhibitors, (B) 0.1 m M okadaic acid phosphatase inhibitor or 100 m M wortmannin or 50 nM staurosporine kinase inhibitors. A + + Doxorubic in + + Etopos ide F L A G P-actin B + Staurosporine - Wortmannin - O k a d a i c A c i d F L A G p-actin 104 Figure 5-2 P 3 3 I N G l b is subject to phosphorylation in vivo and in vitro. (A) F L A G epitope was used to immunoprecipitate pSS11"4011' from M M R U cells expressing F L A G -p33 I N G l b and treated with 1 ug/ml doxorubicin and mock-treated (left lane) or treated with calf intestinal alkaline phosphatase (CIP) (right lane). Immunoprecipitates were resolved by SDS-PAGE and subjected to immunoblotting using antibodies specifically raised against phospho-serine residues or against pSS™0"3. (D) Autoradiogram of [ 3 2P]-metabolic labeling of GST-p33 I N G l b recombinant protein exposed to doxorubicin-treated M M R U cell extracts. A + CIP Phospho-serine p33 INGlb B - + + GST -p33 I N G 1 b + - + [ 3 2P]ATP 105 ,INGlb Figure 5-3 Mass spectrometry analysis of AspN-digested recombinant GST-p33' The p 3 3 I N G l b protein was exposed to cell extracts of untreated (A-B) or doxorubicin-treated (C-D) M M R U cells prior to digestion. The peptide DT AGNS G K A G A corresponding to amino acids 121-131 has a predicted mass of 947 in a non-phosphorylated form (black arrows) and of 1046 in its phosphorylated form (grey arrows). (B-D) CIP-treatment induced de-phosphorylation of serine 126. (D) The 1044.8 m/z peak was reduced to 964 m/z due to the loss of a 80 HPO3 group (black arrows). 100 90 80 £ > 70 C 60 <D "+-» £ 50 * 40 30 20 800 946.1 + H 1045.0+ H B 100, 90! 80 •«) C 60 0) 4-* £ 50 946.1 + H 880 960 1040 Mass (m/z) 1120 1200 880 960 1040 1120 1200 Mass (m/z) 880 960 1040 1120 Mass (m/z) 960 1040 1120 1200 Mass (m/z) 106 Figure 5-4 Generation of phospho-serine 126 antisera and D N A damage-induced phosphorylation of endogenous p33 I N G l b . (A) Antibodies were generated and affinity purified to specifically recognize phosphorylated serine 126. ELISA of immobilized phosphorylated (black line) or non-phosphorylated (grey line) synthetic peptide containing serine 126 of p 3 3 I N G l b incubated with increasing dilutions of the polyclonal rabbit anti p-S126 antiserum. (B) Western analysis of bacteria-produced recombinant p33 I N G l b and in vitro phosphorylated p 3 3 I N G l b using crude cell extracts of untreated or doxorubicin-treated cells. (C-D) SDS-PAGE analysis of endogenous phosphorylation of serine 126 following exposure to combined 20 ug/ml cycloheximide and 20 mJ/cm U V B (C) or 1 ug/ml doxorubicin (D). (E) SDS-PAGE analysis of endogenous phosphorylation of serine 126 upon treatment with cycloheximide alone. 2 . 5 2 . 0 1 .5 1 .0 0 . 5 # # # # # £ / ^  / / / Ab Dilution 0 1 5 15 30 60 min P-Ser126 p-actin B - - + Doxorubicin - + + Cell Extract + + + p33 I N G 1 b P-Ser126 p 3 3 I N G 1 b D 0 1 5 15 30 60 180 min P-Ser126 P-actin 0 1 5 15 30 60 180 min P-Ser126 —m~m.m*—*mMmmm+~— mm p-actin 107 Figure 5-5 Site-directed mutagenesis of serine 126 to abolish phosphorylation. Panel below confirms the site-directed mutagenesis of serine 126 to alanine of the pCIneo-INGJb-FLAG plasmid by D N A sequencing. Asn Ser G l y R f l C f l G C G G C Asn Ala G l y flflCGCRGG G 108 Figure 5-6 Phosphorylation of serine 126 does not affect protein sub-cellular localization. M M R U cells were transfected with pCmeo-/7VG/6-FLAG, or pCIneo-7M/76-S126A-FLAG, and protein sub-cellular localization was examined by immunofluorescent staining with an anti-FLAG antibody. FLAG Hoescht Merged 109 Figure 5-7 Abolishment of serine 126 phosphorylation shortens the half-life of p33 I N G l b protein. (A) M M R U cells were transfected with pCIneo-/7VG/&-FLAG or pCIneo-/A rG76-S126A-FLAG and treated 24 h later with 20 ug/ml cycloheximide to prevent de novo protein synthesis. Cells were harvested at various time points after the addition of cycloheximide to the media and cell lysates were subjected to western blot analysis of wild-type or S126A mutant p 3 3 1 N G l b protein levels. (B) Densitometric analysis of p 3 3 I N G l b protein levels upon correction for total protein loading. Wild-type p 3 3 i N G i b w a g found t Q h a y e a h a i f . l i f e o f 1 6 g h compared to 5.7 h for the S126A mutant. Values are based on three independent experiments. R = correlation coefficient. wt S126A 0 1 3 6 12 24 0 1 3 6 12 24 h FLAG p53 [3-actin B 5.0 <= 3.0 2.5 y=-0.0413x +4.605 R=0.9663 I y=-0.1209x +4.605 R=0.9788 10 Time (h) 15~ 20 25 110 Figure 5-8 Serine 126 is phosphorylated by Chkl upon genotoxic stress but by Cdkl under normal conditions. (A) KinaseProfiler™ kinase assay using a peptide corresponding to amino acids 118-134 of the p 3 3 I N G l b protein as the substrate. Results are expressed as percentage over a control reaction containing 30% phosphoric acid and based on three independent reactions. (B-C) Kinase assay of immunoprecipitated Cdkl (B) and Chkl (C) using a p 3 3 r N G l b peptide corresponding to amino acids 119-135 as substrate. Both kinases were isolated from untreated or M M R U cells exposed to 1 ng/ml doxorubicin for 24 h or 30 min following 20 mJ/cm2 U V B . X-axis corresponds to the reaction time. (D) Immunoprecipitated Cdkl and Chkl from untreated, U V - or doxorubicin- treated M M R U cells used in (B) and (C). (E) Western analysis of endogenous serine 126 phosphorylation in M M R U cells exposed to 1 ug/ml doxorubicin for 24 h in the presence of the kinase inhibitors kenpaullone and staurosporine. (F) Western analysis of the endogenous serine 126 phophorylation levels in nuclear extracts from M M R U cells exposed to 1 ug/ml doxorubicin for 24 h and transfected with A T M siRNA. (G) Western analysis of the effect of the Cdkl inhibitor roscovitin on wild-type P 3 3 I N G l b half-life. I l l ~ 35 B 14 12 CO 10 o * 8 I. 8 0 4 2 0 14 12 to 10 o * 8 1 8 ° 4 CaMKII Cdkl- Chkl CK2 GSK3a Cyclin B OiUV-B • Doxorubicin D Untreated 30 S<jUV-B • Doxorubicin [] Untreated 90 ML. 1 120 min 30 G + - Doxorubicin - + UV Cdkl Chkl + + + + Doxorubicin - Kenpaullone + Staurosporine P-Ser126 p-actin + Doxorubicin + ATMsiRNA P-Ser126 Ponceau 0 1 3 6 12 24 h FLAG p-actin 112 Figure 5-9 Proposed model of p 3 3 I N G l b regulation through serine 126 phosphorylation under normal conditions (A) and following genotoxic stress (B). Under normal cell cycle progression, Chkl is inactive during G 2 phase, thus allowing Cdc25 de-phosphorylation and activation of C d k l . In a negative feedback loop, the Cdkl-cyclin B complex phosphorylates and stabilizes p33 I N G l b , which in turn down-regulates CCNB1 and contributes to the inactivation of Cdk l . Upon genotoxic stress A T M / A T R activate Chk l , which then inhibits Cdc25 leading to Cdkl phosphorylation and inactivation. Activated Chkl further phosphorylates pSS1™0"5 on serine 126 to prolong its half-life, which in turn contributes to the activation of the p53 tumour suppressor and enhances the repair of D N A damage. A B ATM .) (D / 113 Figure 5-10 Proteasome-independent degradation of p33 I N G l b . (A) Levels of S126A p33 I N G l b were followed at various points in time in cycloheximide-treated M M R U cells as previously described. (B) The levels of S126A p 3 3 I N G l b remained unchanged in cycloheximide and proteasome inhibitor MG132-treated cells compared to cells treated with cycloheximide alone, suggesting that p33 1 N G l b is not degraded by the proteasome. However, the p53 protein which is known to be degraded by the proteasome was stabilized by the proteasome inhibitor MG132. (C) Like p53, p 3 3 1 N G l b did incorporate ubiquitin moities but levels of ubiquitinated p 3 3 I N G ! b did not increase upon inhibition of the proteasome. M M R U cells were co-transfected with either empty vector or plasmids encoding wild-type or S126A p 3 3 I N G l b and a plasmid encoding His-tagged ubiquitin. Proteins that incorporated His-Ub were affinity purified using N i - N T A beads and resolved by SDS-PAGE. Levels of ubiquitinated p53 increased upon treatment with the proteasome inhibitor MG132 but decreased upon cycloheximide treatment. However, there was no accumulation of wild-type or S126A FLAG-tagged pSS™ 0" 3 upon inhibition of the proteasome, suggesting that p 3 3 I N G l b can be ubiquitinated but not degraded by the proteasome. This assay does not discriminate between mono- and poly-ubiquitination. 114 A B 0 1 3 6 12 24 hours 0 1 3 6 12 24 hours F L A G F L A G p53 p53 3-actin [3-actin c Vector Wt S126A #' if y # # > # # y & & . a _ , — p53 mm FLAG Table 5-1 NetPhosK 1.0 kinase prediction for p 3 3 I N G l b serine 126. Kinase Score Cdkl 0.50 CaM-II 0.44 GSK3 0.44 CKI 0.36 DNA-PK 0.35 CKII 0.34 P K G 0.32 A T M 0.31 p38MAPK 0.27 P K A 0.26 PKC 0.26 RSK 0.23 Cdk5 0.15 PKB 0.08 116 5.5 References Bakkenist C.J. and Kastan M . B . Initiating cellular stress responses. 2004. Cell 118:9-17. Blom N . , Sicheritz-Ponten T., Gupta R., Gammeltoft S. and Brunak S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. 2004. Proteomics 4:1633-49. Campos E.I., Chin M . Y . , Kuo W.H. and L i G. Biological functions of the ING family tumour suppressors. 2004. Cell Mol Life Sci 61:2597-613. Cheung K.J . Jr, Mitchell D., Lin P. and L i G. The tumour suppressor candidate p33(INGl) mediates repair of UV-damaged DNA. 2001. Cancer Res 61:4974-7. Feng X . , Hara Y . and Riabowol K . Different HATS of the I N G l gene family. 2002. Trends Cell Biol 12:532-8. Hirao A. , Kong Y . Y . , Matsuoka S., Wakeham A. , Ruland J., Yoshida H. , Liu D., Elledge S.J. and Mak T.W. D N A damage-induced activation of p53 by the checkpoint kinase Chk2. 2000. Science 287:1824-7. Hoch J.A. Two-component and phosphorelay signal transduction. 2000. Curr Opin Microbiol 3:165-70. Ito A. , Kawaguchi Y . , Lai C.H., Kovacs J.J., Higashimoto Y . , Appella E. and Yao T P . MDM2-HDAC1-mediated deacetylation of p53 is required for its degradation. 2002. Embo J 21:6236-45. Kataoka H. , Bonnefin P., Vieyra D., Feng X. , Hara Y . , Miura Y . , Joh T., Nakabayashi H., Vaziri H. , Harris C C . and Riabowol K. INGl represses transcription by direct D N A binding and through effects on p53. 2003. Cancer Res 63:5785-92. Manni I., Mazzaro G., Gurtner A. , Mantovani R., Haugwitz U . , Krause K. , Engeland K. , Sacchi A. , Soddu S. and Piaggio G. N F - Y mediates the transcriptional inhibition of the cyclin B l , cyclin B2, and cdc25C promoters upon induced G2 arrest. 2001. J Biol Chem 276:5570-6. Mone M.J. , Bernas T., Dinant C , Goedvree F.A., Manders E .M. , Volker M . , Houtsmuller A.B. , Hoeijmakers J.H., Vermeulen W. and van Driel R. In vivo dynamics of chromatin-associated complex formation in mammalian nucleotide excision repair. 2004. Proc Natl Acad Sci U S A 101:15933-7. 117 Nakamura S., Roth J.A. and Mukhopadhyay T. Multiple lysine mutations in the C-terminal domain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. 2000. Mol Cell Biol 20:9391-8. Shieh S.Y., Ahn J., Tamai K. , Taya Y . and Prives C. The human homologs of checkpoint kinases Chkl and Cdsl (Chk2) phosphorylate p53 at multiple D N A damage-inducible sites. 2000. Genes Dev 14:289-300. Takahashi M . , Seki N . , Ozaki T., Kato M . , Kuno T., Nakagawa T., Watanabe K. , Miyazaki K. , Ohira M . , Hayashi S., Hosoda M . , Tokita H. , Mizuguchi H. , Hayakawa T., Todo S. and Nakagawara A. Identification of the p33(INGl)-regulated genes that include cyclin B l and proto-oncogene D E K by using cDNA microarray in a mouse mammary epithelial cell line N M u M G . 2002. Cancer Res 62:2203-9. Takeda D.Y. , Wohlschlegel J.A. and Dutta A. A bipartite substrate recognition motif for cyclin-dependent kinases. 2001. J Biol Chem 276:1993-7. Tsang F.C., Po L.S., Leung K . M . , Lau A. , Siu W.Y. and Poon R.Y. INGlb decreases cell proliferation through p53-dependent and -independent mechanisms. 2003. FEBS Lett 553:277-85. Vegesna R.V., Wu H.L., Mong S. and Crooke S.T. Staurosporine inhibits protein kinase C and prevents phorbol ester-mediated leukotriene D4 receptor desensitization in RBL-1 cells. 1988. Mol Pharmacol 33:537-42. Xu C.F., Lu Y . , Ma J., Mohammadi M . , and Neubert T.A. Identification of phosphopeptides by M A L D I Q-TOF MS in positive and negative ion modes after methyl esterification. 2005 Mol Cell Proteomics 4:809-18. Zhao B., Bower M.J. , McDevitt P.J., Zhao FL, Davis ST. , Johanson K.O. , Green S.M., Concha N.O. and Zhou B.B. Structural basis for Chkl inhibition by UCN-01. 2002. J Biol Chem 277:46609-15. 118 6 CONCLUDING R E M A R K S The ING family of acetyltransferase and deacetylase co-factors, are increasingly capturing the attention of the scientific community. Various lines of evidence suggest that these proteins enable H A T and H D A C activity onto nucleosomal histones, crucial events to chromatin remodeling leading to the regulation of transcription, repair, cell cycle and apoptosis. The founding member, the p 3 3 I N G l b protein is no exception. This is an exceptional protein that demonstrates versatility as it can associate with both the p300 HAT and the Sin3 H D A C complex. PSS™0115 further plays a critical role in the p53 pathway. This work described the development of a critical tool to the study of the p33 I N G l b protein. Antibodies are used to detect, label and purify specific targets; technicalities that would prove difficult without their use. The development of polyclonal antisera specific for the p33 1 N G l b protein was the first step in a series of experiments that describe its role in normal and diseased conditions. Human malignant melanoma can become a devastating disease i f it is not detected in its early stage soon after development. This disease is also increasingly found in young adults. Unfortunately melanoma tumours tend to display highly heterogeneous phenotypes and, in many cases, are also chemo-refractory. Hence efficient treatments need to be developed to treat this disease. For this reason it is important to fully understand how and why melanoma arises and identify the cellular components that are involved. It is currently believed that melanocytes can undergo neoplastic transformation upon the inactivation of pathways that activate the retinoblastoma protein and the 119 activation M A P K and/or disruption of MAPK-interacting proteins. Although a high proportion of melanoma cases were reported to harbor constitutively active B-Raf or N -Ras a major target leading to Rb inactivation has yet to be identified. In contract to most human cancers, the most obvious candidate, the p53 tumour suppressor protein is rarely inactivated in human melanoma. For this reason this thesis investigates the status of the INGl gene in human melanoma. After all pSS™0 1 1 5 physically cooperates with p53 and through up-regulation of p21 W a f l , contributes to the activation of the Rb cell cycle regulator. In this study two major findings are discussed. First pSS™ 0 1 1 5 is often located in an altered sub-cellular compartment in melanoma cells. Since pSS^ 0 1 1 1 associated with HATs and HDACs to activate and repress nucleosomal DNA, this could surely contribute to the inactivation of this tumour suppressor. This phenomenon is not however restricted to melanoma as it is also reported in various human malignancies. The factors contributing to this phenomenon remain however obscure and would definitely be of interest to the understanding of cancer biology. The second observation is that INGl is mutated in nearly a fifth of the melanoma biopsies studied. Two mutational 'hot spots' were further found to be detrimental to the D N A repair functions associated with the p33 I N G l b protein. Of particular interest, half of the melanoma-stricken individuals that did harbor INGl mutations seemed much more susceptible to the disease than those with the wild-type counterpart. It would be interesting, albeit laborious to obtain a large number of biopsies to statistically confirm this observation. In the later part of the thesis p 3 3 I N G , b was found to be a phospho-protein and serine 126 phosphorylation responsive to genotoxic stress. This is the first critical observation, 120 which describes a regulatory mechanism for the pSS^ 0 1 1 1 protein. Although serine 126 phosphorylation increases upon cellular exposure to U V or doxorubicin, likely through Chkl , further studies are needed, and are currently underway, to help understand the role of serine 126 phosphorylation by Cdkl-cyclin B. The observations presented in this thesis are based on asynchronous cells and we are currently pursuing this study in synchronized cells through cell cycle progression. Since p 3 3 I N G l b is reported to repress cyclin BI expression, chapter 5 describes a potential model in which at the G2-M phase boundary, upon activation of Cdkl by cyclin BI the Cdkl-cyclin BI complex promotes p33 I N G l b activity by phosphorylating serine 126 and therefore prolonging the protein half-life. This may therefore translate in a negative feedback loop through which p 3 3 r N G l b may contribute to destabilize the Cdkl-cyclin BI complex for further entry into G\/GQ. Alternatively, but not necessarily mutually exclusively, phosphorylated p 3 3 I N G l b may also contribute to the chromatin remodeling events required for mitosis i f the half-life of the protein is increased at the G2/M boundary. Regardless of the outcome, future results on this matter will prove invaluable to the understanding of serine 126 phosphorylation and to the role of p 3 3 I N G l b in the cell cycle. It is also interesting to note that mass spectrometric analysis of p 3 3 I N G l b detected potential acetylated residues, giving plenty to study in the future. In closing, this work significantly contributes to the knowledge and understanding of the p 3 2 I N G l b protein, particularly in melanoma. The presented results are exciting and promising, but further raise important questions and new venues that further need to be explored. There are now over 110 hits on an 'ING1' search on the PubMed search 121 engine where there were less than 25 publications on the protein when I first started this work. Histone acetylation is essential to cellular viability and is thought to be a basic and fundamental chromatin regulatory mechanism. Future studies on p 3 3 I N G l b and other ING proteins will surely prove fantastically complex but at the time promising and exciting. 

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