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Expression profile and molecular functions of the tumor suppressor p33ING1 Cheung Jr., K-John J. 2003

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EXPRESSION PROFILE AND MOLECULAR FUNCTIONS OF THE TUMOR SUPPRESSOR  p33  ING1  by  K-JOHN J . C H E U N G J R . B.A., Simon Fraser University, 1994 M . S c , Simon Fraser University, 1999  A T H E S I S SUBMITTED IN PARTIAL F U L F I L L M E N T O F THE REQUIREMENTS OF THE D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Department of Medicine; Experimental Medicine Program) W e accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A January, 2003 © K-John J . Cheung Jr., 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT  The biological functions of the tumor suppressor gene, ING1, have been studied extensively in the last few years since it was cloned. Four alternatively spliced forms of ING1, named p47 ,  p33 ,  ING1  p27 ,  ING1  ING1  and p24 , ING1  have been identified and  found to share many biological functions with those of p53. Some of these isoforms have previously been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity.  Functions, such as cell cycle  arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins.  In this thesis, we sought to characterize further a number of  important biological functions of the p33  ING1  expression of ING1  isoform. W e first investigated how the  is regulated in normal and stress conditions.  Using a p53-  knockout mouse model and various cell lines differing in p53 status and cell type, we found that the expression of p33  ING1  is independent of p53 status and induced by  ultraviolet (UV) irradiation in a dose-/time-dependent and tissue-specific manner. These findings subsequently prompted us to investigate if p33  ING1  plays a role in UV-  stress response, such as repair of UV-damaged DNA. Using both in vitro (host-cellreactivation assay) and in vivo (radioimmunoassay) methods, we found that  p33  ING1  enhances the repair of UV-damaged DNA in collaboration with p53 in melanoma cells and that G A D D 4 5 may participate in the process. Next we investigated the molecular pathways of p33  ING1  enhancement in UV-induced apoptosis in melanoma  cells using various survival and apoptotic overexpression of p33  ING1  assays.  W e demonstrated  that  increases the apoptotic rate in melanoma cells after UV  ii  irradiation and that p53 has a synergistic effect on this process. Moreover, we found that p33  ING1  enhances the expression of endogenous Bax and alters mitochondrial  membrane potential, suggesting that p33  ING1  cooperates with p53 in UV-induced  apoptosis via the mitochondrial cell death pathway in melanoma cells. examined the role of p33  ING1  previous findings  Lastly, we  isoform in melanoma chemosensitivity because  indicate that the  isoform  chemosensitivity in human fibroblasts.  p24  is capable of enhancing  ING1  Using a number of survival and apoptotic  assays to quantitate  cell death in melanoma cells, we showed that neither  overexpressing p33  alone nor coexpression of p33  ING1  ING1  and p53 had an effect on  the frequency of cell death induced by the chemodrug, camptothecin. W e therefore demonstrated that p33  ING1  does not enhance camptothecin-induced cell death in  melanoma cells. In conclusion, we have elucidated in this thesis some of the novel functions of p33  ING1  and the importance of this gene in the context of tumor  suppression.  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iv  List of Tables  vi  List of Figures  vii  List of Abbreviations  ix  Acknowledgements  x  Chapter 1 INTRODUCTION  1  1.1  1 1 4 5 5 7 8 10 11 11 15 20 21 25 29  1.2  1.3  Cancer 1.1.1 Skin cancer Tumor suppressor genes 1.2.1 p53 1.2.1.1 Structure and function 1.2.1.2 Cell cycle arrest 1.2.1.3 Apoposis 1.2.1.4 DNA repair 1.2.2 ING1 1.2.2.1 Isolation and characterization 1.2.2.2 Tumor suppressive functions 1.2.2.3 Expression profile 1.2.2.4 The ING1 family 1.2.2.5 Expression and mutational analyses in tumors General hypothesis and objectives  Chapters 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10  MATERIALS AND METHODS  Animals Cell lines and cell culture Plasmids Antibodies Transfection Determination of transfection efficiency U V B irradiation Light microscopy Western blot analysis Trypan blue exclusion assay  iv  31 31 31 32 32 33 33 33 36 36 36  2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20  S R B cell survival assay Reverse transcriptase-polymerase chain reaction (RT-PCR) Northern blot analysis Immunohistochemistry Host-cell-reactivation assay Radioimmunoassay Immunoprecipitation Propium iodine (PI) staining Flow cytometry Mitochondrial transmembrane potential detection  Chapter 3  E X P R E S S I O N O F p33  ING1  IS I N D E P E N D E N T O F p53  37 37 38 39 39 40 41 42 42 42 44  3.1  Rationale and hypothesis  44  3.2  Results and discussion  47  Chapter 4  p33  MEDIATES R E P A I R O F U V - D A M A G E D DNA  56  4.1 Rationale and hypothesis 4.2 Results and discussion Chapter 5 p33 E N H A N C E S UVB-INDUCED A P O P T O S I S IN MELANOMA CELLS  56 59 68  5.1 5.2  68 70  ING1  ING1  Rationale and hypothesis Results and discussion  Chapter 6  p33 D O E S NOT E N H A N C E C A M P T O T H E C I N INDUCED C E L L D E A T H IN M E L A N O M A C E L L S ING1  87  6.1  Rationale and hypothesis  87  6.2  Results and discussion  89  Chaper7  CONCLUSIONS  98  7.1  Summary  98  7.2  Future directions  100  REFERENCES  103  A P P E N D I X I C U R R I C U L U M VITAE OF A U T H O R  123  v  LIST OF TABLES  Table 1.1  Relative percentages of transfection efficiencies of cell lines used in the study  vi  35  LIST OF FIGURES  Figure 1.1  Genomic structure of human ING1 and its alternatively spliced m R N A variants, p47 , ING1  Figure 1.2  p33 , ING1  p27 , ING1  13 p24  and  ING1  Predicted amino acid sequences of the four ING1 isoforms, p47 , ING1  p33 ,  p27 ,  ING1  and p24  ING1  14  based on c D N A  ING1  sequences from GenBank Figure 2.1  Visual assessment of transfection efficiency  Figure 3.1  Analyses of p33 mouse organs  Figure 3.2  Western blot analysis of p 3 3 * expression levels in +/+ selected p53 and p53' mouse organs.  49  Figure 3.3  Immunohistochemical analysis of p33 p53 + / + and p53v" mice  51  Figure 3.4  p33  Figure 3.5  Western blot analysis of p33 mouse fibroblasts  Figure 4.1  p33  ING1  is UV-inducible in a dose- and time-dependent manner  60  Figure 4.2  p33  ING1  enhances UV-damaged DNA repair  61  Figure 4.3  p33  -mediated DNA repair is p53-dependent  63  Figure 4.4  ING1 physically interacts with G A D D 4 5 , but does not transcriptionally upregulates G A D D 4 5 , X P A , o r X P B  65  Figure 5.1  Effect of p33  71  Figure 5.2  Synergistic effect of p33 death in M M R U cells  Figure 5.3  Effect of p33  Figure 5.4  p33 alters mitochrondrial membrane potential and increases Bax expression  ING1  34  m R N A expression of p53  +/+  and p53'  A  /WG  48  A  ING1  ING1  induction by UVB irradiation in keratinocytes ING1  ING1  in the brain of  ING1  on UVB-induced cell death in M M R U cells and p53 on UVB-induced cell  ING1  ING1  expression in UV-irradiated  on UVB-induced cell death in M E W O cell line  ING1  vii  52 54  76  79 82  Figure 6.1  Survival rate of CPT-treated R P E P cells transfected with p33  ING1  and antisense  90  p33  ING1  Figure 6.2  Microscopic images of CPT-treated R P E P transfectants  92  Figure 6.3  Quantitation of cell death by flow cytometry  93  Figure 6.4  Effect of p33  94  Figure 6.5  Effect of p33 and p53 co-expression on melanoma chemosensitivity  ING1  on M M R U cell survival after C P T treatment  ING1  viii  96  LIST OF ABBREVIATIONS  aa AML Apaf-1 BCC BER CAN CAT Cdk CPD CPT DEK DMEM ESCC FBS FISH GSE HAT HNSCC HRP IAP ING1  LOH MDM2  NER NF-kB NHEK OSCC PBS PCNA PI PIG PUMA PVDF Rb RNA I RT-PCR  sec SMAC SRB SSCP TBP UV WT  Amino acid Acute myelogenous leukemia Apoptotic protease activating factor-1 Basal cell carcinoma Base excision repair Cain (Cancer intron on nine) Chloramphenicol acetyltransferase Cyclin-dependent kinase cyclobutane pyrimidine dimers Camptothecin D E K oncogene (DNA binding) Dulbecco's modified eagle's medium Esophageal squamous cell cancer Fetal bovine serum Fluorescent in situ hybridization Genetic suppressor elements Histone acetytransferase Head and neck squamous cell carcinomas Horseradish peroxidase Inhibitors of apoptosis Inhibitor of growth 1 Loss of heterozygosity Mouse double minute 2 Nucleotide excision repair Nuclear factor kappa B Normal human epithelial keratinocytes Oral squamous cell carcinoma Phosphate buffered saline Proliferating cell nuclear antigen Propium iodine p53-induced genes p53-upregulated modulator of apoptosis Polyvinylidene difluoride Retinoblastoma R N A interference Reverse transcriptase-polymerase chain reaction Squamous cell carcinoma Second mitochondria-derived activator of caspase Sulforhodamine B Single-stranded conformation polymorphism T A T A box-binding protein Ultraviolet Wild-type  ix  ACKNOWLEDGEMENTS  I am truly indebted to my senior supervisor, Dr. Gang Li, because he always welcomed me into his lab, taught me practical science, and always thinks so highly of me. I hope that my diligence, productivity, and scientific contributions to your lab helped pay off my debt to you. I thank Dr. Vincent Ho for supporting me financially during my early period in the lab and for being a member of my committee.  I am  thankful to Dr. William Jia for his valuable collaboration in our research projects and unconditional scientific advice to me and other lab members.  I thank Dr. Lewei  Zhang for participating in my studies as a committee member and for being both understanding and accepting of my career decisions. You are a very kind person and I hope I will not disappoint you. I would also want to express my thanks to Dr. Chris Ong for his invaluable scientific advice in our knockout project and my research thesis, as well as being a member on my committee.  I want to sincerely  express my gratitude to my parents for providing opportunities for me to turn my life around, for their generous financial assistance during these years of studies, and most importantly for raising me.  I thank my brother, K-Johnson, for being my  brother, a trustworthy friend, and for having the same sense of humor. Without him I will have no one to laugh with. Although the future will always be a mystery, the past is known. I am very grateful to Cindy who has done so much for me in the past, who has endured the rough times with me, and who still remains to be my true friend. I am glad to have met you always.  Lastly, I would like to express my appreciation to  the Canadian Institutes of Health Research, the Canadian Dermatology Foundation,  x  and the Roman M. Babicki Fellowship, for their backbone support to my research. Missing any of the people or organizations mentioned here, my studies in Dr. Gang Li's Lab would not have materalized. you my life-long motto: "De Oppresso  I thank you once again and here share with Liber", from the U.S. Army Special Forces.  xi  CHAPTER 1. INTRODUCTION  1.1  Cancer  An estimated 136,900 new cases of cancer and 66,200 deaths from cancer will occur in Canada by the end of 2002.  Based on current incidence rates, 38% of  women and 4 1 % men will develop cancer during their lifetimes. Cancer is now the second leading cause of death in Canada and based on the prevalent trends, its incidence is expected to grow to 70% by the year 2015 (http://www.cancer.ca/).  1.1.1  Skin Cancer  Cancer of the skin is the most common among all types of cancers. Specifically, it accounts for over 40% of all human cancers (Boni et al., 2002).  Based on their  underlying biology and clinical behavior, skin cancers are classified according to the cell of origin.  For instance, melanoma arises from melanocytes, the pigment-  producing cells in the skin, and non-melanoma skin cancer originates from the keratinocytes, the major cell type of the epidermis. There are two common types of non-melanoma skin cancer, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).  A small number of tumors arise from other cell types of the  epidermis or dermis including merkel cells and endothelial cells (Volgelstein et al., 1998). The major causative factor in human skin cancer incidence is exposure to the sun (Fitzpatrick etal., 1985; Armstrong etal., 2001; Cleaver et al., 2002). Sunlight is composed of a continuous spectrum of electromagnetic radiation divided into three  1  main regions of wavelengths: UV, visible, and infrared (Soehnge et al., 1997). UV radiation contains the wavelengths from 200-400 nm and is further divided into three sections: U V A (320-400 nm), UVB (280-320 nm), and U V C (200-280 nm). U V C is essentially blocked from reaching the earth's surface due to absorption by the atmospheric ozone layer, although some accidental exposure occurs from manmade sources, such as germicidal lamps (Soehnge et al., 1997; Cleaver et al., 2002). U V A and UVB radiation both reach the earth's surface in amounts sufficient to have important biological consequences from exposure of the skin and eyes (Soehnge et al., 1997; Cleaver et al., 2002). Wavelengths in the U V B region of the solar spectrum are absorbed into the skin, producing erythema, burns, and eventually skin cancer (Soehnge et al., 1997). Although U V A is the predominant component of solar UV radiation to which we are exposed, it is weakly carcinogenic (Soehnge etal., 1997). UV radiation is absorbed by DNA maximally within the range of 245-290 nm (Tornaletti et al., 1996) and is capable of creating mutagenic lesions in DNA between adjacent pyrimidines in the form of dimers. These dimers are of two main types: cyclobutane dimers (CPD) between adjacent thymine or cytosine residues, and pyrimidine (6-4) photoproducts between adjacent pyrimidine residues. Although both lesions are potentially mutagenic, the cyclobutane dimer is believed to be the major contributor to mutations in mammals (Tornaletti et al., 1996); the (6-4) photoproducts are repaired much more quickly in mammalian cells (Mitchell et al., 1989).  2  The commonest skin malignancy in many Caucasian populations is nonmelanoma skin cancer (Volgelstein et al., 1998). Approximately 80% of the nonmelanoma tumors are B C C , with the remaining 20% being S C C . B C C s rarely metastasize and although S C C s have a low rate of metastasis, overall fatality for S C C s is low. Surgical therapy or radiotherapy is highly effective. Genetically, p53 mutations have been shown to be common in both B C C and S C C . The mutational spectrum in non-melanoma skin cancer with frequent C->T and CC->TT transitions strongly supports UV radiation as the causative mutagen (Brash et al., 1991; 1996; Hollstein et al., 1991; Dumaz et al., 1994). Although B C C and S C C show frequent p53 mutation and ras mutations have been described in both tumor types, loss of heterozygosity (LOH) studies show clear differences between the two tumor types. In B C C , allelic loss is uncommon and is almost entirely confined to a region on 9q (Quinn et al., 1994). By contrast, allelic loss in S C C has been found to be common on chromosomes 3, 9, 13, and 17 (Quinn et al., 1994). Melanoma accounts for about 4% of skin cancer cases, but causes about 80% of skin cancer deaths (Boni et al., 2002).  The incidence of melanoma is  increasing more rapidly than any other tumor  (Rigel et al., 1996a; 1996b).  Moreover, melanoma metastasizes rapidly to other organs and there is no effective treatment for metastatic melanoma. Patients with metastatic melanoma have poor prognosis, with a 5-year survival less than 10% (Roses et al., 1991). Unlike B C C and S C C , p53 mutation is only observed in 15-25% melanoma biopsies, suggesting that p53 mutation is not an early step in melanoma development (Sparrow et al., 1995a; 1995b; Weiss et al., 1995). The most commonly observed abnormality in  3  melanoma is L O H and homozygous deletion at 9p21 (Vogelstein et al., 1998). Deletion mapping of 9p21 indicates that the locus encodes the tumor suppressor gene p16 , INK4  a negative growth regulator,  (Vogelstein et al., 1998). cyclin-dependent  which  induces cell cycle  arrest  p16 is part of a growth control pathway that involves  kinases, cyclins, and the  retinoblastoma  gene  product  Rb  (Vogelstein et al., 1998). Genes other than p16 have also been implicated in the formation of melanoma.  An example is the cyclin-dependent kinase 4 (cdk4), a  target of p16 inhibitory activity (Vogelstein et al., 1998). Furthermore, a very recent study by Davies et al. (2002) identifies somatic missense mutations in the oncogene B R A F , which renders it to have elevated kinase activity, in 66% of malignant melanoma biopsies. Evidence accumulated so far strongly suggests that there are likely many more genes involved in the development of melanoma.  1.2  Tumor Suppressive Genes  Cancers arise as a result of an accumulation of inherited and/or somatic mutations in two  broad classes of genes: proto-oncogenes and tumor  (Macleod, 2000).  suppressor genes  Tumor suppressor genes distinguish themselves from proto-  oncogenes in that their loss, rather than gain, of function contributes to the altered phenotype of cancer cells (Vogelstein et al., 1998).  More than a dozen tumor  suppressor genes have been localized and identified through various experimental approaches, such as linkage analysis and LOH (Vogelstein et al., 1998).  Their  principal responsibility is to prevent uncontrollable cellular proliferation in response to stress stimuli encountered during tumorigenic progression by activating cell cycle  4  arrest, apoptosis, induction of differentiation,  cellular senescence, inhibition  of  angiogenesis, and repair of damaged DNA.  The molecular functions of tumor  suppressor genes, p53 in particular, have been well characterized while many others are only at the initial stage of being understood. One of the best examples of genes that have not been investigated thoroughly is the tumor suppressor ING1, which was cloned in 1996. The process of successfully establishing a candidate gene as an authentic tumor suppressor can be complicated and laborsome. False claims are often made that are based entirely on the discovery of certain tumor suppressive functions of the gene in vitro. By definition, both alleles of the gene must be found to be inactivated by somatic and/or inherited mutations in cancer, and give rise to phenotypes that resemble the loss of tumor suppression. Going by this definition, the two recently identified p53 family members, p63 and p73, would not yet be classified as true tumor suppressors as mutation frequencies in human cancer were extremely low (Soussi et al., 2001).  1.2.1  p53  1.2.1.1 Structure and Function The p53 gene, first described in 1979, maps to 17p13.1 and encodes a 393-amino acid, 53 kD nuclear phosphoprotein (Kress et al., 1979; Lane et al., 1979; Linzer et al., 1979; Finlay et al., 1989).  It consists of 11 exons and spans over 20 kb of  genomic region (Lamb et al., 1986).  5  The p53 protein can be divided roughly into three major domains, encompassing the amino-terminal  region containing the activation domain, the central core  containing its sequence-specific DNA-binding domain, and the carboxyl-terminal domain (Ko et al., 1996). through  its activation domain.  multi-functional  p53 binds in vitro to several proteins  For instance, it interacts with many general  transcription factors such as the TATA box-binding protein (TBP) component of the general transcription factor TFIID (Horikoshi et al., 1995). The binding of p53 in this region to MDM2 has also been found to be crucial in the regulation of p53 half-life (Lin et al., 1994). The central core region of p53 contains the sequence-specific DNA-binding domain involved in the regulation of transcription of target genes such as p 2 1  l V a /  \ a cyclin-dependent kinase inhibitor that can activate both G1 and G 2 cell  cycle arrests (Harper et al., 1993; Agarwal et al., 1995; Bates et al., 1998; Balint et al., 2001). More than 90% of the missense mutations in p53 have been reported to reside in this sequence-specific DNA-binding domain (Levine et al., 1997).  The  carboxyl terminus of p53 functions in two manners: 1) it contains a region that promotes tetramerization of p53 protein products, which is important in the normal functioning of p53 as a tetramer (Jeffrey et al., 1995); and 2) it has basic amino acid residues that bind to DNA and R N A readily with some sequence or structural preferences, thought to be important in the regulation of p53's ability to bind to specific DNA sequences at its central core (Lee et al., 1995). p53 has been shown to be frequently mutated in sporadic cancer (WallaceBrodeur et al., 1999).  Specifically, more than 50% of human malignancies of  epithelial, mesenchymal, haematopoietic, lymphoid, and nervous origin analyzed to  6  date were shown to contain an altered p53 gene. Germline mutations in p53 result in Li-Fraumeni syndrome, a hereditary cancer susceptibility syndrome predisposing individuals to various cancers (Wallace-Brodeur et al., 1999). Since its isolation, p53 has been shown to exhibit a wide range of molecular functions, such as cell-cycle arrest, apoptosis, differentiation, angiogenesis, genetic stability, and DNA repair (Levine et al., 1997). To further investigate the role of p53 in carcinogenesis, two transgenic models were put forth.  Lavigueur et al. (1989) generated transgenic  mice by introducing mutant p53 gene fragments from tumor cell lines into mouse embryos. Donehower et al. (1992) created p53-deficient mice by introducing a null mutation of the p53  gene in murine embryonic stem cells. Mice lacking the  endogenous p53 gene or carrying a mutant p53 transgene have a near normal embryonic development but are prone to the spontaneous development of a variety of neoplasms (Lavigueur et al., 1989; Donehower et al., 1992). After chronic U V B exposure, both mutant p53 transgenic and p53-deficient mice developed more tumors than wild-type (wt) control mice (Li et al., 1995a; 1998). This outcome was later attributed to decreased DNA repair efficiency and reduced apoptosis in these transgenic mice (Li etal., 1996; 1997; Tron etal., 1998a; 1998b).  1.2.1.2 Cell Cycle Arrest The ability of p53 to transactivate downstream targets in growth arrest at specific checkpoints in the cell cycle has been well documented. target genes, p2'\  Waf1  Of the numerous p53  is believed to play a critical role in the induction of cell cycle  arrest (El-Deiry et al., 1993). It has been shown to activate both G1 and G2 arrest in  7  response to p53 induction (Harper et al., 1993; Agarwal et al., 1995; Bates et al., 1998; Balint et al., 2001). For instance, in p53-dependent G1 arrest, it is executed by transcriptionally elevating the expression of p21 ,  subsequently inactivating  Waf1  Cdk2/cyclin E complexes, and preventing phosphorylation of Rb (Xiong et al., 1993). The ultimate consequence is that cell cycle progression is inhibited from G1 to S phase and the cells are collected in late G 1 . Another target of p53 that contributes to G2 arrest is 14-3-3a (Hermeking et al., 1997). The 14-3-3a family proteins have been implicated in signal transduction and cell cycle control (Muslin et al., 2000) partly by binding to p53 and activating its sequence-specific DNA binding (Waterman et al., 1998). 14-3-3a may also represent a positive feedback loop to p53 to prevent cell cycle progression in damaged cells.  1.2.1.3 Apoptosis One of first lines of evidence that p53 is involved in apoptosis came from the study by Yonish-Rouach et al. (1991) in which they observed phenotypic characteristics of apoptosis upon transfection of wt p53 into a clone of p53' ' mouse myeloid cell line. /  Numerous apoptotic genes that are transcriptionally activated by p53 have been identified (Vousden, 2000). The very first p53 apoptotic target identified was the Bax gene, which belongs to the pro-apoptotic Bcl-2 family (Miyashita et al., 1995). Recently, other pro-apoptotic members of this family named Noxa and P U M A (p_53upregulated modulator of apoptosis) have since been identified as p53 targets (Oda et al., 2000; Nakano et al., 2001; Yu et al., 2001).  These proteins localize to  mitochondria and induce mitochondrial membrane potential and cytochrome c  8  release, thereby  activating the  apoptotic  protease activating  factor-1  (Apaf-  1)/caspase-9 apoptotic cascade (Bossy-Wetzel et al., 1999). It is interesting to note that Apaf-1 has also recently been found to be a transcriptional target for p53 (Moroni et al., 2001).  Apaf-1 complexes with cytochrome c after release from  mitochondria to form the apoptosome (the functional apoptotic unit), which causes an A T P - or dATP-dependent conformational change that allows the complex to bind to procaspase-9 and activates its self-proteolytic activity (Kaufmann et al., 2000). The active caspase-9 then activates other caspases such as caspase-3 and - 7 followed by caspase-6 (Rodriguez et al., 1999; Stennicke et al., 1999; Kaufmann et al., 2000). S M A C (second mitochondria-derived activator of caspase), also a protein involved in mitochrondria-induced apoptosis, is the second mitochondrial protein identified after cytochrome c which promotes apoptosis by activating caspase-3 and - 9 via binding to the inhibitors of apoptosis (IAP) and consequently removing their inhibitory activity (Chai et al., 2000; Du et al., 2000).  Change of mitochondrial  membrane potential during apoptosis may also be mediated by several genes encoding redox-controlling enzymes such as PIGs (p53-induced genes) (Polyak et al., 1997). It is speculated that reactive oxygen species produced by PIGs render damage to mitochondria, which in turn trigger apoptosis (Li etal., 1999). In addition, p53 has been implicated in the membrane death receptor-induced pathway of apoptosis. It has been found to up-regulate Fas and D R 5 , both death receptors, and FasL, a death receptor ligand (Ashkenazi et al., 1998). Activation of death  receptors by their  ligands results in trimerization  9  and recruitment  of  intracellular adapter molecules, which initiate the caspase (such as caspase 8) cleavage cascade and apoptosis (Ashkenazi etal., 1998).  1.2.1.4 DNA Repair In addition to prevent uncontrollable cell proliferation by inducing cell cycle arrest and apoptosis, one of the other major functions of the tumor suppressor gene p53 is the repair of damaged DNA.  Evidence indicates that cells lacking normal p53  function are deficient in nucleotide excision repair (NER), which primarily repairs UVinduced DNA damage (Ford et al., 1995; Wani et al., 1999) and base excision repair (BER), which removes bases damaged by alkylating agents, oxygen-free radicals, and hydrolysis (Offer et al., 2001; Zhou et al., 2001). The C-terminus of p53 has been found to bind to different forms of damaged DNA, such as single-stranded DNA, ends of double-strand  breaks, and DNA with insertion, deletion, and  mismatches (Balint et al., 2001).  Furthermore, p53 has been shown to associate  with several components of the repair machinery, including X P B and X P D (Balint et al., 2001). One of the most-studied p53 target genes that participates in DNA repair is GADD45.  G A D D 4 5 has been demonstrated to bind to proliferating cell nuclear  antigen (PCNA), and inhibit replicative DNA synthesis, therefore permitting DNA repair to proceed (Smith et al., 1994). GADD45' ' /  fibroblasts have defects in N E R  similar to those seen in p53 ~ fibroblasts (Smith et al., 2000) and G/ADD45-deficient v  mice show elevated carcinogenesis induced by radiation and genomic instability comparable to that observed in p53 deficient mice (Hollander et al., 1999).  10  ING1  1.2.2  1.2.2.1 Isolation and Characterization of Human ING1 Using subtractive hybridization between cDNAs from a non-transformed mammary epithelial cell line (184A1) and eight breast cancer cell lines (MCF-7, BT-474, Hs578T, ZR-75, MD-MB-468, MD-MB-435, and BT-20), and subsequent selection of short c D N A fragments capable of promoting neoplastic transformation upon injection into nude mice, Garkavtsev and colleagues (1996) isolated the ING1 gene (inhibitor of growth 1). The theory is that these randomly fragmented cDNAs, termed genetic suppressor elements (GSEs), should interfere with the activity of tumor suppressors by either blocking protein production through antisense sequences or by abrogating function  in a dominant  negative fashion through  truncated  sense fragments  (Roninson etal., 1995). The human ING1 gene contains three exons, named 1a, 1b, and 2, and two introns (Gunduz et al., 2000).  It produces at least four m R N A variants from three  different promoter regions (Gunduz etal., 2000; Saito et al., 2000; Jager etal., 1999) (Figure 1.1).  The p 4 7  , W G  ' transcript consists of exons 1b and 2, while the  transcript consists of exons 1a and 2. The third spliced form, p24 , ING1  of a truncated p47  ING1  variant, p27 , ING1  p33  ING1  is composed  messenger with the first A T G codon in exon 2. A fourth ING1  was recently detected (Gunduz et al., 2000; Jager et al., 1999). Its  transcript contains part of the coding sequence in exon 1b and exon 2. Analysis of ING1  promoters has been performed using restriction enzyme  digest at specific sites and luciferase reporter constructs (Gunduz et al., 2000).  11  Figure 1.1 shows the restriction enzyme sites that contain the three promoters for the p33 , ING1  p47 ,  and p24  ING1  ING1  alternative spliced forms.  Specifically, region  between the Xbal and Ncol sites contains the promoter for p33 ,  whereas the  ING1  promoters for p47  and p24  ING1  ING1  are present within the Notl and Pf1 Ml sites, and  the Pf1 Ml and Smal sites, respectively.  The promoter for p27  has yet been  ING1  identified. Initial examination of the cDNA structure of ING1 33,350 daltons, named p33  ING1  (Garkavtsev et al., 1996).  that, due to a cloning error, the reported sequence of p33  ING1  predicted a protein of It was later discovered was truncated at the N-  terminus and encoded a protein of 210-amino acid (aa) and 23,656 daltons, named p24 , ING1  which is the shortest known isoform of ING1  Cheung et al., 2001a).  (Garkavtsev et al., 1999;  In other words, most studies on p33  correction were actually done with the c D N A construct of p24'  and not that of  NG1  p33  /WG  prior to the  ING1  ' . The other three currently known isoforms of ING1 are p47  46,751 daltons), p33  ING1  ING1  (279-aa and 31,843 daltons), and p27  ING1  (422-aa and (235-aa and  27,000 daltons) (Gunduz et al., 2000; Saito et al., 2000; Jager et al., 1999; Garkavtsev et al., 1999) (Figure 1.2).  All four ING1 protein isoforms share an  identical C-terminus with a conserved P H D finger motif (a C4HC3-type zinc finger spanning 50-80-aa residues), which is implicated in transcriptional  regulation  (Gunduz et al., 2000; Saito et al., 2000; Jager et al., 1999; Garkavtsev et al., 1999). Two possible nuclear localization signal motifs (which generally consist of positively charged amino acids, such as lysine and arginine) in the isoforms are also present.  12  Figure 1.1 Genomic structure of human ING1 and its alternatively spliced m R N A variants, p47 , p33 , p27 , and p24 . • Coding sequence in exon. • Noncoding sequence in exon. Restriction enzyme sites indicate positions of the three promoters on ING1. Promoters for p33 , p47 , and p 2 4 are present within the Xbal and Ncol sites, the Notl and Pf1 Ml sites, and the Pf1 Ml and Smal sites, respectively. Promoter for p27 has not been analyzed. ING1  ING1  ING1  ING1  ING1  ING1  /A/Gr  ING1  Xbal Ncol  Notl PflMI Smal I  Exon 1 a  1  k  b  Exon 2  Exon 1 b  • p33  ING1  p47 ING1 p24 :  13  ING1  ING1  p27  I  Figure 1.2 Predicted amino acid sequences of the four ING1 isoforms, p47 p33 \ p27 , and p24 based on cDNA sequences from GenBank (accession numbers: AF181849, AF181850, AF149723, and AB031269, respectively). The metal-chelating residues in the P H D finger domain are represented by white type on black. Underlined residues indicate possible nuclear localization signal motifs. ING  p  47  ING1  mG  '  -  ING1  MSFVECPYHSPAERLVAEADEGGPSAITGMGLCFRCLLFSFSGRSGVEGGRVDLNVFGSLGLQPWIGS SRCWGGPCSSALRCGWFSSWPPPSRSAIPIGGGSRGAGRVSRWPPPHWLEAWRVSPRPLSPLSPATFGRGFIAVA MLHCVQ  68 14  3  6  MEILKELDECYERFSRETDGAQKRRMLHCVQ  31  MLSPANGEQLHLVNYVEDYLDSIESLPFDLQRNVSLMREIDAKYQEILKELDECYERFSRETDGAQKRRMLHCVQ  75  VIPGLWARGRGCSSDRLPRPAGPARRQFQAASLLTRGWGRAWPWKQILKELDECYERFSRETDGAQKRRMLHCVQ  218  RALIRSQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTVGNSGKVGADRPNGDAVAQSDKPNSKRS  81  RALIRSQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTVGNSGKVGADRPNGDAVAQSDKPNSKRS  106  RALIRSQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTAGNSGKAGADRPKGEAAAQADKPNSKRS  150  RALIRSQELGDEKIQIVSQMVELVENRTRQVDSHVELFEAQQELGDTAGNSGKAGADRPKGEAAAQADKPNSKRS  293  RRQRNNENRENASSNHDHDDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYBLBNQVSYGEMIG  156  RRQRNNENRENASSNHDHDDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYBLBNQVSYGEMIG  181  RRQRNNENRENASSNHDHDDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYBLBNQVSYGEMIG  225  RRQRNNENRENASSNHDHDDGASGTPKEKKAKTSKKKKRSKAKAEREASPADLPIDPNEPTYBLBNQVSYGEMIG  368  BDNDEBPIEWFBJFSBVGLNHKPKGKWYBPKBRGENEKTMDKALEKSKKERAYNR  210  BDNDEBPIEWFBFSBVGLNHKPKGKWYBPKBRGENEKTMDKALEKSKKERAYNR  235  BDNDEBPIEWFBFSBVGLNHKPKGKWYBPKBRGENEKTMDKALEKSKKERAYNR  279  BDNDEBPIEWFBFSBVGLNHKPKGKWYBPKBRGENEKTMDKALEKSKKERAYNR  443  14  Fluorescent in situ hybridization (FISH) localized ING1 to 13q33-34, while radiation hybrid mapping showed that ING1  is linked to the cytogenetic marker  S H G C - 5 8 1 9 , which resides at 13q34 (Garkavtsev et al., 1997a; Zeremski et al., 1997) . Immunofluorescence using the ING1 antibody indicated that ING1 protein products are primarily localized in the nucleus (Garkavtsev etal., 1997a).  1.2.2.2 Tumor Suppressive Functions Overexpression of the antisense  construct  p24  promotes  construct inhibits cell growth, while  ING1  cell transformation  its  (Garkavtsev et al., 1996).  Specifically, flow cytometry analysis shows that normal fibroblasts transfected with p24  are arrested in the G0/G1 phase of the cell cycle. It appears that  ING1  p24 ING1  mediated growth arrest requires the participation of functional p53 (Garkavtsev et al., 1998) , which is the most frequently mutated gene in human tumors (Hollstein et al., 1991; Wallace-Brodeur et al., 1999) and one of the most extensively studied tumor suppressors involved in a myriad of anti-tumor functions, such as cell-cycle regulation, apoptosis, senescence, angiogenesis, and DNA repair (Somasundaram et al., 2000; Giaccia et al., 1998; May et al., 1999; Li et al., 1996; 1997; Tron et al., 1998a; 1998b). Overexpression of p24  ING1  the p2\"  Waf1  can increase p53-dependent activation of  promoter (Garkavtsev et al., 1998). p 2 1  l/Va  " is.a cyclin-dependent kinase  (Cdk) inhibitor and a well-known downstream target of p53 involved in negative growth  regulation  by  inhibiting  both the  kinase activity  of  Cdk-2 and  the  phosphorylation of the retinoblastoma (Rb) protein (El-Deiry et al., 1993; Harper et al., 1993). Elevated expression of p 2 1  l V a f t  15  could, therefore, account for the ability of  p24  ING1  to prevent cell proliferation  in concert with activated p53.  Further  underscoring the functional dependency between ING1 and p53 is the finding that both  proteins  can  physically associate with  immunoprecipitation (Garkavtsev et al., 1998).  one  another  as  indicated  by  The binding between the two  proteins may alter the conformation of and therefore mediate the activity of p53 as a transcription factor.  It is however uncertain which products of ING1  form the  complex, as information on its isoforms was unknown at the time and the antibody used likely recognized all protein products. A recent study by Leung and colleagues (2002) however provided strong evidence that the isoform p33  ING1  is capable of  binding to the region of p53 that MDM2 binds to in the regulation of p53 stability. Finally, inhibition of p24  ,NG1  expression has also been found to promote anchorage-  independent growth in murine breast epithelial cells as well as focus formation in murine fibroblast cells (Garkavtsev et al., 1996). In addition to its involvement in growth control, p24  ING1  play a role in senescence. p24  ING1  has been shown to  Normal human fibroblasts expressing the antisense  insert can increase their replicative life by 8-10% (Garkavtsev et al., 1997b).  The expression of ING1 protein and mRNA was found to be higher in senescent cells compared to young proliferation-competent human diploid fibroblasts.  This  study also examined ING1 expression throughout the cell cycle, where ING1 protein decreased from GO to G 1 , increased in late G 1 , reaching maximum in S phase, followed by a decrease in G2. The lack of information on ING1 isoforms, however, made it impossible to distinguish which protein and m R N A products were being monitored.  16  ING1 has also been found to modulate apoptosis. Elevated expression of p24  enhances  ING1  serum  starvation-induced  cell  death  in  p19  mouse  teratocarcinoma and NIH 3T3 cells (Helbing et al., 1997). Apoptosis by activation of c-myc, a transcriptional activator, has been well-documented (Hotti et al., 1999; Juin et al., 1999; Nesbit et al., 2000; Kato et al., 1990). p24  ING1  Coexpression of c-myc and  dramatically enhances the extent of serum starvation-induced apoptosis,  suggesting that p24 apoptotic  pathway.  /A/Gr  -mediated cell death may be synergistic with the c-myc p24  ING1  has  also  been  shown  to  sensitize cells  to  chemotherapeutic agents and radiation, such as etoposide and y-irradiation, in the presence of wt p53 (Garkavtsev et al., 1998). By isolating p33  ING1  from their fetal  brain c D N A library, Shinoura and colleagues (1999) demonstrated that apoptosis can be induced by adenovirus-mediated transfer of both p33  ING1  and p53 in two  glioma cell lines, which do not undergo apoptosis by overexpressing either gene alone.  Scott et al. (2001a) recently showed that ING1  possesses two distinct  nucleolar targeting sequences within the nuclear localization signal region, which promotes the translocation of its encoded products to the nucleolus after U V irradiation. p33  ING1  was also found to contain a common octapeptide motif called the  PCNA-interacting-protein domain at the amino terminus, through which it binds competitively to the interdomain connector loop of P C N A upon UV irradiation (Scott et al., 2001a; 2001b; Warbrick et al., 1998; Tsurimoto et al., 1999). These authors also showed that human fibroblasts overexpressing p33  ING1  have higher percentage  of apoptosis compared to vector controls. A very recent and detailed analysis by Vieyra et al. (2002b) demonstrated that ING1  17  induces apoptosis in an isoform-,  stimulus-, and cell age-dependent fashion.  First, they showed that growth-factor  deprivation-induced apoptosis occurs only in early (young)- but not late (senescentpassaged fibroblasts. This effect was accompanied by an increase in endogenous INGI  ?33  expression of p33 , ING1  T  n  e  y  a ] s o  f  o  u  n  d  t n g t  e c t o p i c  but not the  p47  ING1  isoform, sensitizes young but not senescent cells to UV irradiation- and hydrogen peroxide-mediated  apoptosis.  Coexpression of p33  ING1  and p53  enhanced cell death compared to expression of either one alone. showed that chromatin binding affinity of p33  ING1  ING1  isoforms of ING1, p24  ING1  and p 3 3  / N G  Finally, they  was increased in senescent cells,  which were resistant to apoptosis, suggesting that p33 functions via chromatin remodeling.  significantly  may exert its suppressive  These studies all in all suggest that both  ' , can function as pro-apoptotic regulators.  In one of the most comprehensive investigations of the genes regulated by ING1, Takahashi and colleagues (2002) identified 19 genes as a result of expression of antisense ING1  in mouse mammary epithelial cell line N M u M G .  Using c D N A  microarray, they showed that overexpression of the antisense ING1 construct, which was designed to suppress all isoforms of ING1, stimulated expression of 14 genes, including cyclin B1 and D E K oncogene, whereas 5 genes were transcriptionally repressed.  It is interesting to note that the expression relationship between ING1  and cyclin B1 fits nicely in the context of cell cycle control. For instance, cycline B 1 , which accumulates during G2/M phase of the cell cycle, belongs to the regulatory subunit of the cdc2 protein kinase, and cdc2/cyclin B1 complex is required for mitotic initiation (Elledge et al., 1996). In contrast, ING1 protein level starts to increase at late G 1 , reaching maximum in S phrase followed by a significant decrease in G 2 / M  18  phase (Garkavtsev et al., 1997).  D E K is a nuclear protein that was found to be  fused to an oncogenic protein C A N (short for Cain - cancer intron on nine) in a portion of acute myelogenous leukemia (AML).  D E K is also highly expressed in  human hepatocellular carcinoma compared to normal liver tissues (Kondoh et al., 1999). The molecular mechanistic aspects of different ING1 isoforms were further explored by Skowyra and colleagues (Skowyra et al., 2001). p33  ING1  was shown to  associate with Sin3, S A P 3 0 , H D A C 1 , RbAp48, and other proteins in in vitro deacetylation of core histones, while p24  ING1  and p47  ING1  did not show physical  binding with any of the deacetylation-associated proteins (Skowyra et al., 2001; Vieyra et al., 2002a; Kuzmichev et al., 2002). The unique function of deacetylation is frequently associated with regulating gene expression by chromatin condensation and gene silencing (Struhl et al., 1998; Knoepfler et al., 1999). Further investigation indeed found that p33  ING1  represses transcription in cultured cells using luciferase  and G A L 4 as reporters (Skowyra et al., 2001). The data suggest that the 99 amino acids of the N-terminal of p33  ING1  are responsible for both the association with  deacetylation-associated proteins as well as transcription repression. p24  and  ING1  p47 , ING1  on the other hand, lack the unique N-terminus specific for p33 ,  which  ING1  explains why they do not display those binding and functional properties. In a recent report by Vieyra and colleagues (2002a), the authors demonstrated that p33  ING1  is  able to affect the degree of physical association between proliferating cell nuclear antigen (PCNA) and p300, an association that has been proposed to link DNA repair to chromatin remodeling. Lastly, as mentioned earlier, the p33  ING1  19  isoform has been  shown to physically bind to the p53 protein at the N-terminal region to which MDM2 normally binds (Leung er al., 2002).  It is believed that both p33  ING1  and MDM2  directly compete with each other in order to regulate the stability of p53, hence its protein level.  The weakness of this study however is that the activation and  induction of p53 protein level as a result of enhanced stabilization by p33  were  ING1  assessed  in  a  p53-null  cell  line,  H1299  non-small  cell  lung carcinoma,  overexpressing a transfected p53 plasmid. In other words, the findings may not be relevant in physiologically normal conditions where endogenous p53 is present.  1.2.2.3 Expression Profile ING1 m R N A was found to be ubiquitously expressed in various human tissues as two major bands at 2.2 and 2.5 kb by Northern blot analysis (Shimada er al., 1998). It is not known as to which protein isoforms they represent. Another more recent report also examined a number of human tissues by multiplex R T - P C R that distinguished the different ING1 spliced forms (Saito et al., 2000). Most tissues were found to show various degrees of expression of p24  ING1  In a separate study, p 3 3  /NG  and p 3 3  / W G  ' , but not  p47 . ING1  * mRNA (referred as variant A in their paper) was shown  to be highly expressed in normal human tissues and cancer cell lines, while m R N A (referred as variant B) was weakly expressed (Jager et al., 1999).  p24  ING1  This  observation was further supported by the study of Skowyra and colleagues (2001), in which low abundance of p24  ING1  was detected in HeLa cells.  20  Similar to the human ING1 expression pattern, mouse ING1 (mlNG1) was detected in various tissues (Zeremski et al., 1999).  mRNA  Information on the  expression profile of different mlNG1 isoforms is lacking. While stress stimulus such as the lack of serum in culture media induces ING1 protein expression and subsequently apoptosis (Helbing et al., 1997). Again, no information  is available on which isoform(s) of ING1  was induced in these  studies.  1.2.2.4 The ING1 Family By searching a human E S T database for cDNA with sequence similarity to Shimada and colleagues (1998) isolated a partial ING1-like from a human fetal brain c D N A library. shares 58.9% sequence identity with p 3 3  gene, named  ING1, ING1L,  The ING1L protein is 280-aa long and , W G  ' (Shimada etal., 1998). The C-terminal  PHD-type zinc finger domain is also conserved in ING1L. FISH and radiation hybrid mapping indicate that ING1L  resides at 4q35.1 (Shimada et al., 1998). In terms of  expression profile, Northern blot analysis of normal human tissues shows ubiquitous expression of the 1.3- and 1.5-kb INGL transcripts (Shimada et al., 1998).  Also,  ING1L m R N A expression was found to be significantly higher in colon tumors than in the adjacent normal tissue. ING1L was also cloned by Nagashima et al. (2001), which they termed ING2. Western analysis showed ubiquitous but variable expression of ING1, while ING2 expression was highly variable or absent in many cell lines. Highest expression of ING2 occurred in cell lines with null or mutant p53.  21  The DNA-damaging agents,  etoposide and neocarzinostatin, induced expression of ING2 but not ING1. Like ING1, ING2 was found to negatively regulate cell growth and survival in a p53dependent manner through the induction of G1 cell cycle arrest and apoptosis. ING2 was also found to enhance the transcriptional transactivation activity of p53 and increased acetylation of p53. Western analysis detected ING1 but not ING2 in p53 immunoprecipitates.  It was therefore concluded that ING2 is a DNA damage-  inducible gene that negatively regulates cell proliferation through activation of p53 by enhancing its acetylation. Another member of the ING1 family was cloned by screening a human breast cancer c D N A library and designated as ING2 (the sequence of this gene is different than that cloned by Nagashima and colleagues, though they both are named identically) (Jager et al., 1999). homolog as INGJ.  In this thesis, we will refer to this human  ING1  This 42-aa protein shares 76% amino acid identity with all the  ING1 isoforms examined in the study. All normal human tissues and most breast cancer and melanoma cell lines showed expression of INGJ m R N A (Jager et al., 1999). In an attempt to analyze L O H at 7q31 in head and neck squamous cell carcinomas (HNSCC), another homolog of ING1,  named ING3, was  identified  (Gunduz er al., 2002). ING3 encodes a 418-aa protein which shows high homology with other members of ING1 especially within the P H D zinc finger domain.  The  ING3 gene is relatively large, composing of at least 12 exons spanning over 25 kb genomic distance. Northern blot analysis of the 1.9 kb ING3 transcript was detected in most organs except brain, colon, small intestine, and lung. Mutation analysis of  22  H N S C C showed only one missense mutation in 49 primaries but 50% of the samples demonstrated either decreased or lack of expression of the  mRNA  transcript compared to normal tissues. In Xenopus  Laevis, a gene homologous to the human ING2 was isolated by  Wagner et al. (2001), which they termed xlNG2.  Alignment of xlNG2 and human  ING2 c D N A sequences showed 7 1 % sequence identity within the open reading frame. The xlNG2 gene encodes a 32 kD protein product which was shown to be involved in the development of tadpole in a tissue-specific and hormone-responsive manner. A homolog of human ING1 has also been found in mouse. mlNG1  produces  three transcripts from three different promoters in the same gene (Zeremski et al., 1999). The protein products all have the conserved C-terminal P H D finger region. Two of the transcripts produce a protein of 24 kD in size, but was named  p3'\ , ING1  because it runs as if it was a 31 kD product (Zeremski et al., 1999). This protein is likely the equivalent of the human p24 , ING1  which has growth suppressive function.  The other mouse transcript produces a larger protein, named p37 , ING1  which binds to  and interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. human p33 . ING1  p37  is speculated to be the equivalent of the  ING1  There appears to be no mouse equivalent to the human  p47 . ING1  Using a yeast two-hybrid assay to screen for A1 interacting protein, an antiapoptotic Bcl-2 family member and a direct transcriptional target of nuclear factor kappa B (NF-kB), in mouse mammary glands, Ha and colleagues (2002a) unexpectedly isolated a homolog of the mlNG1  gene, named mINGIh.  23  Four splicing variants of  mINGIh  were discovered and found to enhance cell death upon serum starvation.  This process however can be inhibited by the expression of the A1 protein. It was also found that the nuclear localization signal in the conserved P H D finger domain of mINGIh is split into two regions, much like the human counterparts, and mutation induced in either of the two regions abrogates the apoptotic function (Ha et al., 2002b). Three Saccharomyces  cerevisiae  and  two  Schizosaccharomyces  pombe  protein homologs of ING1 have since been identified and share significant homology (50 to 60% identity) in the C-terminal containing the P H D finger region (Loewith er al., 2000). Introduction of human p33  was shown to rescue phenotypes induced  ING1  by the deletion of the yeast ING1  homologs, such as abnormal  multibudded  morphology, an inability to utilize nonfermentable carbon sources, heat shock sensitivity, slow growth, temperature sensitivity, and sensitivity to caffeine (Loewith et al., 2000), suggesting that ING1 is functionally conserved in both species. addition, the yeast ING1 histone  acetytransferase  In  homologs were found to associate with proteins in the (HAT) complexes, which  have  been  implicated  in  chromatin-mediated transcriptional regulation (Brown et al., 2000; Berger et al., 1999).  Subsequent studies in yeast provided further insight into the mechanistic  involvement of the ING1 yeast homologs in chromatin regulation (Nourani et al., 2001; Choy et al., 2001; Howe et al., 2002).  These are some of the first  investigations that provide a possible link between the human ING1 gene and its molecular mechanisms in tumor suppression through the studying of homologs of other species.  24  1.2.2.5 Expression and Mutational Analyses in Tumors In lymphoid tumor cell lines, decreased expression of ING1 m R N A was observed in 4 of 5 T-cell lines and 5 of 11 B-cell lines, but no mutations were found (Ohmori et al., 1999). A significant decrease in ING1 m R N A expression was also observed in 15 of 20 gastric carcinoma tissues compared to their corresponding normal tissues, with 80% of the 15 tumors showing wt p53 (Oki et al., 1999). Only one missense alteration was found in 1 of 12 gastrointestinal cancer cell lines by sequence analysis.  Rare mutation of ING1 in these tumors suggests that other inactivation  mechanisms may be responsible for the reduced ING1 expression. In colorectal carcinomas, neither L O H nor mutation as indicated by single-stranded conformation polymorphism  (SSCP)  was  present  in 29  primaries  (Sarela et al., 1999).  Consequently, ING1 is unlikely the gene involved in the multistage development of colorectal cancers.  Contrarily, Garkavtsev and colleagues (1996) reported ING1  protein level to be higher in neuroblastoma cell lines compared with diploid fibroblast cells. 1996).  Rearrangement of ING1 was observed in one cell line (Garkavtsev et al., Since cell cultures are usually passaged extensively and genetic changes  may occur during long-term propagation, the expression and mutational analysis of ING1  in neuroblastoma biopsies would be required to confirm the findings by  Garkavtsev's group. In one of the largest studies of ING1 mutation analysis, Toyama et al. (1999) reported that 44% of 452 breast cancer primaries and all of 10 breast cancer cell lines have marked decreases in ING1  mRNA expression.  25  Fifty-eight percent of  breast primaries with decreased ING1 had metastasized to regional lymph nodes. Despite many cases of abnormal ING1 expression, mutation in this gene is very rare. Only one germline missense mutation at codon 95 (0.27%) and three silent alterations at codon 188 (4.8%), codon 166 (0.27%) and codon 228 (0.27%) were detected in 377 primary breast cancer carcinomas, but no mutations were found in 10 breast cancer cell lines and 65 primary ovarian cancer tissues.  Reduced  expression of ING1 in breast cancers was supported by the study of Tokunaga and colleagues (2000), in which 70% of 24 breast cancer tissues had significantly less ING1 m R N A expression. It was also found that 9 of 15 tumors with decreased ING1 stained negative for p53 protein. The basis for ING1 suppression in breast cancers remains unknown. LOH was found at 13q34 in 20 of 44 head and neck squamous cell carcinoma ( H N S C C ) primaries, but the ING1 gene was not affected (Sanchez-Cespedes et al., 2000). Sequence analysis showed no somatic mutations, indicating that ING1 is not a tumor suppressor target in H N S C C . suggests otherwise.  A separate study on H N S C C , however,  Gunduz and colleagues (2000) reported that 23 of 34 (68%)  informative cases of H N S C C showed L O H at 13q34. Using primers specific for exon 1a and exon 2, three missense and three silent mutations in ING1 were detected in the 23 tumors with L O H . Missense mutations were found within the P H D finger domain and nuclear localization motif (Gunduz et al., 2000), which may affect the function of all the ING1 variants sharing the common C-terminus. One study investigated ING1 mutation status in oral squamous cell carcinoma ( O S C C ) using S S C P but found no mutation in any of the 71 primaries, suggesting  26  that the ING1  gene may not be important in the development of this disease  (Krishnamurthy et al., 2001). In an attempt to find out if the ING1 gene was involved in the pathogenesis of human esophageal squamous cell cancer ( E S C C ) , Chen and colleagues (2001) examined 31 cases of E S C C for expression levels and mutation status of ING1.  Results show that ING1 protein expression was absent in all E S C C  samples compared to controls and 4 missense mutations were found. Surprisingly, the tumors with the mutations in ING1 levels of the protein.  showed no correlation to the expression  It is, however, interesting to note that all 4 of the missense  mutations occurred within the P H D finger domain and nuclear localization motif of ING1, and may be responsible for the development of some of the E S C C s .  It was  further found that there was no statistically significant correlation between mutation status and prognosis in the patients with the E S C C condition. Most studies above, however, did not distinguish the expression of different ING1 variants.  The first study that addressed this issue was Jager et al. (1999).  They found that variant A (or p33 ) ING1  was clearly overexpressed in all six breast  cancer cell lines, eight melanoma cell lines, and a breast cancer primary, while variant B (or p24 ) ING1  1999).  was only present in four breast cancer cell lines (Jager et al.,  The study also implicated ING1 as a breast cancer antigen.  One recent  report by Nouman et al. (2002a) found that, upon examining 145 patients with childhood acute lymphoblastic leukemia, loss of p33  ING1  protein expression in nuclei  occurred in 78% of the tissue samples. This loss was accompanied by an increased cytoplasmic expression of p33 . ING1  Interestingly, it was found that a significant  number of patients who had lost nuclear expression had a better  27  prognosis  compared to those without the loss. Another study by Ito et al. (2002) showed that the levels of p33  ING1  mRNA transcript were not significantly different between 3 A M L  cell lines and 10 A M L biopsies. Furthermore, neither point mutations nor deletions in ING1 were found, suggesting that p33  ING1  may not play a role in the development  of A M L . In melanoma, Nouman and colleagues (2002b) analyzed a group of 67 melanocytic lesions and demonstrated a consistent trend of nuclear to cytoplasmic compartment shift of the p33  ING1  malignancy.  protein in samples with increasing degrees of  Specifically, while none of the benign melanocytic nevi showed  complete loss of nuclear p 3 3  / W G t  expression, almost 50% of the invasive malignant  melanoma samples did. This decreased or loss of nuclear expression was again associated with an increase of cytoplasmic expression of p33 . ING1  In contrast with  other expression and mutation studies of tumors, Campos et al. (2002) showed that instead of a decrease in expression, p33  ING1  protein and m R N A levels were found to  be elevated in all 14 melanoma cell lines compared to normal melanocytes. However, only one missense mutation at codon 260, within the P H D finger domain, occurred in one cell line. It was postulated that the observed increase in expression of p33  ING1  may be due to other types of modifications such as DNA methylation.  Lastly, a study undertaken by Bromidge et al. (2002) showed that, using primers specific for p33  ING1  and p47  transcripts, the two transcript levels did not differ  ING1  significantly from each other in both normal tissues and tissues of hematological malignancies. Though the p33  ING1  transcript was most abundant in comparison to  the other two, sequence analysis showed no mutation.  28  Taken together, it appears that ING1 is rarely mutated but its expression is often suppressed in human tumors. Several theories can be put forth to explain the reduced expression of ING1.  One possible explanation could be that, since the  ING1 gene and flanking regions are G C rich, methylation of the ING1 promoter may result in reduced expression (Gunduz et al., 2000). This is similar to the case of the so-called "metastasis suppressor", E-cadherin, where methylation of both alleles of the gene contributes to a significant reduction or even absence of expression in leukemia (Melki et al., 2000). inactivation.  Biological agents such as viruses may also cause  For example, most cases of cervical cancer are associated with  infection of human papillomavirus (Zur Hausen et al:, 1991), which produces the E6 protein to bind to and subsequently accelerate the degradation of p53 (Scheffner et al., 1990). In addition, downregulation by upstream regulators may also contribute to  reduced  expression.  For  instance,  amplification  of  MDM2  results  in  overexpression of its product, which then inactivates p53 (Oliner et al., 1992; Momand et al., 1992; Barak et al., 1993). The upstream regulators of ING1 however remain to be identified.  1.3 General Hypothesis and Objective W e hypothesized that the expression of p33  ING1  is dependent on the status of p53 in  normal and stress conditions, and overexpression of p33  ING1  enhances UV-damaged  DNA repair, UV-induced apoptosis, and camptothecin-induced cell death. The primary objective of this study was to further our understanding of the tumor suppressive role of the ING1  alternative spliced variant, p33 , ING1  29  in stress  conditions.  W e started by looking at the expression profile of p33  INU1  in various  mammalian organs and in skin cells after exposure to UV irradiation (Chapter 3). Next, using primarily melanoma cell lines, we investigated the role of p33  ING1  in  repair of UV-damaged DNA (Chapter 4), in UV-induced apoptosis (Chapter 5), and in chemosensitivity (Chapter 6).  30  CHAPTER 2. MATERIALS AND METHODS  2.1  Animals  p53  +/+  and p53" mice were purchased from Taconic Inc. (New York). p53' mice A  A  carried a disrupted, nonfunctional p53 gene, created by homologous recombination in an embryonic stem cell line and by microinjection of the stem cells into 3.5-day old C57BL/6 blastocysts (Donehower etal., 1992).  2.2  Cell Lines and Cell Culture  Normal Human Epithelial keratinocytes (NHEK) were obtained from the Tissue Bank of Vancouver General Hospital. They were maintained in Keratinocyte-SFM medium (Canadian Life Technologies, Burlington, ON). A human H a C a T keratinocyte (kindly provided by Dr. N.E. Fusenig, DKFZ, Heidelberg, Germany) and three human melanoma cell lines, M M R U , R P E P , and M E W O , were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Canadian Life Technologies, Mississauga, ON), 100 units/ml penicillin and 100 pg/ml streptomycin (Canadian Life Technologies, Mississauga, ON) at 37°C in a 5% C0  2  atmosphere.  M M R U and R P E P were kindly provided by Dr. H. R. Byers  (Boston University School of Medicine, Boston, MA), and M E W O by Dr. A. P. Albino (Memorial Sloan-Kettering Cancer Center, New York, NY).  The p53  status of  HaCaT, M M R U , R P E P , and M E W O was previously determined (Tilgen et al., 1983; Li et al., 1995b). HaCaT and M E W O contain mutant p53, while M M R U and R P E P contain wt p53.  Dermal fibroblasts of p53  31  +/+  and p53' mice were isolated from 4A  week old mice. The mice were sacrificed by cervical dislocation and a 2 x 2 cm skin biopsy were dissected from the dorsal area. The hair was removed and the skin biopsy was disinfected with 2.5% betadine for 1 min, followed by 1 min in 70% ethanol, and washed with phosphate buffered saline (PBS) twice. The skin tissue was then minced and incubated in D M E M containing 200 units/ml collagenase (Sigma, St Louis, MO) at 37°C for 6 h. The digested skin tissue was centrifuged at 1000 rpm for 10 min and the pellet washed with pre-warmed D M E M twice. The cells were resuspended in D M E M containing 10% F B S and incubated at 37°C in a 5% C0  2  atmosphere.  2.3  Plasmids  Plasmids used for transfection included pCI-vector, p C I - p 3 3 p33  ING1  /WG7  , pCI-antisense  (kind gifts from Dr. K. Riabowol, University of Calgary, Calgary, A B ) , p G F P -  N2 (Clontech, Windsor, ON), p E C H which contains a wt p53 cDNA, and pED1 which contains a point mutation in the human p53 cDNA that changes Cys-135 to serine (kind gifts from Dr. S. Benchimol, University of Toronto, Toronto, ON), and pCMVcat (kind gift from Dr. L. Grossman, Johns Hopkins University, Baltimore, MD).  2.4  Antibodies  Antibodies used for Western blotting were anti-ING1 rabbit polyclonal antibody (Pharmingen, Mississauga, ON), anti-p-actin goat monoclonal antibody, anti-p53 DO-1  mouse  monoclonal, anti-Bax polyclonal antibody,  32  anti-GADD45 mouse  monoclonal, anti-XPA rabbit polyclonal, anti-XPB rabbit polyclonal antibodies (Santa Cruz, Santa Cruz, CA), secondary IgG (Calbiochem, San Diego, CA).  2.5  Transfection  Cells were transfected at 40-50% confluency with Effectene reagent (Qiagen, Mississauga, ON) at a ratio of 1 u.g plasmid DNA to 25 uJ Effectene in a 60mm petri dish with approximately 2 X 10 cells. 6  2.6  Determination of Transfection Efficiency  Transfection efficiency of a particular cell line was determined by first, introducing a GFP-bearing plasmid, p G F P - N 2 , with Effectene; second, assessing the number of green fluorescence emitting cells/ the total number of cells (fluorescent and nonfluorescent) counted X 100%. Figure 2.1 shows microscopic images of M M R U cells transfected with the p G F P - N 2 plasmid. Table 1 shows percentages of transfection efficiencies of the cell lines used in this study.  2.7  UVB Irradiation  Medium was removed and the cells were exposed to UVB (280-320nm) using a bank of four unfiltered  FS40 sunlamps (Westinghouse, Bloomfield, NJ).  The  intensity of the UV light was measured by the IL 700 radiometer fitted with a W N 320 filter and an A127 quartz diffuser (International Light, Newburyport, MA).  Medium  was replaced and cells were incubated in a 5% C 0 incubator at 37°C after U V B 2  irradiation.  33  Figure 2.1 Visual assessment of transfection efficiency. M M R U cells were transfected with p G F P - N 2 plasmids and visualized under an inverted fluorescent and white light microscope 24 h post-transfection. Photographs were taken at 400X magnification.  Fluorescent  Fluorescent + Light  pGFP-N2  34  Table 1.1 Relative percentages of transfection efficiencies of cell lines used in the study. Percentages were determined by counting the number of cells emitting green fluorescence/ total number of cells (fluorescent and non-fluorescent) counted X 100%. Results were derived from three independent experiments.  Cell line  Transfection Rate (Percentage)  MMRU  65.0 ± 7 . 7  RPEP  60.0 + 8.3  MEWO  42.5 + 5.9  35  2.8  Light Microscopy  Cell morphology was visualized using an inverted scope (Nikon, Tokyo, Japan) and images were taken using a digital camera (Minolta, Richmond, BC).  2.9  Western Blot Analysis  Cells were harvested by scraping and lysed with the triple detergent lysis buffer containing 50 mM Tris-CI pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecylsulfate, 1% Nonidet P-40, 100 ug/ml phenylmethylsulfonyl fluoride, 1 ug/ml aprotinin, 1 ug/ml leupeptins, and 1 ug/ml pepstatin A. Concentrations of proteins were determined by the DC Protein Assay (Bio-Rad, Mississauga, ON).  Fifty  micrograms of proteins per lane were separated on 10% polyacrylamide/SDS gels and  electroblotted  onto  polyvinylidene difluoride  (PVDF)  filters.  Filters  were  incubated with primary antisera for 1 h, followed by three washes in P B S for 5 min each, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antisera for 1 h at room temperature. The signals were detected with the E C L Western blotting detection system (New England Biolab, Guelph, ON).  2.10  Trypan Blue Exclusion Assay  The floating dead cells in the medium were collected and those that remained attached to the plates were removed, collected by trypsinization, and mixed with the floating dead cells.  They were then counted using a hematocytometer in the  presence of 0.4% trypan blue reagent (Sigma, Mississauga, ON).  36  2.11  SRB Cell Survival Assay  Cells were grown in 24-well plates. After treatment at 80% confluency, the medium was removed and the cells were fixed with 500 pi 1:1 acetone/methanol for 10 min at -20°C, air-dried, and stained with 500 ul of sulforhodamine B (SRB) (0.4% w/v in 1% acetic acid) for 20 min at room temperature. After four washes with 1% acetic acid, the cells were air-dried, and then incubated at room temperature with 500 pi of 10 mM Tris (pH 10.5) for 5 min with gentle shaking to solubilize the bound dye. Spectrophotometric readings were then taken at 550 nm for 100 pi aliquots.  2.12  Reverse Transcriptase-Polymerase  Total  R N A was  Mississauga,  extracted  ON)  spectrophotometry.  and  by TriZol the  Chain Reaction (RT-PCR) reagent  concentrations  (Canadian Life Technologies, were  determined  by  UV  Five micrograms of total R N A was reverse-transcribed into  c D N A in the presence of 10 units/pl of S U P E R S C R I P T II RNase H" RT (Canadian Life Technologies, Mississauga, ON), 5X first strand buffer (250 mM Tris-HCL, pH 8.3, 375 mM K C L , 15 mM MgCI ), 100 mM DTT, 10 mM dNTP Mix (10 mM each of 2  d A T P , d G T P , d C T P and dTTP at pH 7.0), and 2 pmole of the random oligo primer (Canadian Life Technologies, Mississauga, ON) in a total volume of 20 pi. The RT mix was then incubated at 42°C for 2 min. The reaction was inactivated by heating at 70°C for 15 min. The 100 pi of P C R reaction contained 10% of the first strand reaction, 10X P C R Buffer (200 mM Tris-HCL, pH 8.4, and 500 mM KCI), 50 mM MgCI ,  Mix,  10  GATCCTGAAGGAGCTAGACG-3')  and  2  10  mM  dNTP  37  pM 10  of mM  the of  the  forward reverse  primer primer  (5'(5'-  A G A A G T G G A A C C A C T C G A T G - 3 ' ) , and 5 units/ul of the Taq DNA polymerase. Amplification was carried out as follows: 1) initial denaturation at 94°C for 3 min, 2) denaturation at 95°C for 45 sec, 3) annealing at 50°C for 1 min, 4) polymerization at 72°C for 2 min, 5) repeat of step 2 to step 4 for 40 cycles, and 6) final polymerization at 72°C for 5 min.  Samples were then electrophoresed on 1% agarose gels  containing 0.5 |ag/ml of ethidium bromide and visualized under UV light. Reaction mix with p C I - p 3 3  /,VGr  plasmid DNA was used as a positive control.  For semi-  quantitative P C R , 2 uJ of the cDNA samples from reverse transcription were diluted at 1/10 and 1/100. They were then amplified by P C R as described above.  2.13  Northern Blot Analysis  Total R N A was extracted by TriZol reagent and the concentrations were determined by UV spectrophotometry.  Samples were heated to 65°C and run on 1% agarose  gels containing formaldehyde and 0.5 ug/ml ethidium bromide.  After separation,  capillary transfer to nitrocellulose was performed overnight at room temperature and its efficiency assessed by U V light. The blot was then baked for 2 h in a vacuum oven at 80°C. Pre-hybridization was carried out by incubating the blot with a mixture containing 6 X S S P E , 5XDenhardt's reagent, 0.5% S D S , and 100 ug/ml yeast tRNA for 1 h at 65°C. The p33  ING1  probe was first made by amplifying a 577 bp fragment  by P C R using primer 1 ( 5 ' - G A T C C T G A A G G A G C T A G A C G - 3 ' ) and primer 2 (5'A G A A G T G G A A C C A C T C G A T G - 3 ' ) and then labeling it with a - P [ d C T P ] (10 mCi/ml) 32  according to the manufactured protocol in the Random Primers D N A Labeling System (Canadian Life Technologies, Mississauga, ON). Hybridization was carried  38  out by incubating the blot with the labeled probe at 65°C for 16-24 h. Filters were washed with 2 X S S C / 0 . 1 % S D S once for 15 min at room temperature and then three washes for 20 min each at 65°C.  Blots were visualized on X-ray films after an  overnight exposure.  2.14  Immunohistochemistry  All biopsies were frozen-sectioned at six-microns and mounted onto saline-coated slides.  They were then fixed in cold acetone for 2 min.  Using the ImmunoCruz  Staining Systems (Santa Cruz Biotechnology Inc, Santa Cruz, CA), serial sections were first blocked with horse serum for 20 min, then incubated with  p33  ING1  polyclonal antibody at 1:500 dilution for 2 h at room temperature, followed by two washes in P B S each for 2 min. Next, the sections were incubated with biotin-labeled anti-rabbit secondary antibody with avidin-biotin-peroxidase complex for 30 min, followed by two washes in P B S each for 2 min and staining with the H R P substrate containing DAB chromogen and peroxidase substrate for 30 sec to 5 min. Sections were immediately dehydrated two times in 95% ethanol for 10 min each, twice in 100% ethanol for 10 sec each, and three times in xylenes for 10 sec each. Sample slides had permanent mounting medium added, were covered with glass coverslips, and observed under a light microscope. Negative controls were done with exactly the same protocol described here except without the primary antibody.  2.15  Host-cell-reactivation Assay  The pCMVcat plasmid contains a gene encoding chloramphenicol acetyltransferase  39  (cat) under the transcriptional control of the immediate early promoter of the human cytomegalovirus. Samples of pCMVcat plasmid D N A were irradiated at 40, 80, or 480 m J / c m using an UV-crosslinker at 50 ug/ml final concentration and used for 2  transfection. 40 h after transfection, cells were harvested and the cell pellets were resuspended in 50 ul of 0.25 M Tris-CI (pH 8.0) and 5 mM EDTA. Cell-free extracts of the transfected cells were made by three repeated freeze-thawings (liquid nitrogen to freeze, 37°C to thaw), heated to 65°C for 10 min, centrifuged at 12,000g for 10 min, and the cleared supernatants were then used for C A T assays.  The  assay reaction mixtures contained 7.5 (al of 5 mM chloramphenicol, 50 ul of cell-free extract, 1 pi of 2.5 mM [ H]acetyl-CoA, and 16.5 pi of d H 0 . Reaction mixtures were 3  2  incubated at 37°C for 90 min. Following incubation, 200 ul of ice-cold ethyl acetate was added, tubes were shaken and centrifuged at 12,000 g for 5 min. After quick freezing the aqueous phases in a dry ice/ethanol bath, the organic phases were removed and extracted with 200 ul of distilled water. Organic phases were dried to completion and radioactivity was determined in a scintillation counter. Determinants were performed  in triplicates. Controls included transfection with undamaged  plasmid D N A and mock transfection without plasmid DNA.  2.16  Radioimmunoassay  Antisera were raised against DNA dissolved in 10% acetone and irradiated with U V B light under conditions that have been shown to produce cyclobutane pyrimidine dimers (CPDs) exclusively. 2-5 ug of heat-denatured sample D N A was incubated with 5-10 pg of poly(deoxyadenylate-deoxythymidylic acid) (labeled to >5 X 1 0  40  8  cpm/pg by nick translation with [ P]dTTP) in a total volume of 1 ml of 10 mM Tris 32  (pH 7.8), 150 mM NaCI, 1 mM EDTA, and 0.15% gelatin (Sigma, St. Louis, MO). Antiserum was added at a dilution that yielded 30-60% binding to labeled ligand, and, after incubation overnight at 4°C, the immune complexes were precipitated with goat anti-rabbit immunoglobulin (Calbiochem, San Diego, CA) or carrier serum from nonimmunized rabbits ( U T M D A C C , Science Park/Veterinary Division, Bastrop, TX). After centrifugation, the pellet was dissolved in tissue solubilizer ( N C S , Amersham, Piscataway, NJ) and mixed with ScintiSafe (Fisher, Hampton, NH) containing 0.1% glacial acetic acid, and the  3 2  P was quantified by liquid scintillation spectrometry.  Under these conditions, antibody binding to an unlabeled competitor antibody binding to the radio-labeled ligand. through  a  standard  (dose-response)  6  Sample inhibition is extrapolated  curve  photoproducts in 10 bases (i.e., CPDs/mb).  inhibits  to  determine  the  number  of  For the standard, we used double-  stranded salmon testis DNA (Sigma, Mississauga, ON) irradiated with increasing doses of U V C light and heat-denatured, aliquoted, and kept frozen at -20°C. Rates of photoproduct induction were quantified using nonimmunological enzymatic and biochemical techniques and determined to be 0.81 CPDs/mb/mJ/cm . 2  2.17  Immunoprecipitation  Cells were grown to - 8 0 % confluency in 100 mm tissue culture dishes and harvested for their lysates.  They were incubated with anti-ING1 antibody or a  nonspecific control anti-lnterleukin-12B rabbit polyclonal antibody (Santa Cruz, Santa Cruz, C A ) at 4°C for 1 h, then with protein A sepharose at 4°C overnight. The  41  beads were washed three times with solubilization buffer prior to boiling for 5 min. The precipitates were then resolved by electrophoresis, followed by Western analysis as described in section 2.9.  2.18  Propium Iodine (PI) Staining  Cells grown  on coverslips in 35 mm  plates were fixed with  1 ml of  1:1  acetone:methanol at -20°C for 10 min and allowed to air dry. Cells were then rehydrated with P B S for 2 min before staining with 50 ug/ml PI and 20 |ig/ml RNase A at 25°C in the dark for 10-30 min. Coverslips were washed twice with P B S , let air dry in the dark, mounted onto slides, and visualized under a fluorescent microscope (Nikon, Tokyo, Japan) for apoptotic bodies.  2.19  Flow Cytometry  Transfected cells were collected by trypsinization and pelleted by centrifugation at 2,000 X g for 5 min.  Cell pellets were then resuspended in 1 ml of hypotonic  fluorochrome buffer (0.1% Triton X-100, 0.1% sodium citrate, 25 ug/ml R N a s e A , and 50 ug/ml PI). After incubation at 4°C overnight, the samples were analyzed by flow cytometry to determine the percentage of subdiploid DNA.  2.20  Mitochondrial Transmembrane Potential Detection  Disruption of the mitochondrial transmembrane potential was detected using a MitoCapture™ Apoptosis Detection Kit (Calbiochem, S a n Diego, C A ) . The assay was performed according to the manufacturer's specifications. Briefly, cells were  42  grown in 35 mm plates and irradiated at 80% confluency. Following treatment, the medium was removed and the cells were incubated with 2 ml of  diluted  MitoCapture™ solution at 37°C in a 5% C 0 incubator for 15 min. After incubation, 2  the dye solution was removed and the cells were washed twice with 1 ml of the prewarmed incubation buffer.  The cells were then observed immediately under a  fluorescent microscope.  43  CHAPTER 3. EXPRESSION OF p33  ING1  3.1  IS INDEPENDENT OF p53  Rationale and Hypothesis  p53, a nuclear protein, regulates a number of downsteam targets such as p 2 1  vva  ^,  G A D D 4 5 , Bax, and Bcl-2. Studies from more than two decades indicate that p53 is a key mediator of cell cycle regulation, apoptosis, DNA repair, senescence, and sensitization to chemotherapeutic agents (Bond et al., 1994; Bunz et al., 1998; Li et al., 1997; 2000; Miyashita etal., 1994; Smith etal., 1994). Regarded as a "guardian of genome", p53 holds the title of being the most frequently mutated gene known to date (Hollstein et al., 1991). Evidence suggests that loss of normal p53 function is associated with cell transformation in vitro and the development of neoplasms in vivo (Finlay et al., 1989).  Under genotoxic stress conditions, p53 protein levels rapidly  increase in the cell. The accumulation of p53 induces the expression of p 2 1 potent inhibitor  of cyclin-dependent  kinase activity,  which  progression (Shaulsky et al., 1991; El-Deiry et al., 1993).  inhibits  l v a f t  , a  cell cycle  It has also been  demonstrated that p53 maintains genomic stability by enhancing DNA repair and apoptosis. W e recently demonstrated that loss of wt p53 function results in reduced DNA repair and apoptosis in mouse keratinocytes and fibroblasts after UV irradiation (Li etal., 1996; 1997; 1998a; Tron etal., 1998a; 1998b). A s a result of reduced DNA repair and apoptosis, mice with abnormal p53 function either by gene knockout or overexpression of  mutant p53 are predisposed to  development (Li etal., 1995a, 1998a).  44  UV-induced skin cancer  Our understanding of the biological function of /A/67 has improved over the last few years. One of the reasons that this gene product has gained increasing attention from the biological community is that, though it has no structural similarity with p53, both gene products share many of the tumor suppressive functions, including growth arrest, apoptosis, senescence, and sensitization to drug treatment. As well, overexpressed ING1 has been reported to physically associate with p53, further pointing to the importance of its role in carcinogenesis (Garkavtsev et al., 1998). Current studies show that overexpression of ING1 inhibits cell growth while chronic expression of ING1 antisense constructs promotes cell (Garkavtsev et al., 1996; 1998).  transformation  In addition, it was found that the function of cell  growth control is dependent on the activity of both ING1 and p53, and p21  waf1  has  been shown to be their downstream effector (Garkavtsev et al., 1998; Shinoura et al., 1999). Although ING1 shares functional similarities with p53, it is not known how the expression of ING1 is regulated. Since p53 is a well-known transcriptional factor for many downstream targets (El-Deiry et al., 1993; Miyashita et al., 1995; OwenSchaub et al., 1995), we hypothesized that p53 is necessary for the regulation of the p33  ING1  isoform expression in both normal and stress environment. Most studies  on ING1 came primarily from in vitro analysis using long-term cultured cell lines. Since many genetic changes may occur in this type of system, we chose to use the p53 knockout mouse model for this study. To investigate if p33  ING1  could be induced under stress conditions where p53  is frequently upregulated (Hall et al., 1993; Li et al., 1998a), we exposed fibroblasts  45  from p53  +/+  and p 5 3 " mice, NHEK, and a keratinocyte cell line (HaCaT), to U V B and v  compared the p33  ING1  immortalized,  protein levels in these cells.  non-tumorigenic  human  keratinocyte  HaCaT is a spontaneously cell  line  that  behaves  phenotypically like its normal counterpart in terms of patterns of growth and differentiation (Boukamp et al., 1988).  Besides the advantage of being similar in  many respects with its normal counterpart, this cell line lacks the functional p53 gene, allowing the study of the relationship between p53 and its associates. Another reason for using keratinocytes is that they are the primary target of U V B in the skin. As such, the data derived from them will be biologically relevant.  46  3.2  Results and Discussion  Studies that have examined the relationship between ING1 and p53 have mostly been done in vitro (Garkavtsev et al., 1998; Shinoura et al., 1999). Since p53 is a transcription factor that is known to initiate a whole host of molecular events by transactivating genes, we investigated if p53 could be the upstream regulator of  p33  ING1  p53'  A  p33  ING1  by first examining whether  was expressed in organs from p53  p33  ING1  mice. Results from R T - P C R showed that  liver, lung, heart, and skin of both p53  p33  ING1  quantitative R T - P C R indicated that heart of p53  +/+  +/+  p53' ' /  and  +/+  and  was expressed in the brain,  mice (Figure 3.1A).  Semi-  mRNA levels were virtually equal in the The data suggest that p33  and p53~ mice (Figure 3.1 B).  ING1  A  expression is independent of p53 status.  To further confirm p53-independent  expression of ING1, we used Northern blot analysis to compare m R N A levels in the brain, liver, lung, heart, skin, kidney, testis, and thymus of p53  and  Our results showed that there was no significant difference in  p33  +/+  expression between  p53  +/+  and  p53'  A  p53' ' /  ING1  mice.  mRNA  mice in all eight organs examined (Figure  3.1C). Next, we investigated whether p53 status affects p 3 3 post-transcriptional level. W e compared lung, heart, and skin between  p53  +/+  and  p33  ING1  p53' '  no substantial difference in the levels of p 3 3  /  / N G )  / W G )  expression at the  protein levels in the brain, liver, mice. Figure 3.2 shows that there is  protein between p53  +/+  and p53"  A  mice in all five organs examined. Recently, three other isoforms of the ING1 gene, which encode 47, 27, and 24 kD proteins, have been found (Saito et al., 2000). The anti-ING1 antibody  we used detected the 33 kD isoform predominantly. T o  47  Figure 3.1 Analysis of p33 m R N A expression of p53 and p53" organs. (A) R T - P C R analysis of m R N A level in different organs of p53 and p53~ mice. C 1 , negative control without R N A in the reaction. C 2 , positive control with pCI- p 3 3 ' plasmid D N A in the reaction. (B) Semi-quantitative R T - P C R analysis of p33 m R N A level in the heart of p53 and p53~ mice. A series of dilutions of the p 3 3 c D N A was performed and comparison was made between the p53 and p53~ groups. (C) Northern blot analysis of p33 m R N A expression levels in selected p53 and p53" organs. The 18S rRNA was used as an internal control. ING1  +/+  A  +/+  A  , W G  ING1  +/+  A  / W G I  +/+  ING1  +/+  A  Brain +/+  -/-  Liver +/+  -/-  Lung +/+  p33ING1  48  -/-  Heart +/+  -/-  Skin +/+  -/-  C1 C2  A  Figure 3.2 Western blot analysis of p33 expression levels in selected p53 p53" organs. (3-actin was used as internal control. A  Brain +/+ P33ING1  Actin  Liver  -/-  +/+  -/-  Lung +/+  mm  *mmm <  m  •» •  49  Heart -/-  +/+  -/-  Skin +/+  -/-  and  determine if ING1 expression was independent of p53 using an independent method, we  used immunohistochemical  expression in the brain between the p53  +/+  p53  +/+  and p53'  A  staining to and p53'  A  mice have similar levels of p33  ING1  look  at  p33  ING1  protein  groups. Our results show that  (Figure 3.3).  p53 plays a significant role in responding to DNA damage. hypothesis that p33  ING1  To test the  may also be a downstream target of p53 in stress conditions,  we exposed normal N H E K cells and the HaCaT cell line, which carries mutated p53 alleles (Lehman ef al., 1993), to U V B irradiation and examined the levels of ING1 protein.  Our results demonstrate that the amount of p33  protein is induced in  ING1  both N H E K and HaCaT cells after U V B irradiation (Figure 3.4A and B). densitometry, a 2-fold increase in the level of p 3 3 mJ/cm  2  ,WG  Using  * protein in N H E K cells at 40  and approximately 4-fold increase in HaCaT cells at 100 m J / c m were 2  observed. It was also noted that the expression of p33  ING1  in HaCaT cells is dose-  dependent, with a maximum induction at 80 m J / c m . The observation of similar 2  levels of p33  ING1  induction in N H E K and HaCaT cells by UV irradiation further  confirms p53-independent expression of To confirm that UV induces p33  ING1  p33 . ING1  expression at the transcriptional level and  that this was not due to post-translational modifications, p33  ING1  examined in HaCaT cells over a time-course.  m R N A levels were  Figure 3.4C shows that  p33  ING1  m R N A levels increase with time, starting at 4 h and peaking at 24 h. Using human fibroblasts, Garkavtsev and colleagues (1998) found that the DNA damaging agent adriamycin, did not induce ING1 expression. To determine if UV-induced p 3 3  I N G 1  in keratinocytes was tissue-specific, we exposed dermal  50  Figure 3 . 3 Immunohistochemical analysis of p33 in the brain of p 5 3 and p 5 3 " mice. (A) p 5 3 . (B) p53-'\ Arrows indicate p33 expressing neural cells Scale bar, 25 um. +/+  ING1  +/+  ING1  -/-  +/+ IK  '*  X *  A  B  51  4*  :..  Figure 3.4 p33 induction by UVB irradiation in keratinocytes. Proteins were extracted from the cells 24 h after U V B irradiation and subjected to Western blot analysis. (A) N H E K were exposed to UVB at 0 and 40 m J / c m . (B) HaCaT cells were exposed to U V B at 0, 20, 40, 80, and 100 m J / c m . p-actin was used as an internal control. (C) Northern blot analysis of p 3 3 * expression levels in HaCaT over a time-course. HaCaT cells were exposed to U V B at 80 m J / c m and total R N A was collected at 0, 1, 2, 4, 8, 24, and 48 h. The 18S rRNA was used as an internal control. ING1  2  2  ,WG  2  NHEK  40 mJ  wmmm  P33ING1  Actin  B  HaCaT 0  20  40  80  100 mJ P33ING1  -^"^p^  0  1  U P r  2  4  Actin  ^UPr  8  24  48 h p33ING1  18s  52  fibroblasts isolated from p 5 3 that neither p53  +/+  +/+  nor p53'  and p53" mice to 40 m J / c m of U V B . Our data show /_  2  fibroblasts exhibit any change in p33  A  expression  ING1  (Figure 3.5), suggesting that UV-induction of p33  ING1  is cell type-specific.  In this study, we found that wt p53 is not required for the expression of p33  ING1  in both normal and stress conditions, although their partnership has been  shown to be required for exerting biological effects such as growth inhibition and apoptosis (Garkavtsev et al., 1998; Shinoura et al., 1999). Similarly, Zeremski et al. (1999) reported that there was a lack of effect of p53 status on ING1 levels in murine p33  mammary gland cell lines under normal and stress conditions. Induction of by UV irradiation in human keratinocytes suggests a role of p33  ING1  in cellular stress  ING1  response and skin carcinogenesis. UV induction of p33  seen in keratinocytes suggests two possibilities: 1)  ING1  p33  ING1  is capable of responding to stress in terms of expression independent of p53  but cannot perform its cellular activities without functional p53, and 2) p 3 3  /WG  * is  capable of responding to stress and carries out its functions independent of p53. Further functional studies are needed to decipher the role of p33  ING1  stress response.  in the cellular  Because we have previously shown that p53 gene knockout  predisposes animals to UV-induced skin cancer development, reduces UV-induced DNA repair, and lowers the apoptotic rate (Li et al., 1995a; 1996; 1997; 1998a; Tron er al., 1998a), investigations on how p33  ING1  cooperates with p53 in keratinocytes to  modulate cell cycle arrest, DNA repair, and apoptosis will further determine the functional significance of p33  ING1  in the UV-induced stress response in particular,  and in skin carcinogenesis in general. To support the notion that p33  ING1  53  plays a  Figure 3.5 Western blot analysis of p33 expression in UV-irradiated murine fibroblasts. p 5 3 and p53" dermal fibroblasts were exposed to U V B at 0 and 40 mJ/cm . B-actin was used as an internal control. G  + / +  A  2  +/+ Fb  40  -/- Fb  0  40 mJ p33ING1  VHJMMP  ^liiiiM^  wnf0itt^  54  A^ctin  role in skin carcinogenesis, we have recently found that UV-induced murine squamous carcinomas express significantly higher levels of p33  ING1  p53 status in comparison to normal skin (data not shown).  regardless of  Although, current  mutational studies indicate that very few alterations are found in the ING1 gene in breast, ovarian, and gastric cancers (Toyama er al., 1999; Ohmori et al., 1999; Oki et al., 1999), ING1  mutation may only be specific to UV-induced skin cancers.  Future studies on ING1 mutational status in skin tumors will help reveal the nature of its ING1 induction. In conclusion, our results indicate that p33  ING1  p53 status in normal and stress conditions.  55  expression is dependent on  CHAPTER 4. p 3 3  4.1  ING1  MEDIATES REPAIR OF UV-DAMAGED DNA  Rationale and Hypothesis  Exposure to solar UV radiation (UVA and UVB) that penetrates the  earth's  atmosphere causes DNA damage in the skin (Griffiths et al., 1998). If the lesions are not properly removed, mutations and ultimately carcinogenic transformation may occur.  Consequently, an array of DNA repair systems has evolved in the cell to  maintain the integrity and stability of the genetic material. One of the most important and versatile  repair mechanisms is nucleotide excision repair (NER),  which  eliminates a wide variety of DNA damage, including the common UV-induced lesions, cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (Maeda et al., 2001).  The sequence of the N E R process consists of multiple-steps: lesion  recognition, strand incision, damaged nucleotide displacement, gap filing by DNA polymerization, and ligation (Emmert et al., 2000). Three rare autosomal recessive inherited  human  disorders  are  associated with  dysfunctional  NER  activity:  xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy, all of which are characterized by a high sensitivity to sunlight (Emmert et al., 2000; van Oosten et al., 2000). Loss of p53 function leads to decreased N E R of specific lesions. Several independent groups observed that cells lacking functional p53 exhibited defective repair of UV damage and were more sensitive to UV irradiation than their wt p53 counterparts (Smith etal., 1995; Ford etal., 1995; 1998; Havre etal., 1995; Zhan et al., 1996; Li etal., 1996; 1998a; Cistulli etal., 1998; Chang etal., 1999; El-Mahdy er  56  al., 2000; McKay er al., 2000). For instance, disruption of wt p53 function with either the human papillomavirus E6 oncoprotein or a mutant p53 transgene results in reduced repair of UV-induced DNA damage (Smith et al., 1995; Ford et al., 1998). Furthermore, DNA repair efficiency is reduced in fibroblasts from Li-Fraumeni patients with germline p53 mutations (Ford et al., 1995). p53 has also been shown to enhance y-radiation-induced DNA repair (Mallya er al., 1998).  Using  p53  transgenic mouse models, we previously demonstrated that repair of UV-induced DNA damage is decreased in mouse keratinocytes isolated from transgenic mice which carry mutant alleles of p53 and p53 knockout mice, both in vivo and in vitro (Li etal., 1996; 1997; Tron etal., 1998a). The exact mechanism by which p53 promotes DNA repair remains to be elucidated.  A likely possibility is the activation of, among other genes, the  downstream target, G A D D 4 5 . The gene was originally cloned on the basis of rapid induction in cells by UV light (Fornace et al., 1998). G A D D 4 5 enhances N E R (Smith et al., 2000).  Later it was shown that  Most evidence suggests that the  G A D D 4 5 response is p53-dependent (Zhan etal., 1996; Kastan etal., 1992). Smith et al. (1994) demonstrated that GADD45 stimulates N E R and inhibits entry of cells into S phase.  Furthermore, antisense DNA directed against G A D D 4 5 m R N A  reduced the repair of UV-damaged DNA (Smith et al., 1996).  Data from Hall and  colleagues (1995) have suggested that the DNA repair response is mediated by the binding of G A D D 4 5 to P C N A , a protein involved in cell cycle regulation and DNA repair.  Other lines of evidence suggest that p53 may directly participate in N E R  through a protein-protein interaction with components such as X P B and X P D ,  57  subunits of TFIIH involved in N E R , or by binding to sites of DNA damage (Wang et al., 1994; 1995).  Bakalkin and colleagues (1994) show that p53 has the ability to  bind to single-stranded DNA ends and catalyze DNA renaturation and strand transfer.  In vivo evidence demonstrates that when Xpc homozygous mutant (Xpc ") v  mice were crossed with p53'  A  mutants, the rate of development of UV-induced skin  cancer correlated with genotype as follows: Xpc' /p53' A  > Xpc' /p53 A  (n=14) > Xpc' /p53  Hence, inactivation of both p53  (n=19).  +/+  A  A  +A  (n=22)  alleles augments the  development of skin cancer in mice (Friedberg et al., 2001). More recently, studies suggest that there exists a physical association between p53 and B R C A 1 and B R C A 2 , both of which are important for cellular response to DNA damage by interacting with RAD50 and RAD51 (Zhong et al., 1999; Chai et al., 1999; Marmorstein etal., 1998). Recent findings that the tumor suppressor candidate /A/67 shares similar biological functions with p53 (Garkavtsev et al., 1998; Shinoura et al., 1999) and the two proteins physically bind to each other (Garkavtsev et al., 1998) led us to speculate that ING1 irradiation.  may also participate  UV-induction of p33  ING1  in cellular stress response to UV  seems to be a common phenomenon in  epidermal cells as we have shown that p33  ING1  is upregulated at the transcriptional  level in N H E K and a keratinocyte cell line, HaCaT (Cheung et al., 2000). hypothesized that p33  ING1  We  enhances repair of UV-damaged DNA in the presence of  functional p53.  58  4.2  Results and Discussion  W e first examined if p33  ING1  would respond to U V B in a human melanoma cell line,  M M R U , which contains wt p53 (Li et al., 1995b). W e found that there was a clear induction of p 3 3  , W G 7  protein with increasing UV doses (Figure 4.1 A and B). To test  the possibility that the induction was due to transcriptional regulation, we examined the R N A levels at various time points after UVB irradiation. induced p33  ING1  W e found that U V B -  was indeed a result of transcriptional control (Figure 4.1 C). These  results indicate that p 3 3  ,WG  * was induced in a dose- and time-dependent manner  after U V B irradiation. To study if p33  ING1  mediates DNA repair, we used the host-cell-reactivation assay  where a UV-damaged plasmid containing the chloramphenicol acetyltransferase reporter gene (pCMVcat) was co-transfected with either vector, p33 , ING1  p33  or antisense  expression vector into M M R U cells. The activity of the reporter gene was  ING1  used as an indicator of the extent of repair. overexpressing the p33  ING1  Our data demonstrated that cells  construct had significant increase in the repair rate of the  UV-damaged plasmid compared to the vector and antisense controls (p=0.01, 40mJ; p=0.01, 80mJ, student t-test) (Figure 4.2A).  This enhancement in repair was  maintained in conditions even when severely UV-damaged C A T plasmids (at 480 mJ/cm ) were used (p=0.01, t-test) (Figure 4.2A). To confirm the results from the 2  host-cell-reactivation assay, we performed radioimmunoassay for global genomic repair. The levels of the major UVB-induced photoproducts, C P D , were monitored in M M R U cells overexpressing p33 . ING1  The results  59  showed that the repair rate of  Figure 4.1 p 3 3 is UV-inducible in a dose- and time-dependent manner. (A) Western analysis of UVB-induced p 3 3 protein expression in M M R U cells. Cells were irradiated with U V B at 0, 10, 20, 40, and 80 m J / c m and harvested after 24 h incubation. An anti-ING1 antibody was used for primary antibody incubation and pactin was used as loading control. Lane C represents lysate from M M R U cells overexpressing the p C I - p 3 3 plasmid, confirming that the bands induced by UV irradiation were the p 3 3 ^ protein. (B) Densitometry of p 3 3 induction in (A). (C) Northern analysis of UVB-induced p 3 3 m R N A in M M R U cells. Cells were irradiated with U V B at 40 m J / c m and harvested at 0, 2, 4, 8, 12, and 24 h after UV exposure. The p 3 3 probe was first made by amplifying a 577 bp fragment by P C R using primer 1 ( 5 ' - G A T C C T G A A G G A G C T A G A C G - 3 ' ) and primer 2 (5'A G A A G T G G A A C C A C T C G A T G - 3 ' ) and then labeling it with a - P [ d C T P ] (10 mCi/ml) according to the manufactured protocol in the Random Primers DNA Labeling System (Canadian Life Technologies). 18s rRNA was used as loading control. I N G 1  I N G 1  2  ING1  G 1  I N G 1  I N G 1  2  I N G 1  32  10  20  40  80 mJ  Actin  60  Figure 4.2 p 3 3 enhances UV-damaged DNA repair. (A) Effect of p 3 3 on repair of UV-damaged plasmid DNA by host-cell-reactivation assay. Undamaged or UV-damaged pCMVcat plasmids were co-transfected with vector (V), p C I - p 3 3 (p33 ) or pCI-antisense p33 (AS) into M M R U cells and incubated at 37°C with 5% CO2 for 40 h. C A T activity was determined by scintillation counting and expressed as: net dpm damage dose/net dpm zero dose. Experiments were performed in triplicates. Shown are representatives of two independent sets of experiments. (B) Effect of p33 on repair of UV-damaged genomic D N A by radioimmunoassay. M M R U cells transfected with vector or p33 plasmids were irradiated with UVB at 20 mJ/cm and genomic DNA harvested at 0, 4, 24, and 48 h. The percentage of remaining C P D was then measured using antisera specific for C P D (data presented as average of two independent experiments). ,/VG7  //VCj7  /A/G7  ING1  ING1  ING1  2  A  61  C P D was nearly doubled in p33  //VG7  -transfected cells compared to the vector-  transfected control cells 24 h post-UV irradiation (Figure 4.2B). A s expected, the p53 protein was induced in a UV dose-dependent manner (Figure 4.3A). To examine the relationship between p 3 3  / W G 7  and p53 in D N A repair,  we disrupted the activity of endogenous wt p53 in M M R U cells by introducing the pED1 construct containing a dominant-negative mutantp53 (Johnson era/., 1991; Li et al., 2000). To confirm pED1 expression in the cells, an anti-p53 antibody, which recognizes both wt and mutant p53 proteins, was used. An elevated level of p53 was seen in pED1-transfected M M R U compared to the vector control, indicating Similar levels of p33  successful transfection (Figure 4.3B).  between p E D 1 -  ING1  transfected and control cells were observed (Figure 4.3B), eliminating the possibility that the overexpressed mutant p53 might block the expression of p33 . ING1  Using the  host-cell-reactivation assay, we noted that the repair enhancement of p33  ING1  was  dramatically suppressed in pED1 (mut p53)-transfected cells but restored in p E C H (wt p53)-transfected cells (Figure 4.3C), suggesting that p33  ING1  requires the  presence of p53 to repair damaged DNA. To  study the  examined if p33  pathways involved in p33  /A/G7  -mediated  DNA repair, we  is the upstream regulator of G A D D 4 5 , X P A , and X P B , all of  ING1  which have been shown to have significant involvement in D N A repair (Smith et al., 2000).  W e found that there was no change in expression in any of the  aforementioned proteins in M M R U cells overexpressing p33  ING1  indicating that p33  ING1  (Figure 4.4A),  is not the upstream regulator of them. To test the possibility  62  Figure 4.3 p33 -mediated DNA repair is p53-dependent. (A) Western analysis of p53 protein expression in UVB-irradiated M M R U cells. Cells were irradiated with UVB at 0, 10, 20, 40, and 80 m J / c m and harvested after 24 h incubation. A n antip53 antibody was used for primary antibody incubation and B-actin was used as loading control. (B) Western analysis of p53 and p33 proteins in M M R U cells transfected with the dominant-negative mutant p53 (pED1) expression vector. (C) Effect of p53 on p33 -mediated DNA repair. Host-cell-reactivation assay was performed on M M R U cells transfected with UV-damaged (40 mJ/cm ) pCMVcat plasmid and control vector (V), p33 , pED1 (mut p53), p33 + p E D 1 , p E C H (wt p53), or p33 + p E C H . 40 h later, C A T activity was measured using the undamaged pCMVcat as control. Experiments were performed in triplicates. Shown is a representative of two independent sets of experiments. //VG7  2  ING1  //VGr  2  ING1  )NG1  ING1  63  0  10  20  40  80 mJ p53  Actin  V  pED1 p53 p33ING1  mm  ^  Actin  &  J*  &  ^  <8>  64  Figure 4.4 ING1 physically interacts with G A D D 4 5 , but does not transcriptionally upregulate G A D D 4 5 , X P A , or X P B . (A) Effect of p33 on the expression of G A D D 4 5 , X P A , and X P B proteins. M M R U cells were transfected with vector alone (V), p33 , or antisense p33 (AS) expression vectors. 24 h after transfection, cells were harvested and their lysates were analyzed by Western blotting using antiG A D D 4 5 , anti-XPA, and anti-XPB antibodies, p-actin served as loading control. (B) Co-immunoprecipitation of ING1 with X P A , X P B , and G A D D 4 5 . M M R U total cell lysates were immunoprecipitated with a nonspecific control antibody (lane 1) or with the anti-ING1 antibody which recognizes different isoforms of ING1 (lane 2). Lane 3 indicates whole cell lysate control. Antibodies against X P A , X P B , and G A D D 4 5 were then used in Western analysis. The physical binding between ING1 and G A D D 4 5 was observed in three separate experiments. ING1  ING1  ING1  V  p33ING1  AS p33ING1 GADD45 XPA XPB Actin  B  1  2  3 !  GADD45 XPA XPB  65  that ING1 may physically associate with GADD45, X P A , and X P B , we performed immunoprecipitation and found that there was a weak physical association, as indicated by the intensity of the signal, between ING1 and G A D D 4 5 (Figure 4.4B). No binding was observed between ING1 and X P A / X P B (Figure 4.4B). For the first time, we demonstrated that overexpression of p33  ING1  enhances  N E R of both UV-damaged genomic DNA and exogenous plasmid DNA, further supporting the notion that p33  ING1  is a tumor suppressor. Nucleotide excision repair  is a crucial stress-response mechanism to maintain genomic stability. UV radiation damages DNA primarily in the forms of C P D and (6-4) photoproducts.  These  photoproducts are repaired by N E R , which involves a complex series of proteins that orchestrate the identification and removal of damaged DNA, addition of nucleotides, and finally re-ligation of the DNA strand (Sancar et al., 1994). If UV-induced DNA photoproducts are not promptly removed, they will in turn lead to mutation and skin carcinogenesis.  For instance, xeroderma pigmentosum patients who have defects  in N E R suffer a 1000-fold increase in skin cancer incidence (Kraemer et al., 1994). Wt p53 binds to and modulates X P B and X P D (Wang et al., 1995), two components of the TFIIH transcription unit which possesses helicase, A T P a s e and kinase activity (Wang er al., 1994). However, our results demonstrate that  p33  ING1  does not transcriptionally regulate or physically bind to X P A and X P B (Figure 4.4). The physical association between ING1 and G A D D 4 5 (Figure 4.4B) suggests that ING1 may be a crucial component in the GADD45-mediated nucleotide excision repair pathway.  The fact that GADD45 is upregulated by p53 and that p 3 3  , W G 7  requires the participation of functional p53 in DNA repair (Figure 4.3) further  66  supports the close functional association between the tumor suppressor ING1 and GADD45.  Increasing evidence has indicated that G A D D 4 5 is essential in UV-  damaged DNA repair and genome stability (Smith et al., 2000; Smith et al., 1996; Hollander et al., 1999).  Recently, an interesting report shows that G A D D 4 5 can  recognize UV-altered chromatin state and modulate DNA accessibility to repair proteins such as DNase I and T4 endonuclease V (Carrier et al., 1999). of interest to exploit the mechanistic role of p33  ING1  It would be  in this GADD45-mediated repair  process, since recent evidence also suggests that p33  ING1  plays a role in chromatin  remodeling (Skowyra et al., 2001; Vieyra et al., 2002a; 2002b; Kuzmichev er al., 2002). In conclusion, our results strongly support the hypothesis that  p33  ING1  enhances N E R of UV-damaged DNA in the presence of functional p53. Since there is a strong causal relationship between UV exposure and melanoma formation, loss or inactivation of p33  ING1  can potentially contribute to neoplastic development.  67  CHAPTER 5. p33'  5.1  NG7  ENHANCES UVB-INDUCED APOPTOSIS IN MELANOMA CELLS  Rationale and Hypothesis  The incidence of melanoma is rising at a rate second only to lung cancer in women (Chin et al., 1998; Mackie et al., 1998).  It is estimated that the incidence of  melanoma has increased by 15-fold in the past 60 years (Glass et al., 1989; Koh et al., 1995).  Melanoma is among the most deadly cancers as it can rapidly  metastasize to other organs and its 5-year survival rate still remains at less than 10% (Koh et al., 1991; Roses et al., 1991).  Epidemiological studies strongly  implicate UV radiation as the main environmental risk factor for melanoma (Mackie er al., 1998; Gilchrest et al., 1999).  It is well-known that UV irradiation causes  damage to DNA, which can lead to mutation and carcinogenesis if the DNA damage is not removed promptly.  W e and others have previously shown that the tumor  suppressor p53 plays a crucial role in the process of removal of UV-induced DNA damage either by nucleotide excision repair or apoptosis (Li et al., 1996; 1997; 1998a; Tron er al., 1998a; Ziegler et al., 1994).  However, mutational analysis of  melanoma biopsies revealed that p53 mutation occurs in only approximately 15% of human melanomas (Zerp et al., 1999; Akslen et al., 1998; Sparrow et al., 1995b; Weiss et al., 1995), suggesting that other genes might be involved  in the  development of melanoma. Due to its functional similarity with p53, we investigated the role of p33  ING1  cellular stress response to UV irradiation.  W e previously found that  in p33  ING1  expression was induced in a dose/time dependent manner after UV irradiation in  68  both keratinocyte and melanoma cells (Cheung et al., 2000; 2001b).  W e also  demonstrated that the ability of melanoma cells to repair UV-induced DNA damage could be enhanced by the presence of p33 . ING1  lend credibility to the idea that p33  ING1  irradiation.  Studies from other groups further  has a significant role in stress response to UV  For example, Scott et al. (2001a) recently demonstrated that ING1  possesses two distinct nucleolar targeting sequences (NTS) within the nuclear localization signal region, which promotes the translocation of its encoded products to the nucleolus after UV irradiation. p33  ING1  was also found to contain a common  octapeptide motif called the PCNA-interacting-protein (PIP) domain at the amino terminus, through which it binds competitively to the interdomain connector loop of P C N A upon UV irradiation (Scott et al., 2001b; Warbrick et al., 1998; Tsurimoto et al., 1999). These authors also found that human fibroblasts overexpressing  p33  ING1  have a higher percentage of apoptosis compared to cells receiving vector controls. In order to investigate the molecular pathways of p33  ING1  enhancement in UV-  induced apoptosis in biologically relevant cells, we hypothesized that p33  ING1  p53 synergistically enhance UV-induced cell death in melanoma cells.  69  and  5.2  Results and Discussion  Information on the role of ING1  in cellular stress response to UV irradiation is  lacking. There are only four studies to date indicating that ING1 has a role in such condition.  Specifically, the expression of p33  ING1  was found to be induced by UV  irradiation in a dose-/time-dependent and tissue-specific manner (Cheung et al., 2000; 2001b).  Overexpression of the p33  isoform appeared to enhance DNA  ING1  repair in melanoma cells and apoptosis in fibroblast cells (Cheung er al., 2001b; Scott et al., 2001a; 2001b). The p33  isoform has also been shown to translocate  ING1  to the nucleolus and bind to P C N A after UV irradiation (Scott et al., 2001a; 2001b). To further investigate the role of p33  in UV-induced apoptosis, we transfected a  ING1  melanoma cell line, M M R U , with either p33  ING1  or antisense p33  ING1  expression  vector. Western blot analysis confirmed the expression of these plasmids (Figure 5.1A), suggesting successful transfection.  Using the trypan blue exclusion assay,  we determined the cell death rate of p33  -overexpressing M M R U cells after U V B  /A/G7  irradiation in comparison to cells transfected with vector alone or antisense and found that overexpression of p33  ING1  p33 , ING1  consistently enhanced cell death at various  doses of U V B (p=0.01, 40mJ; p=0.02, 80mJ; p=0.03, 120mJ; t-test) (Figure 5.1 B). Induction of apoptosis by UV was confirmed by PI staining. The chromatin in cells undergoing apoptosis became condensed to form apoptotic bodies in the nuclei, a typical feature of apoptosis (Figure 5.1 C). Quantitative data of PI staining indicate that p33 apoptotic  ,A/G7  -overexpressing M M R U cells present significantly more condensed  bodies than  the  controls (p=0.004, t-test) (Figure 5.1 D).  70  Figure 5.1 Effect of p 3 3 ' on UVB-induced cell death in M M R U cells. (A) Western blot analysis of p33 expression in M M R U cells transfected with pCI-vector (V), pCI-p33 (p33 ), and pCI-antisense p 3 3 (AS). A n anti-p33 polyclonal antibody was used for primary antibody incubation and B-actin as loading control. (B) Cell death assay by trypan blue exclusion of UVB-irradiated M M R U cells transfected with pCI-vector (V), pCI-p33 (p33 ), and pCI-antisense p 3 3 (AS). 24 h after transfection, M M R U cells were irradiated with U V B at 0, 40, 80, and 120 m J / c m . Trypan blue exclusion assay was then performed 24 h after U V B irradiation. Data represent mean + S D from triplicate plates. The experiment was repeated twice with similar results. (C) PI staining images of apoptotic cells after UVB irradiation. M M R U cells were transfected with p C I - p 3 3 and exposed to 80 m J / c m as above. 24 h after UVB irradiation, the cells were stained with PI, and images were taken using a fluorescent microscope. Cells without U V B irradiation were used as control. (D) Quantitative data from PI staining of UVB-irradiated M M R U cells transfected with pCI-vector (V), p C I - p 3 3 (p33 ), and pCIantisense p33 (AS). Data represent mean + S D from triplicate plates. (E) Microscopic images of UVB-irradiated M M R U cells transfected with vector (V), pCIp33 (p33 ), and pCI-antisense p 3 3 (AS). 24 h after transfection, M M R U cells were irradiated with U V B . Photographic images were taken 24 h after U V B irradiation. (F) Quantitation of cell death by flow cytometry. M M R U cells were irradiated with U V B at 0 and 80 m J / c m 24 h after transfection. Cells were collected by trypsinization 24 h after UVB irradiation and analyzed by flow cytometry. Experiments were performed twice with similar results. ,A/b  ,/VG7  7  / W G 7  ING1  /A,G7  /A/G7  / N G 7  ING1  2  /A/G7  2  /WG7  ING1  ING1  /WG7  ING1  2  71  ING1  72  UV  P33ING1  73  OmJ  80mJ  p33!NG1 4.9%  5.7%  0 mJ .  O  200  j L.  400  600  k 800 1000  Lu, _J 0 200 400  600  600 1000  200  0  400  600  800  1000  CO  31.7%  44.8%  25.5%  ©  80 m J  §  0  200  400  600  800 1000  0  200  400  ^ A 600  S00 1000  DNA Content  74  o CM  L  0  200  .  400  600  800  1000  Similarly, microscopic images show that there is significantly less spindle-shaped live cells in M M R U cells overexpressing p33  24 h after U V B irradiation compared  ING1  to vector- and antisense-control cells (Figure 5.1E). To further confirm the role of p33  ING1  in UV-induced apoptosis, we performed flow cytometry analysis, and the  results indicate that cells overexpressing p33  displayed more sub-G1 population  ING1  compared to the controls (Figure 5.1 F), which is consistent with the results from the trypan blue assay and PI counts. W e have previously shown that the tumor suppressor p53 plays an essential role in cellular stress response to UV irradiation, such as enhancement of DNA repair and promotion of apoptosis (Li et al., 1996; 1997). Recent findings that the tumor suppressor ING1 physically binds to p53 (Garkavtsev et al., 1998) and that adenovirus-mediated transfer of p33  ING1  with p53 synergistically induced apoptosis  in glioma cells (Shinoura et al., 1999) led us to hypothesize that p33  ING1  and p53  may work together in the enhancement of UV-induced apoptosis in melanoma cells. W e transfected M M R U cells with vector, p33 ,  p E C H (wt p53), or p33  ING1  ING1  + pECH,  and exposed to U V B irradiation at 80 mJ/cm . Using flow cytometry analysis, we 2  found that overexpression of wt p53 alone had no effect on UV-induced cell death in M M R U cells (Figure 5.2A), similar to the findings by Shinoura et al. (1999) that overexpression of wt p53 alone did not significantly induce apoptosis in glioma cells. However, co-expression of p33  ING1  t-test) (Figure 5.2A).  and p53 shows synergistic enhancement (0.001,  This cooperation between p33  ING1  and p53 has also been  observed in other cellular responses such as nucleotide excision repair of UVdamaged DNA (Cheung et al., 2001b). To confirm that p53 is required for  75  p33 ING1  Figure 5.2 Synergistic effect of p 3 3 and p53 on UVB-induced cell death in M M R U cells. (A) Cell death assessment using flow cytometry on UVB-irradiated M M R U cells transfected with pCI-vector (V), pCI-p33 (p33 ), p E C H (wt p53), and p C I - p 3 3 + p E C H . M M R U cells were irradiated with UVB at 0 and 80 m J / c m 24 h after transfection. Sub-G1 population represents dead cells. Experiments were performed in triplicate. (B) Trypan blue exclusion assessment of UVB-irradiated M M R U cells transfected with pCI-vector (V), pCI-p33 , p E C H , p C I - p 3 3 + p E C H , and pCI-p33 + pED1 (mut p53). The procedures for transfection and U V B irradiation were performed as in (A). Percentages of dead cells were determined 24 h post U V B irradiation. (C) Western analysis of p53 protein expression in M M R U cells transfected with pCI-vector (V), pCI-p33 and pCI-antisense p33 (AS). Western analysis was performed 24 h after transfection. p-actin was used as a loading control. ,A/G7  ,/VG7  /Afe7  /A/G7  2  ,A/G7  /WG7  ,A/G7  /A/G7  76  ING1  70 - • v • P33ING1  60  H wt p53  50  • p33ING1 + wtp53  40 - • p33ING1+ mut p53 30 20 10 0 0  80  UVB dose (mJ/cm ) 2  V  p33ING1  AS p33ING1 mamumm p53 Actin  77  induced apoptosis, we transfected M M R U cells with vector, p33 \ INU  p33  ING1  + p E C H , or p33  p E C H (wt p53),  + pED1 (mut p53). Trypan blue exclusion assay was  ING1  performed after exposure to UVB irradiation at 80 mJ/cm . The results show that, 2  similar to those of flow cytometry analysis, significant enhancement in cell death was observed in cells transfected with p33  + p E C H (p=0.002, t-test) (Figure 5.2B).  ING1  However, this synergy was diminished when the wt p53 plasmid (pECH) was replaced by a mutant p53 expression vector (pED1) (p=0.13, t-test) (Fig 5.2B). To further confirm that cooperation between p 3 3 not due to the ability of p33  ING1  / N G 7  and p53 in UV-induced apoptosis is  to elevate p53 expression, p53 protein levels were  determined in M M R U cells transfected with vector, p33 ,  and antisense p33  ING1  Western blotting. The results indicate that p33  ING1  by  ING1  does not induce the expression of  p53 protein (Figure 5.2C). To further provide evidence of p53-dependence of p33  ING1  in UV-induced  apoptosis, we transfected a melanoma cell line M E W O that contains mutant p53, with vector, p33 , ING1  or antisense p33  and irradiated the cells with U V B . Twenty-  ING1  four hours later, the percentage of cell death was determined by trypan blue exclusion. Our results indicate that overexpression of p33  ING1  did not enhance U V B -  induced cell death in the absence of functional p53 (p=0.90, 20mJ; p=0.37, 40mJ; p=0.41, 80mJ; p=0.12, 120mJ; t-test) (Figure 5.3A). Flow cytometry analysis also supported the data from trypan blue assay (Figure 5.3B). These results were further supported by microscopic images showing similar survival among cells transfected with vector, p33 , ING1  and antisense p33  ING1  24 h after U V B irradiation (Figure 5.3C).  To eliminate the possibility that this observed lack of enhancement by  78  p33  ING1  Figure 5 . 3 Effect of p33 on UVB-induced cell death in M E W O cell line. (A) M E W O cells were transfected with pCI-vector (V), pC\-p33 (p33 ), and pCIantisense p33 (AS). 24 h post-transfection, cells were irradiated with U V B at 0, 20, 40, 80, and 120 m J / c m . Trypan blue exclusion assay was then performed 24 h after U V B irradiation. Experiments were performed in triplicate. (B) Twenty-four hours after transfection, cells were exposed to 120 m J / c m of U V B and sub-G1 population was determined by flow cytometry 24 h post U V B irradiation. (C) Selected microscopic images of UVB-irradiated M E W O cells transfected with vector (V), p C I - p 3 3 (p33 ), and pCI-antisense p33 (AS). (D) Western analysis of p33 protein expression in M E W O and M M R U cells. (3-actin was used as a loading control. ING1  ING1  ING1  ING1  2  2  /A/G7  ING1  ING1  ING1  B 0 mJ c  3  O  o  120 mJ  0  200  400  600  800  1000  0  200  400  600  800  1000  DNA Content  79  0  200  400  600  800  1000  0 mJ  120 m J  V  \ •  •,  •*•'- A  ; .,\  p33ING1  AS  D MEWO  MMRU •  ^Ml^^^B  wrfb^HW  p33ING1  ^^^^ Actin  80  overexpression was not due to the presence of lower endogenous  p33  INU1  expression in the M E W O cell line compared to M M R U , we assessed the levels of p33  protein in both cell lines using western analysis. Figure 5.3D shows that  ING1  there is no obvious difference in p33  ING1  expression between these two cell lines.  Activation of the mitochondrial cell death pathway has been shown to be involved in UV-induced apoptosis (Antonsson et al., 2001; Green et al., 1998). To investigate if p33  ING1  activates the mitochondrial apoptosis pathway after U V B  irradiation, cells transfected with p 3 3  , W G 7  or vector alone were irradiated with 80  m J / c m of U V B and stained with a cationic dye using the MitoCapture™ Apoptosis 2  Detection kit 24 h after UVB irradiation. aggregates  in the  mitochondria  in  The cationic dye accumulates and  healthy  cells and  emits  an  orange-red  fluorescence. In apoptotic cells, the dye remains in the cytoplasm due to alteration in mitochondrial membrane potential, and emits a green fluorescence. Stained cells were visualized under a fluorescent microscope.  Figure 5.4A shows that control  cells stained orange-red while green fluorescent staining (apoptotic cells) was observed in cells exposed to U V B . There is a significantly higher percentage of green-stained cells in p33  ,A/G7  -transfected cells compared to vector controls (25.1%  vs 12.7%) (Figure 5.4B). These data suggest that p33  ING1  enhances UVB-induced  apoptosis by altering mitochondrial membrane potential. Studies have revealed that Bax is a p53 downstream target and involved in the mitochondrial apoptosis pathway (Shimizu er al., 1999).  Bax and  other apoptotic inducers are capable of facilitating the opening of the voltage dependent ion channel in the outer mitochondrial membrane, therefore inducing the  81  Figure 5.4 p33 alters mitochrondrial membrane potential and increases Bax expression. (A) Disruption of the mitochondrial transmembrane potential was detected using the MitoCapture™ Apoptosis Detection Kit (Calbiochem). Shown here are representative images of M M R U cells transfected with pCI-vector (V) and pCI-p33 , and irradiated with U V B . Live cells are indicated by red fluorescence and apoptotic cells by green fluorescence. (B) Quantitation of apoptotic cells in (A). A total of 500 cells were counted from randomly selected fields. Percentage of dead cells was determined from the number of apoptotic cells, represented by green fluorescent cells, over the total number of cells counted. The experiment was repeated twice with similar results. (C) Western analysis of Bax protein. M M R U cells transfected with pCI-vector (V), p C I - p 3 3 , and pCI-antisense p33 (AS) were irradiated with UVB at 0 and 80 mJ/cm . 24 h later, lysates were prepared and western analysis was performed using an anti-Bax polyclonal antibody for primary antibody incubation and p-actin as loading control. (D) Densitometry of Bax induction in (C). ING1  /A/G7  /NG)  2  82  ING1  83  0 mJ V  80 m J  p33ING1  AS  V  p33ING1  AS  Bax Actin  0  80  UVB dose (mJ/cm2)  84  release of cytochrome c and promoting a chain reaction ultimately leading to cell death (Shimizu et al., 1999). p33  ING1  Recently, Nagashima et al. (2001) reported that  can upregulate Bax promoter in colorectal carcinoma R K O cells.  investigate if p33  activates endogenous Bax and to confirm the role of p33  ING1  ING1  To in  the mitochondrial apoptotic pathway, we compared the levels of Bax protein in cells transfected with vector, p33 , ING1  irradiation.  and antisense p33  ING1  before and after U V B  Our results demonstrate that cells overexpressing p33  ING1  have higher  Bax expression compared to vector and antisense controls, while U V B irradiation increases Bax expression in all three groups but most significantly in cells overexpressing p 3 3  / N G 7  (Figure 5.4C and D).  Taken together, our data provide support to the hypothesis that this alternatively spliced form, p33 , ING1  enhances UVB-induced apoptosis in melanoma  cells and that this enhancement requires the participation of p53. It is interesting to note that p33  ING1  is also capable of enhancing repair of UV-damaged DNA. S o  under what different circumstances does p33  ING1  cell death? Presumably, p 3 3  / W G 7  participate in DNA repair or induce  may work in a manner similar to p53, where DNA  repair is a dominant function at relatively low doses of UV irradiation, while induction of apoptosis becomes the main stress response mechanism at high UV doses (Li et al., 1998a). In this report, we have also shown that p 3 3  / W G 7  upregulates the expression of  endogenous Bax protein and alters mitochondrial membrane potential. Since only about 15% of melanoma cases contain p53 mutation, other genetic alterations must occur during the course of melanoma development.  85  With the ever increasing  evidence that p33  ING1  plays an important role in cellular stress response, such as  DNA repair and apoptosis, to UV irradiation, mutation and/or abnormal expression of the p33  ING1  gene may be a crucial step during melanoma development.  More  recently, we observed increased expression, and several missense and silent mutations of ING1 in human melanoma cell lines (Campos et al., 2002). Although p33  the mutation rate in these culture cells was relatively low but their pattern of expression and mutation follow that of p53.  ING1  p53 protein has been shown to be  overexpressed in majority of human melanoma biopsies (Stretch et al., 1991; Lassam et al., 1993), while mutation of the p53 gene occurs only in 15-25% melanomas. Nevertheless, the degree of p53 overexpression is shown to be closely associated with tumor invasion, chemoresistance, and poor prognosis (Sparrow et al., 1995a; E s s n e r e f a / . , 1998; Whiteman etal., 1998). Our  novel finding  that  p33  cooperates with  ING1  p53 to  activate  mitochondrial pathway not only provides a better understanding of the p33  ING1  the  role in  UV stress response, but may also open another avenue for the prevention and treatment  of the highly chemo- and radio-resistant life-threatening  melanoma.  86  disease  -  CHAPTER 6. p 3 3  /NGt  DOES NOT ENHANCE CAMPTOTHECIN-INDUCED  CELL DEATH IN MELANOMA CELLS  6.1  Rationale and Hypothesis  Cutaneous malignant melanoma is a severe and life-threatening skin cancer. Currently there is no effective treatment for metastatic melanoma.  One of the  obstacles in melanoma treatment is its resistance to chemotherapy. Recently, it has been shown that apoptosis is a common mode of action for various anticancer drugs, such as camptothecin (CPT), and that the expression of apoptotic genes, such as p53, mediates chemosensitivity in melanoma cells (Li et al., 1998b; 2000). However, the factors that determine chemosensitivity in melanoma are poorly understood. C P T is a naturally occurring alkaloid compound identified during the 1960s in a screen of plant extracts for antitumor drugs (Wall et al., 1995). C P T belongs to a class of DNA-damaging agents that bind irreversibly to the DNA-topoisomerase I complex, inhibiting the reassociation of DNA after cleavage by topoisomerase I and traps the enzyme in a covalent linkage with DNA.  The enzyme complex is  ubiquinated and eliminated by the 26S proteosome, therefore depleting cellular topoisomerase I (Desai et al., 1997). W e selected C P T as a testing agent in this study due to its relative novelty and the fact that some of its derivatives, such as topotecan and irinotecan, are FDA-approved and demonstrating promising results in clinical trials. A s a class of chemotherapeutic agents, they also possess some of the best anticancer/toxicity ratios among experimental drugs (Saleem et al., 2000).  87  Information on the role of ING1 in chemosensitivity is lacking. Only one study provided evidence that the p24  ING1  isoform is capable of enhancing chemosensitivity  in human fibroblasts containing wt p53 after exposing to etoposide. To determine if the p33  ING1  p33  ING1  isoform also had a role in chemosensitivity, we hypothesized that  overexpression enhances CPT-induced cell death in melanoma cells.  88  6.2  Results and Discussion  In this study, we investigated whether p 3 3 melanoma cells.  / N G 7  enhances CPT-induced cell death in  W e first confirmed that the melanoma cell line, R P E P , was  transfectable and that the p33  ING1  construct could be expressed (Figure 6.1A). Next,  we compared the cell survival rate among cells overexpressing p33  ING1  (vector and antisense p33 ) ING1  and controls  after treatment with various doses of C P T .  Results  from the S R B assay indicated that after 24 h of drug treatment, there was no significant difference in cell survival among the three groups (p=0.24, 25nM; p=0.75, 100nM; p=0.95, 400nM; t-test) (Figure 6.1 B). No significant difference was observed between p33  /WG7  -expressing cells compared to the controls at the 48 h time point as  well (p=0.62, 25nM; p=0.22, 100nM; p=0.71, 400nM; t-test) (Figure 6.1 C).  In  addition, there is no significant change in cell morphology among three experiment groups (Figure 6.2). To further confirm the observations from the S R B cell survival assay, we used flow cytometry analysis to assess the frequency of CPT-induced cell death in cells transfected with vector alone, p33 , ING1  or antisense p33 .  Results  ING1  from flow cytometry analysis were consistent with those of S R B staining (Figure 6.3), indicating that overexpression of the isoform p33  ING1  or suppression of p33  ING1  by  antisense has little or no effect on cell death induced by C P T in melanoma cells. This is further confirmed by results from the S R B cell survival assay using another melanoma cell line, M M R U (Figure 6.4). To eliminate the possibility that there may be insufficient levels of p53 expression in the cells we used, we investigated if co-expression of both p33  ING1  and  p53 genes would enhance CPT-induced cell death in R P E P melanoma cells. Figure  89  Figure 6.1 Survival rate of CPT-treated R P E P cells transfected with p33 and antisense p33 . (A) Western blot analysis of p33 expression in R P E P cells transfected with pCI-vector (V), p C I - p 3 3 ^ (p33 ), and pCI-antisense p33 (AS). An anti-p33 polyclonal antibody was used for primary antibody incubation and p-actin as loading control. (B) Visual representation of S R B staining on transfected R P E P cells after 24 h treatment with C P T at 0, 25, 100, and 400 nM. (C) Spectrophotometry readings of S R B assay from (B); 48 h time point was also added. Experiments were performed in triplicate. INU1  ING1  ING1  G7  ,A/GJ  90  ING1  ING1  V  p33ING1  p33ING1  ^  Actin  91  AS  Figure 6.2 Microscopic images of CPT-treated R P E P transfectants. R P E P cells were transfected with vector (V), pCI-p33 ' (p33 ), and pCI-antisense p33 (AS) for 24 h and then treated with 0 or 400 nM C P T . Cells were viewed and photographed using a Nikon microscope with a 10X objective. ,A/G  Control  ING1  ING1  400 nM  92  Figure 6.3 Quantitation of cell death by flow cytometry. Sub-G1 population ING1 represents dead cells. V, vector; p33 , p C I - p 3 3 ; A S , pCI-antisense p 3 3 ; Ctl, no C P T treatment; C P T , 400 nM treatment for 24 h. ,A/GI  V  p33ING1  DNA C o n t e n t  93  ,A/G)  AS  Figure 6.4 Effect of p 3 3 on M M R U cell survival after C P T treatment. Cells were transfected with vector (V), p33 (p33 ), or antisense p 3 3 (AS) for 24 h and then treated with 0, 50, 200, or 400 nM of C P T . Cell survival was determined by S R B assay. Data represent mean + S D from three independent experiments. / W G )  ING1  ING1  94  , N G 7  6.5 shows that overexpression of both constructs, p 3 3  ,A/b7  and p E C H (containing  human wt p53), had no enhancement on CPT-induced cell death compared to either one alone. p33 , ING1  Taken together, our data indicate that the alternatively spliced form,  does not enhance chemosensitivity in melanoma cells after C P T treatment. Since the cloning of ING1 in 1996, there has been significant progress in  terms of establishing this gene as a tumor suppressor and deciphering relationship with p53.  its  Numerous tumor suppressive functions of ING1 have been  observed, including G1 cell cycle arrest (Garkavtsev et al., 1996), anchoragedependent growth (Garkavtsev et al., 1996), senescence (Garkavtsev et al., 1997), apoptosis (Helbing et al., 1997; Shinoura et al., 1999), DNA repair (Cheung et al., 2001), and chemosensitivity (Garkavtsev et al., 1998). ING1  The interesting fact that  produces a number of variants and that these variants may have different  effects illustrates the intricacy of the biological function of ING1  (Skowyra et al.,  2001; Zeremski etal., 1999). Human melanoma is highly resistant to chemotherapy.  W e previously  showed that overexpression of mutant p53 in a wt p53 melanoma cell line rendered more resistance to C P T treatment (Li et al., 2000). Similarly, Kim et al. (2001) found that introduction of wt p53 enhanced chemosensitivity in a poorly  differentiated  human thyroid cancer cell line in the presence of adriamycin.  Nguyen and  colleagues (1996) also showed that the transfer of wt p53 into cisplatin-treated H1299 cells, in which p53 is homozygously deleted, resulted in up to 60% inhibition of cell proliferation compared to controls. However, p53-enhanced chemosensitivity  95  Figure 6.5 Effect of p33 and p53 co-expression on melanoma chemosensitivity. R P E P cells were transfected with vector (V), p C I - p 3 3 (p33 ), p E C H (p53), or p C I - p 3 3 ' + p E C H for 24 h, and treated with 0 or 400 nM of C P T for 24 h. Flow cytometry was performed to quantitate the sub-G1 cells. Ctl, no C P T treatment; C P T , 400 nM treatment for 24 h. The experiment was repeated twice with similar results. ING1  /A/G7  ING1  ,A/G  V  P33ING1  p53  DNA Content  96  p33ING1+p53  appears to be cell-type and/or drug specific. For example, a study by Zhu et al. (2001) demonstrated that p73, but not p53, is capable of sensitizing M C F 7 cells to apoptosis induced by a number of chemotherapeutic agents. And in our study, we showed that overexpression of wt p53 in a cell line with normal p53 function also did not enhance CPT-induced cell death, indicating that p53 status may only be partially responsible for melanoma chemoresistance. Recent findings that loss of Apaf-1 (a p53 downstream target) expression by hypermethylation  also contributes  to  melanoma chemoresistance (Soengas et al., 2001) further suggest the complexity of the molecular pathway of chemosensitivity. Although p33  ING1  has been shown to  cooperate with p53 to exert a number of tumor suppressive effects, this cooperation does not seem to exist in CPT-induced cell death in melanoma cells.  97  Chapter 7. CONCLUSIONS  7.1  Summary  The biological functions of the tumor suppressor gene, ING1, have been studied extensively in the last few years since it was cloned in 1996 by Garkavtsev and colleagues. p27 ,  Four alternatively spliced forms of ING1, named p47 ,  and p24 ,  ING1  p33 ,  ING1  ING1  ING1  have been identified and some found to share many biological  functions with those of p53. Some of these isoforms have previously been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, and chemosensitivity. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. In this thesis, we sought to further characterize the various aspects of the  p33  ING1  isoform. W e first investigated how the expression of ING1 is regulated in normal and stress conditions. expression of p33  ING1  in p33  ING1  Using a p53-knockout mouse model, we examined if the  is dependent on p53. W e found that there was no difference  m R N A and protein levels between p53  +/+  and p53"~ murine organs. In 7  addition, when normal human epithelial keratinocytes and a keratinocyte cell line, HaCaT, which lacks wt p53 function, were exposed to U V B irradiation, the expression levels of p33  ING1  were elevated in both normal human epithelial  keratinocytes and HaCaT cells. It is interesting, however, that U V B irradiation did not induce p33  ING1  expression in dermal fibroblasts isolated from p53  +/+  and p53"  mice. Based on our findings, we therefore concluded that the expression of is independent of p53 status. UV induction of p33  ING1  98  A  p33  ING1  in keratinocytes suggests that  p33  may play a role in cellular stress response and skin carcinogenesis. The  ING1  finding that the expression of the p 3 3  W G ?  isoform is induced by UV irradiation in a  dose-/time-dependent and tissue-specific manner prompted us to investigate if p33  plays a role in UV-stress response, such as repair of UV-damaged DNA.  / W G 7  W e found that overexpression of p33  ING1  enhances repair of UV-damaged DNA and  that p53 is required for the repair process in melanoma cells. Furthermore, physical binding between ING1 and G A D D 4 5 was detected by immunoprecipitation. observations suggest that p33  cooperates with p53 in nucleotide excision repair  ING1  and that G A D D 4 5 may be one of its components. molecular pathways of p33  ING1  These  Next we investigated the  enhancement in UV-induced apoptosis in biologically p33  relevant cells using melanoma cell lines. W e found that overexpression of increased while the introduction of an antisense p33  ING1  ING1  plasmid reduced the  apoptosis rate in melanoma cells after UVB irradiation. W e also demonstrated that enhancement of UV-induced apoptosis by p33  ING1  p53. Moreover, we found that p33  ING1  again required the presence of  enhanced the expression of endogenous Bax  and altered mitochondrial membrane potential. These observations strongly suggest that p33  ING1  cooperates with p53 in UV-induced apoptosis via the mitochondrial cell  death pathway in melanoma cells. p24  ING1  Previous findings indicate that the isoform  is capable of enhancing chemosensitivity in human fibroblasts.  investigate  p33  if the  overexpressed p33  ING1  ING1  isoform  is also  involved  in chemosensitivity,  in melanoma cells and assessed for cell death  treatment with camptothecin.  To we after  Results from cell survival assay and flow cytometry  analysis show no significant difference among cells transfected with vector,  99  p33 , ING1  and antisense p33 . ING1  Furthermore, co-transfection of the p33'  NtJ1  and  p53  constructs had no effect on the frequency of cell death, indicating that there is no synergistic effect between the two tumor suppressors in camptothecin-induced cell death in melanoma cells, which is in contrast to previously observed collaboration between p33  ING1  that p33  ING1  and p53 in DNA repair and apoptosis. W e therefore demonstrate  does not enhance camptothecin-induced cell death in melanoma cells.  Taken altogether, we have elucidated in this thesis some of the novel functions of p33  ING1  and the importance of this gene in the context of tumor suppression. With  further exploration, we hope to eventually transfer our knowledge of this gene from the laboratory bench to the bedside of our cancer patients.  7.2  Future Directions  To further elucidate the functions of the tumor suppressor gene ING1, scientific tools are needed.  better  For instance, although we were able to consistently  demonstrate relatively high expression of the wt p33  plasmid in our cell lines, the  ING1  upper limit of transfection efficiency is reached at 70% in one cell line. In most other cell lines, the efficiencies are usually much lower. To circumvent this limitation, we are currently developing the adenoviral infection approach in an attempt to improve expression efficiency of exogenous plasmid DNA in many of our melanoma cell lines as well as other cell lines of different origin. W e are also interested in establishing cell lines expressing wt p33  ING1  and antisense p33  ING1  by stable transfection.  Although these techniques are very well established and relatively simple to perform, and their reagents are widely available, one major drawback is that high levels of  100  forced expression of wt p33  ING1  may not represent physiologically normal conditions  in the cell and that the antisense p33  construct consistently appears weak in  ING1  suppressing the levels of endogenous p33  ING1  in many instances. To overcome this  problem, we have recently considered using an in vivo approach in which we attempt to knock out the ING1 gene on 13q34 by homologous recombination in mice. The generation of knock-out mice will not only allow us to investigate tumor suppressive functions, such as DNA repair and stress-induced apoptosis, of ING1 in the most physiologically relevant setting, it will assist in answering the ultimate question of whether the ING1 mammals.  An alternative  gene has any effects on normal development of and  recently developed technique  for  disrupting  endogenous genes in mammalian cells is the R N A interference (RNA i) method. Initially used in the nematode Caenorhabditis  elegans  in the early 1990s (Fire et al.,  1991), this technique employs double-stranded R N A with sequence specific against the target gene m R N A and silences gene expression by base-pairing with the homologous mRNA, consequently targeting it for degration by specific enzymes (Hammond et al., 2001). This approach has been considered to be a very effective way of suppressing gene expression in comparison to the use of antisense plasmid transfection. The ultimate and eventual outcome of basic research is clinical applications. Since numerous types of human cancer have been found to exhibit abnormal expression of the p33  ING1  therapy. p33  ING1  isoform, one may be able to correct such defect by gene  In the case of skin cancer, our laboratory has recently demonstrated that is overexpressed in 100% of melanoma cell lines (Campos et al., 2002) and  101  96%  of  melanoma  counterparts.  primaries  (data  not  shown)  compared to  their  normal  Although only few missense mutations were found, other types of  inactivating mechanisms, such as mutations in the introns that affect the splicing process of the p33  ING1  dysfunctional p33  ING1  transcript, may account for the overexpression of the  protein. Gene therapy using wt p33  may be one option to  ING1  eliminate the maligancy by apoptosis.  Melanoma-specific expression can be  achieved by constructing a vector containing the wt p33  DNA attached to the  ING1  tyrosinase promoter, which is only activated in melanin-producing cells (Bertolotto et al., 1996). To capitalize our finding that overexpression of p33  ING1  in the presence of  UV radiation can cause apoptosis in melanoma cells, a photodynamic therapeutic approach for treatment of melanoma may be utilized.  Photodynamic therapy  employs light and light sensitive agents (such as porphyrins) to cause cell death by generating toxic oxygen species (Karrer et al., 2001). However, in our case, the light sensitive agents would be copies of wt p33  ING1  delivered into the area of target for  UV light application. 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Science  122  1991, 254: 1167-73.  2001  U B C Division of Dermatology Annual Research conference, Vancouver, B C (Topic: p33ING1 role in D N A repair)  2000  U B C Experimental Medicine Annual Student Research Day, Vancouver, B C (Topic: ING1 role in D N A repair)  1999  U B C Experimental Medicine Annual Student Research Day, Vancouver, B C (Topic: p33ING1 involvement in UV-induced stress response and its in vivo relationship with p53)  1999  7 7 International Association for Dental Research (IADR), Vancouver, B C (Topic: Increased allelic loss in early oral dysplasia from high-risk sites)  1999  Frost Road Elementary School, Surrey, B C (Topics: Genetics: Applications, Career, and Training)  1998  British Columbia Cancer Research Center, Vancouver, B C (Topic: L O H profile of oral premalignant lesions in low-/high-risk sites)  1998  Burnaby North Secondary School, Burnaby, B C (Topic: Applications of Genetics)  1997  British Columbia Cancer Research Center, Vancouver, B C (Topic: Genetic susceptibility and human chromosome 11)  th  ABSTRACTS  p33 via bcl-2 and caspase 3. Annual meeting for Society of Investigative Dermatology, Florida, U S A . April 3 0 - M a y 4 , 2003.  1.  Chin M, Cheung K-J, Ho V, Li G . Induction of apoptosis  2.  Cheung K-J, Ho V, Li G . Role of p 3 3  3.  Cheung K-J, Mitchell D, Lin P, Ho V , Li G . p33  role in D N A repair. Annual meeting for Society of Investigative Dermatology, Washington, D C , U S A . May 9-12, 2001.  4.  Luu Y , Cheung K-J, Bush J , Li G . p53 stabilizing compound, CP31398, induces apoptosis in a p53-dependent manner. Annual Meeting of the Society for Investigative Dermatolo Washington D C , May 9-12, 2001.  5.  Campos E l , Cheung K-J, Li G . Mutational analysis of the p 3 3 gene in human melanoma cell lines. Annual Meeting of the Society for Investigative Dermatology, Washington D C , May 2001.  6.  Cheung K-J, Lin P, Ho V, Li G . Role of p 3 3  7.  Cheung K-J, Bush J A , Ho V , Li G . The Role of p 3 3 ' in Cellular Stress Response and its In Vivo Relationship. Annual Meeting of Canadian Society for Investigative Dermatology, Mo June 29-July 2, 2000.  8.  Cheung K-J, Ho V , Li G . p33  ' in UV-induced apoptosis. Annual meeting for Society of Investigative Dermatology, Los Angeles, U S A . May 15-18, 2002. / N G  ING1  / N G ?  ' in D N A repair. Annual Meeting of the Canadian Society for Investigative Dermatology, Halifax, N S , June 28-30, 2001. , W G  m G  involvement in UV-induced stress response and its in vivo p53. Journal of Investigative Dermatology, 114(4), p854. 2000. ING1  relationship with 9.  INh1  Cheung K-J, Zhang L, Rosin M P . Increased allelic loss in early oral dysplasia from high-risk th sites. 77 International Association for Dental Research (IADR), Vancouver, B C , Canada. 199  124  PUBLICATIONS 1. Cheung K-J and Li G. p53 and p33 : Role in nucleotide excision repair of UV-damaged DNA. Book chapter in Comprehensive Series in Photosciences. In press. 2003. INU1  2. Cheung K-J and Li G. The tumour suppressor p33 does not regulate migration and angiogenesis in melanoma cells. International Journal of Oncology. 21(6): 1361-5,2002. ING1  3. Cheung K-J and Li G. p33 enhances UVB-induced apoptosis in melanoma cells. Experimental Cell Research. 279(2): 291-8, 2002. ING1  4. Lin P, Bush J, Cheung K-J, Li G. Tissue-specific regulation of Fas/APO-1/CD95 expression by p53. International Journal of Oncology. 21(2): 261-4, 2002. 5. Luu Y, Bush J, Cheung K-J, Li G. p53 stabilizing compound, CP31398, induces apoptosis by upregulating Bak and activating Caspases-9/3. Experimental Cell Research. 276(2): 214-22, 2002. 6. Cheung K-J and Li G. The tumor suppressor ING1 does not enhance cell death in camptothecin-treated melanoma cells. International Journal of Oncology. 20(6): 1319-22, 2002. 7. Cheung K-J, Mitchell D, Lin P, Li G. The novel tumor suppressor p33 damaged DNA. Cancer Research. 61(13): 4974-77, 2001.  ING1  mediates repair of UV-  8. Cheung K-J and Li G. The tumor suppressor ING1: structure and function. Experimental Cell Research. 268(1): 1-6, 2001. 9. Zhang L, Cheung K-J, Lam WL, Cheng X, Poh C, Priddy R, Epstein J, Le ND, Rosin M. Increased genetic damage in oral leukoplakia from high-risk sites. Cancer. 91(11): 2148-55, 2001. 10. Cheung K-J and Li G. Tissue-specific regulation of Chkl expression by p53. Experimental and Molecular Pathology. 71 (2): 156-60, 2001. 11. Campos E, Cheung K-J, Murray A, Li S, Li G. The novel tumour suppressor gene ING1 is overexpressed in human melanoma cell lines. British Journal of Dermatology. 146(4): 574-80, 2001. 12. Bush J, Cheung K-J, Li G. Curcumin induces apoptosis in human melanoma cell lines through a death receptor/Caspase-8 pathway independent of p53. Experimental Cell Research. 271: 30514,2001. 13. Cheung K-J, Bush J, Jia W, Li G. Expression of the novel tumor suppressor p33 Independent of p53. British Journal of Cancer. 83 (11):1468-72, 2000. LABORATORY • • • • • • •  SKfOIS  Cloning Polymerase chain reaction Western and Northern blotting Slot blotting Propidium iodine staining Annexin V staining Mitocapture apoptosis detection  125  ING1  Is  • • • • • • • • • • • • • • • • • • • •  Flow cytometry Immunohistochemistry Immunoprecipitation Gel electrophoresis Autoradiography DNA sequencing LOH (loss of heterozygosity) assay MN (micronuclei) assay DNA/RNA/Protein extraction DNA fragmentation assay Luciferase assay CAT (Chloramphenicol acetyltransferase) assay for DNA repair SSCP (single-strand conformational polymorphism) Soft agar growth assay with methylene blue staining Cell survival assay with SRB, MTS, and Trypan blue exclusion Differential display Cell/tissue culture Tissue microdissection Light and fluorescent microscopy UV and fluorescent spectrophotometry  126  

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