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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 p33ING1 by K-JOHN J . C H E U N G JR. B.A., Simon Fraser University, 1994 M . S c , Simon Fraser University, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES (Department of Medicine; Experimental Medicine Program) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 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 p47ING1, p33ING1, p27ING1, and p24ING1, 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 p33ING1 isoform. We first investigated how the expression of ING1 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 p33ING1 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 p33ING1 plays a role in UV-stress response, such as repair of UV-damaged DNA. Using both in vitro (host-cell-reactivation assay) and in vivo (radioimmunoassay) methods, we found that p33ING1 enhances the repair of UV-damaged DNA in collaboration with p53 in melanoma cells and that GADD45 may participate in the process. Next we investigated the molecular pathways of p33ING1 enhancement in UV-induced apoptosis in melanoma cells using various survival and apoptotic assays. W e demonstrated that overexpression of p33ING1 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 p33ING1 enhances the expression of endogenous Bax and alters mitochondrial membrane potential, suggesting that p33ING1 cooperates with p53 in UV-induced apoptosis via the mitochondrial cell death pathway in melanoma cells. Lastly, we examined the role of p33ING1 isoform in melanoma chemosensitivity because previous findings indicate that the isoform p24ING1 is capable of enhancing chemosensitivity in human fibroblasts. Using a number of survival and apoptotic assays to quantitate cell death in melanoma cells, we showed that neither overexpressing p33ING1 alone nor coexpression of p33ING1 and p53 had an effect on the frequency of cell death induced by the chemodrug, camptothecin. W e therefore demonstrated that p33ING1 does not enhance camptothecin-induced cell death in melanoma cells. In conclusion, we have elucidated in this thesis some of the novel functions of p33ING1 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 Cancer 1 1.1.1 Skin cancer 1 1.2 Tumor suppressor genes 4 1.2.1 p53 5 1.2.1.1 Structure and function 5 1.2.1.2 Cell cycle arrest 7 1.2.1.3 Apoposis 8 1.2.1.4 DNA repair 10 1.2.2 ING1 11 1.2.2.1 Isolation and characterization 11 1.2.2.2 Tumor suppressive functions 15 1.2.2.3 Expression profile 20 1.2.2.4 The ING1 family 21 1.2.2.5 Expression and mutational analyses in tumors 25 1.3 General hypothesis and objectives 29 Chapters MATERIALS AND METHODS 31 2.1 Animals 31 2.2 Cell lines and cell culture 31 2.3 Plasmids 32 2.4 Antibodies 32 2.5 Transfection 33 2.6 Determination of transfection efficiency 33 2.7 UVB irradiation 33 2.8 Light microscopy 36 2.9 Western blot analysis 36 2.10 Trypan blue exclusion assay 36 iv 2.11 S R B cell survival assay 37 2.12 Reverse transcriptase-polymerase chain reaction (RT-PCR) 37 2.13 Northern blot analysis 38 2.14 Immunohistochemistry 39 2.15 Host-cell-reactivation assay 39 2.16 Radioimmunoassay 40 2.17 Immunoprecipitation 41 2.18 Propium iodine (PI) staining 42 2.19 Flow cytometry 42 2.20 Mitochondrial transmembrane potential detection 42 Chapter 3 E X P R E S S I O N OF p33ING1 IS INDEPENDENT OF p53 44 3.1 Rationale and hypothesis 44 3.2 Results and discussion 47 Chapter 4 p33ING1 MEDIATES REPAIR OF UV-DAMAGED DNA 56 4.1 Rationale and hypothesis 56 4.2 Results and discussion 59 Chapter 5 p33ING1 E N H A N C E S UVB-INDUCED APOPTOSIS IN 68 MELANOMA C E L L S 5.1 Rationale and hypothesis 68 5.2 Results and discussion 70 Chapter 6 p33ING1 DOES NOT E N H A N C E CAMPTOTHECIN- 87 INDUCED CELL DEATH IN MELANOMA C E L L S 6.1 Rationale and hypothesis 87 6.2 Results and discussion 89 Chaper7 CONCLUSIONS 98 7.1 Summary 98 7.2 Future directions 100 R E F E R E N C E S 103 APPENDIX I CURRICULUM VITAE OF AUTHOR 123 v LIST OF TABLES Table 1.1 Relative percentages of transfection efficiencies of cell 35 lines used in the study vi LIST OF FIGURES Figure 1.1 Genomic structure of human ING1 and its alternatively 13 spliced mRNA variants, p47ING1, p33ING1, p27ING1, and p24ING1 Figure 1.2 Predicted amino acid sequences of the four ING1 isoforms, 14 p47ING1, p33ING1, p27ING1, and p24ING1 based on cDNA sequences from GenBank Figure 2.1 Visual assessment of transfection efficiency 34 Figure 3.1 Analyses of p33ING1 mRNA expression of p53+/+ and p53'A 48 mouse organs Figure 3.2 Western blot analysis of p33 / W G * expression levels in 49 selected p53+ / + and p53'A mouse organs. Figure 3.3 Immunohistochemical analysis of p33ING1 in the brain of 51 p53+ / + and p53v" mice Figure 3.4 p33ING1 induction by UVB irradiation in keratinocytes 52 Figure 3.5 Western blot analysis of p33ING1 expression in UV-irradiated 54 mouse fibroblasts Figure 4.1 p33ING1 is UV-inducible in a dose- and time-dependent manner 60 Figure 4.2 p33ING1 enhances UV-damaged DNA repair 61 Figure 4.3 p33ING1 -mediated DNA repair is p53-dependent 63 Figure 4.4 ING1 physically interacts with GADD45, but does not 65 transcriptionally upregulates GADD45, XPA , o r X P B Figure 5.1 Effect of p33ING1 on UVB-induced cell death in M M R U cells 71 Figure 5.2 Synergistic effect of p33ING1 and p53 on UVB-induced cell 76 death in MMRU cells Figure 5.3 Effect of p33ING1 on UVB-induced cell death in M E W O cell line 79 Figure 5.4 p33ING1 alters mitochrondrial membrane potential and 82 increases Bax expression vii Figure 6.1 Survival rate of CPT-treated R P E P cells transfected with 90 p33ING1 and antisense p33ING1 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 p33ING1 on MMRU cell survival after C P T treatment 94 Figure 6.5 Effect of p33ING1 and p53 co-expression on melanoma 96 chemosensitivity viii LIST OF ABBREVIATIONS aa Amino acid AML Acute myelogenous leukemia Apaf-1 Apoptotic protease activating factor-1 B C C Basal cell carcinoma B E R Base excision repair C A N Cain (Cancer intron on nine) CAT Chloramphenicol acetyltransferase Cdk Cyclin-dependent kinase C P D cyclobutane pyrimidine dimers C P T Camptothecin DEK DEK oncogene (DNA binding) DMEM Dulbecco's modified eagle's medium E S C C Esophageal squamous cell cancer FBS Fetal bovine serum FISH Fluorescent in situ hybridization G S E Genetic suppressor elements HAT Histone acetytransferase H N S C C Head and neck squamous cell carcinomas HRP Horseradish peroxidase IAP Inhibitors of apoptosis ING1 Inhibitor of growth 1 LOH Loss of heterozygosity MDM2 Mouse double minute 2 NER Nucleotide excision repair NF-kB Nuclear factor kappa B NHEK Normal human epithelial keratinocytes O S C C Oral squamous cell carcinoma P B S Phosphate buffered saline P C N A Proliferating cell nuclear antigen PI Propium iodine PIG p53-induced genes PUMA p53-upregulated modulator of apoptosis PVDF Polyvinylidene difluoride Rb Retinoblastoma RNA I RNA interference R T - P C R Reverse transcriptase-polymerase chain reaction sec Squamous cell carcinoma S M A C Second mitochondria-derived activator of caspase S R B Sulforhodamine B S S C P Single-stranded conformation polymorphism TBP TATA box-binding protein UV Ultraviolet WT 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. I thank you once again and here share with you my life-long motto: "De Oppresso 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 41% 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: UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm). UVC is essentially blocked from reaching the earth's surface due to absorption by the atmospheric ozone layer, although some accidental exposure occurs from man-made sources, such as germicidal lamps (Soehnge et al., 1997; Cleaver et al., 2002). UVA 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 UVB region of the solar spectrum are absorbed into the skin, producing erythema, burns, and eventually skin cancer (Soehnge et al., 1997). Although UVA 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 non-melanoma skin cancer (Volgelstein et al., 1998). Approximately 80% of the non-melanoma 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 LOH and homozygous deletion at 9p21 (Vogelstein et al., 1998). Deletion mapping of 9p21 indicates that the locus encodes the tumor suppressor gene p16INK4, a negative growth regulator, which induces cell cycle arrest (Vogelstein et al., 1998). p16 is part of a growth control pathway that involves cyclin-dependent 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 BRAF, 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 suppressor genes (Macleod, 2000). 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 multi-functional carboxyl-terminal domain (Ko et al., 1996). p53 binds in vitro to several proteins through its activation domain. 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 G2 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 RNA 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 (Wallace-Brodeur 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 UVB 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. Of the numerous p53 target genes, p2'\Waf1 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 p21Waf1, subsequently inactivating 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 G1. 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 PUMA (p_53-upregulated 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 DR5, both death receptors, and FasL, a death receptor ligand (Ashkenazi et al., 1998). Activation of death receptors by their ligands results in trimerization and recruitment of 9 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 UV-induced 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. GADD45 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 NER similar to those seen in p53v~ fibroblasts (Smith et al., 2000) and G/ADD45-deficient mice show elevated carcinogenesis induced by radiation and genomic instability comparable to that observed in p53 deficient mice (Hollander et al., 1999). 10 1.2.2 ING1 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, Hs-578T, ZR-75, MD-MB-468, MD-MB-435, and BT-20), and subsequent selection of short cDNA 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 mRNA 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 p33ING1 transcript consists of exons 1a and 2. The third spliced form, p24ING1, is composed of a truncated p47ING1 messenger with the first A T G codon in exon 2. A fourth ING1 variant, p27ING1, 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 p33ING1, p47ING1, and p24ING1 alternative spliced forms. Specifically, region between the Xbal and Ncol sites contains the promoter for p33ING1, whereas the promoters for p47ING1 and p24ING1 are present within the Notl and Pf1 Ml sites, and the Pf1 Ml and Smal sites, respectively. The promoter for p27ING1 has yet been identified. Initial examination of the cDNA structure of ING1 predicted a protein of 33,350 daltons, named p33ING1 (Garkavtsev et al., 1996). It was later discovered that, due to a cloning error, the reported sequence of p33ING1 was truncated at the N-terminus and encoded a protein of 210-amino acid (aa) and 23,656 daltons, named p24ING1, which is the shortest known isoform of ING1 (Garkavtsev et al., 1999; Cheung et al., 2001a). In other words, most studies on p33ING1 prior to the correction were actually done with the cDNA construct of p24'NG1 and not that of p 3 3 / W G ' . The other three currently known isoforms of ING1 are p47ING1 (422-aa and 46,751 daltons), p33ING1 (279-aa and 31,843 daltons), and p27ING1 (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 PHD 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 mRNA variants, p47ING1, p33ING1, p27ING1, and p24ING1. • Coding sequence in exon. • Noncoding sequence in exon. Restriction enzyme sites indicate positions of the three promoters on ING1. Promoters for p33ING1, p47ING1, and p24 / A / G r are present within the Xbal and Ncol sites, the Notl and Pf1 Ml sites, and the Pf1 Ml and Smal sites, respectively. Promoter for p27ING1 has not been analyzed. Xbal Ncol Notl Pf lMI Smal I 1 k b I Exon 1 a Exon 1 b • Exon 2 p33 p47 p24 ING1 ING1 ING1 : p27 ING1 13 Figure 1.2 Predicted amino acid sequences of the four ING1 isoforms, p47 p33ING\ p27ING1, and p24ING1 based on cDNA sequences from GenBank (accession numbers: AF181849, AF181850, AF149723, and AB031269, respectively). The metal-chelating residues in the PHD finger domain are represented by white type on black. Underlined residues indicate possible nuclear localization signal motifs. p47m G' - M S F V E C P Y H S P A E R L V A E A D E G G P S A I T G M G L C F R C L L F S F S G R S G V E G G R V D L N V F G S L G L Q P W I G S 6 8 S R C W G G P C S S A L R C G W F S S W P P P S R S A I P I G G G S R G A G R V S R W P P P H W L E A W R V S P R P L S P L S P A T F G R G F I A V A 1 4 3 M L H C V Q 6 M E I L K E L D E C Y E R F S R E T D G A Q K R R M L H C V Q 3 1 M L S P A N G E Q L H L V N Y V E D Y L D S I E S L P F D L Q R N V S L M R E I D A K Y Q E I L K E L D E C Y E R F S R E T D G A Q K R R M L H C V Q 7 5 V I P G L W A R G R G C S S D R L P R P A G P A R R Q F Q A A S L L T R G W G R A W P W K Q I L K E L D E C Y E R F S R E T D G A Q K R R M L H C V Q 2 1 8 R A L I R S Q E L G D E K I Q I V S Q M V E L V E N R T R Q V D S H V E L F E A Q Q E L G D T V G N S G K V G A D R P N G D A V A Q S D K P N S K R S 8 1 R A L I R S Q E L G D E K I Q I V S Q M V E L V E N R T R Q V D S H V E L F E A Q Q E L G D T V G N S G K V G A D R P N G D A V A Q S D K P N S K R S 1 0 6 R A L I R S Q E L G D E K I Q I V S Q M V E L V E N R T R Q V D S H V E L F E A Q Q E L G D T A G N S G K A G A D R P K G E A A A Q A D K P N S K R S 1 5 0 R A L I R S Q E L G D E K I Q I V S Q M V E L V E N R T R Q V D S H V E L F E A Q Q E L G D T A G N S G K A G A D R P K G E A A A Q A D K P N S K R S 2 9 3 R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y B L B N Q V S Y G E M I G 1 5 6 R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y B L B N Q V S Y G E M I G 1 8 1 R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y B L B N Q V S Y G E M I G 2 2 5 R R Q R N N E N R E N A S S N H D H D D G A S G T P K E K K A K T S K K K K R S K A K A E R E A S P A D L P I D P N E P T Y B L B N Q V S Y G E M I G 3 6 8 B D N D E B P I E W F B J F S B V G L N H K P K G K W Y B P K B R G E N E K T M D K A L E K S K K E R A Y N R 2 1 0 B D N D E B P I E W F B F S B V G L N H K P K G K W Y B P K B R G E N E K T M D K A L E K S K K E R A Y N R 2 3 5 B D N D E B P I E W F B F S B V G L N H K P K G K W Y B P K B R G E N E K T M D K A L E K S K K E R A Y N R 2 7 9 B D N D E B P I E W F B F S B V G L N H K P K G K W Y B P K B R G E N E K T M D K A L E K S K K E R A Y N R 4 4 3 14 Fluorescent in situ hybridization (FISH) localized ING1 to 13q33-34, while radiation hybrid mapping showed that ING1 is linked to the cytogenetic marker SHGC-5819, 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 p24ING1 construct inhibits cell growth, while its antisense construct promotes cell transformation (Garkavtsev et al., 1996). Specifically, flow cytometry analysis shows that normal fibroblasts transfected with p24ING1 are arrested in the G0/G1 phase of the cell cycle. It appears that p24ING1-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 p24ING1 can increase p53-dependent activation of the p2\"Waf1 promoter (Garkavtsev et al., 1998). p21 l / V a " 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 could, therefore, account for the ability of 15 p24ING1 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 one another as indicated by immunoprecipitation (Garkavtsev et al., 1998). 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 p33ING1 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, p24ING1 has been shown to play a role in senescence. Normal human fibroblasts expressing the antisense p24ING1 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 mRNA products were being monitored. 16 ING1 has also been found to modulate apoptosis. Elevated expression of p24ING1 enhances 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). Coexpression of c-myc and p24ING1 dramatically enhances the extent of serum starvation-induced apoptosis, suggesting that p24 / A / G r-mediated cell death may be synergistic with the c-myc apoptotic pathway. p24ING1 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 p33ING1 from their fetal brain cDNA library, Shinoura and colleagues (1999) demonstrated that apoptosis can be induced by adenovirus-mediated transfer of both p33ING1 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 UV irradiation. p33ING1 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 p33ING1 have higher percentage of apoptosis compared to vector controls. A very recent and detailed analysis by Vieyra et al. (2002b) demonstrated that ING1 induces apoptosis in an isoform-, 17 stimulus-, and cell age-dependent fashion. First, they showed that growth-factor deprivation-induced apoptosis occurs only in early (young)- but not late (senescent-passaged fibroblasts. This effect was accompanied by an increase in endogenous ?33INGI T n e y a ] s o f o u n d t n g t e c t o p i c expression of p33ING1, but not the p47ING1 isoform, sensitizes young but not senescent cells to UV irradiation- and hydrogen peroxide-mediated apoptosis. Coexpression of p33ING1 and p53 significantly enhanced cell death compared to expression of either one alone. Finally, they showed that chromatin binding affinity of p33ING1 was increased in senescent cells, which were resistant to apoptosis, suggesting that p33ING1 may exert its suppressive functions via chromatin remodeling. These studies all in all suggest that both isoforms of ING1, p24ING1 and p 3 3 / N G ' , 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 NMuMG. Using cDNA 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 DEK 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 B1, 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 G2/M 18 phase (Garkavtsev et al., 1997). DEK is a nuclear protein that was found to be fused to an oncogenic protein CAN (short for Cain - cancer intron on nine) in a portion of acute myelogenous leukemia (AML). DEK 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). p33ING1 was shown to associate with Sin3, SAP30, HDAC1, RbAp48, and other proteins in in vitro deacetylation of core histones, while p24ING1 and p47ING1 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 p33ING1 represses transcription in cultured cells using luciferase and GAL4 as reporters (Skowyra et al., 2001). The data suggest that the 99 amino acids of the N-terminal of p33ING1 are responsible for both the association with deacetylation-associated proteins as well as transcription repression. p24ING1 and p47ING1, on the other hand, lack the unique N-terminus specific for p33ING1, which explains why they do not display those binding and functional properties. In a recent report by Vieyra and colleagues (2002a), the authors demonstrated that p33ING1 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 p33ING1 isoform has been 19 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 p33ING1 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 p33ING1 were 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 mRNA 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 p24ING1 and p 3 3 / W G ' , but not p47ING1. In a separate study, p33 / N G * mRNA (referred as variant A in their paper) was shown to be highly expressed in normal human tissues and cancer cell lines, while p24ING1 mRNA (referred as variant B) was weakly expressed (Jager et al., 1999). This observation was further supported by the study of Skowyra and colleagues (2001), in which low abundance of p24ING1 was detected in HeLa cells. 20 Similar to the human ING1 expression pattern, mouse ING1 (mlNG1) mRNA was detected in various tissues (Zeremski et al., 1999). 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 EST database for cDNA with sequence similarity to ING1, Shimada and colleagues (1998) isolated a partial ING1-like gene, named ING1L, from a human fetal brain cDNA library. The ING1L protein is 280-aa long and shares 58.9% sequence identity with p 3 3 , 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 mRNA 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. The DNA-damaging agents, 21 etoposide and neocarzinostatin, induced expression of ING2 but not ING1. Like ING1, ING2 was found to negatively regulate cell growth and survival in a p53-dependent 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 cDNA 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). In this thesis, we will refer to this human ING1 homolog as INGJ. 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 mRNA (Jager et al., 1999). In an attempt to analyze LOH 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 PHD 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 cDNA sequences showed 71% 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 PHD 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 p24ING1, which has growth suppressive function. The other mouse transcript produces a larger protein, named p37ING1, which binds to and interferes with the accumulation of p53 protein and activation of p53-responsive promoters after DNA damage. p37ING1 is speculated to be the equivalent of the human p33ING1. There appears to be no mouse equivalent to the human p47ING1. 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. Four splicing variants of 23 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 PHD 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 PHD finger region (Loewith er al., 2000). Introduction of human p33ING1 was shown to rescue phenotypes induced 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. In addition, the yeast ING1 homologs were found to associate with proteins in the histone acetytransferase (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 mRNA 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 mRNA 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 LOH 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. Rearrangement of ING1 was observed in one cell line (Garkavtsev et al., 1996). 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. Fifty-eight percent of 25 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 mRNA 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 (HNSCC) 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 . A separate study on H N S C C , however, suggests otherwise. Gunduz and colleagues (2000) reported that 23 of 34 (68%) informative cases of H N S C C showed LOH 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 LOH. Missense mutations were found within the PHD 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 (OSCC) 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 (ESCC), 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 showed no correlation to the expression levels of the protein. It is, however, interesting to note that all 4 of the missense mutations occurred within the PHD 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 p33ING1) was clearly overexpressed in all six breast cancer cell lines, eight melanoma cell lines, and a breast cancer primary, while variant B (or p24ING1) was only present in four breast cancer cell lines (Jager et al., 1999). 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 p33ING1 protein expression in nuclei occurred in 78% of the tissue samples. This loss was accompanied by an increased cytoplasmic expression of p33ING1. Interestingly, it was found that a significant number of patients who had lost nuclear expression had a better prognosis 27 compared to those without the loss. Another study by Ito et al. (2002) showed that the levels of p33ING1 mRNA transcript were not significantly different between 3 AML cell lines and 10 AML biopsies. Furthermore, neither point mutations nor deletions in ING1 were found, suggesting that p33ING1 may not play a role in the development of AML. 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 p33ING1 protein in samples with increasing degrees of malignancy. 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 p33ING1. In contrast with other expression and mutation studies of tumors, Campos et al. (2002) showed that instead of a decrease in expression, p33ING1 protein and mRNA 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 PHD finger domain, occurred in one cell line. It was postulated that the observed increase in expression of p33ING1 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 p33ING1 and p47ING1 transcripts, the two transcript levels did not differ significantly from each other in both normal tissues and tissues of hematological malignancies. Though the p33ING1 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). Biological agents such as viruses may also cause inactivation. 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 We hypothesized that the expression of p33ING1 is dependent on the status of p53 in normal and stress conditions, and overexpression of p33ING1 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, p33ING1, in stress 29 conditions. We started by looking at the expression profile of p33INU1 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 p33ING1 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"A mice were purchased from Taconic Inc. (New York). p53'A mice 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 HaCaT keratinocyte (kindly provided by Dr. N.E. Fusenig, DKFZ, Heidelberg, Germany) and three human melanoma cell lines, MMRU, 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% C 0 2 atmosphere. MMRU 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 MMRU and R P E P contain wt p53. Dermal fibroblasts of p53+ / + and p53'A mice were isolated from 4-31 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 DMEM 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 DMEM twice. The cells were resuspended in DMEM containing 10% FBS and incubated at 37°C in a 5% C 0 2 atmosphere. 2.3 Plasmids Plasmids used for transfection included pCI-vector, pCI-p33 / W G 7 , pCI-antisense p33ING1 (kind gifts from Dr. K. Riabowol, University of Calgary, Calgary, AB), p G F P -N2 (Clontech, Windsor, ON), pECH 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, anti-GADD45 mouse 32 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 6 cells. 2.6 Determination of Transfection Efficiency Transfection efficiency of a particular cell line was determined by first, introducing a GFP-bearing plasmid, pGFP-N2, with Effectene; second, assessing the number of green fluorescence emitting cells/ the total number of cells (fluorescent and non-fluorescent) counted X 100%. Figure 2.1 shows microscopic images of M M R U cells transfected with the pGFP-N2 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 2 incubator at 37°C after UVB irradiation. 33 Figure 2.1 Visual assessment of transfection efficiency. M M R U cells were transfected with pGFP-N2 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 R P E P 60.0 + 8.3 M E W O 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 ECL 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 Chain Reaction (RT-PCR) Total RNA was extracted by TriZol reagent (Canadian Life Technologies, Mississauga, ON) and the concentrations were determined by UV spectrophotometry. Five micrograms of total RNA was reverse-transcribed into cDNA 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 KCL, 15 mM MgCI 2), 100 mM DTT, 10 mM dNTP Mix (10 mM each of dATP, dGTP, dCTP 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 2 , 10 mM dNTP Mix, 10 pM of the forward primer (5'-G A T C C T G A A G G A G C T A G A C G - 3 ' ) and 10 mM of the reverse primer (5'-37 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 pCI-p33 / , V G r 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 RNA 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 UV 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% SDS, and 100 ug/ml yeast tRNA for 1 h at 65°C. The p33ING1 probe was first made by amplifying a 577 bp fragment by P C R using primer 1 (5 ' -GATCCTGAAGGAGCTAGACG-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- 3 2 P[dCTP] (10 mCi/ml) according to the manufactured protocol in the Random Primers DNA 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 2XSSC/0 .1%SDS 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 p33ING1 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 DNA were irradiated at 40, 80, or 480 mJ/cm 2 using an UV-crosslinker at 50 ug/ml final concentration and used for 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 CAT 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 [3H]acetyl-CoA, and 16.5 pi of d H 2 0 . Reaction mixtures were 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 DNA and mock transfection without plasmid DNA. 2.16 Radioimmunoassay Antisera were raised against DNA dissolved in 10% acetone and irradiated with UVB light under conditions that have been shown to produce cyclobutane pyrimidine dimers (CPDs) exclusively. 2-5 ug of heat-denatured sample DNA was incubated with 5-10 pg of poly(deoxyadenylate-deoxythymidylic acid) (labeled to >5 X 10 8 40 cpm/pg by nick translation with [ 3 2P]dTTP) in a total volume of 1 ml of 10 mM Tris (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 (UTMDACC, Science Park/Veterinary Division, Bastrop, TX). After centrifugation, the pellet was dissolved in tissue solubilizer (NCS, 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 inhibits antibody binding to the radio-labeled ligand. Sample inhibition is extrapolated through a standard (dose-response) curve to determine the number of photoproducts in 10 6 bases (i.e., CPDs/mb). For the standard, we used double-stranded salmon testis DNA (Sigma, Mississauga, ON) irradiated with increasing doses of UVC 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, CA) 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 re-hydrated 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 RNase 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, San Diego, CA). 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 2 incubator for 15 min. After incubation, the dye solution was removed and the cells were washed twice with 1 ml of the pre-warmed incubation buffer. The cells were then observed immediately under a fluorescent microscope. 43 CHAPTER 3. EXPRESSION OF p33ING1 IS INDEPENDENT OF p53 3.1 Rationale and Hypothesis p53, a nuclear protein, regulates a number of downsteam targets such as p21 v v a ^ , GADD45, 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 l v a f t , a potent inhibitor of cyclin-dependent kinase activity, which inhibits cell cycle progression (Shaulsky et al., 1991; El-Deiry et al., 1993). It has also been demonstrated that p53 maintains genomic stability by enhancing DNA repair and apoptosis. We 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). As 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 UV-induced skin cancer development (Li etal., 1995a, 1998a). 44 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 transformation (Garkavtsev et al., 1996; 1998). In addition, it was found that the function of cell growth control is dependent on the activity of both ING1 and p53, and p21waf1 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; Owen-Schaub et al., 1995), we hypothesized that p53 is necessary for the regulation of the p33ING1 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 p33ING1 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 p53 v " mice, NHEK, and a keratinocyte cell line (HaCaT), to UVB and compared the p33ING1 protein levels in these cells. HaCaT is a spontaneously immortalized, non-tumorigenic human keratinocyte 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 UVB 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 p33ING1 by first examining whether p33ING1 was expressed in organs from p53 + / + and p53'A mice. Results from RT-PCR showed that p33ING1 was expressed in the brain, liver, lung, heart, and skin of both p53+ / + and p53'/' mice (Figure 3.1A). Semi-quantitative R T - P C R indicated that p33ING1 mRNA levels were virtually equal in the heart of p53+ / + and p53~A mice (Figure 3.1 B). The data suggest that p33ING1 expression is independent of p53 status. To further confirm p53-independent expression of ING1, we used Northern blot analysis to compare mRNA levels in the brain, liver, lung, heart, skin, kidney, testis, and thymus of p53+/+ and p53'/' mice. Our results showed that there was no significant difference in p33ING1 mRNA expression between p53+/+ and p53'A mice in all eight organs examined (Figure 3.1C). Next, we investigated whether p53 status affects p 3 3 / W G ) expression at the post-transcriptional level. We compared p33ING1 protein levels in the brain, liver, lung, heart, and skin between p53+/+ and p53'/' mice. Figure 3.2 shows that there is no substantial difference in the levels of p 3 3 / N G ) 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. To 47 Figure 3.1 Analysis of p33ING1 mRNA expression of p53+/+ and p53"A organs. (A) R T - P C R analysis of mRNA level in different organs of p53+ / + and p53~A mice. C 1 , negative control without RNA in the reaction. C2 , positive control with pCI- p 3 3 , W G ' plasmid DNA in the reaction. (B) Semi-quantitative R T - P C R analysis of p33ING1 mRNA level in the heart of p53+/+ and p53~A mice. A series of dilutions of the p 3 3 / W G I cDNA was performed and comparison was made between the p53+/+ and p53~A groups. (C) Northern blot analysis of p33ING1 mRNA expression levels in selected p53 + / +and p53"A organs. The 18S rRNA was used as an internal control. Brain Liver Lung Heart Skin C1 C2 +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-p33ING1 48 Figure 3.2 Western blot analysis of p33 expression levels in selected p53 and p53" A organs. (3-actin was used as internal control. Brain Liver Lung Heart Skin +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/-P33ING1 mm Actin *mmm <m •» • 49 determine if ING1 expression was independent of p53 using an independent method, we used immunohistochemical staining to look at p33ING1 protein expression in the brain between the p53+/+ and p53'A groups. Our results show that p53+/+ and p53'A mice have similar levels of p33ING1 (Figure 3.3). p53 plays a significant role in responding to DNA damage. To test the hypothesis that p33ING1 may also be a downstream target of p53 in stress conditions, we exposed normal NHEK cells and the HaCaT cell line, which carries mutated p53 alleles (Lehman ef al., 1993), to UVB irradiation and examined the levels of ING1 protein. Our results demonstrate that the amount of p33ING1 protein is induced in both NHEK and HaCaT cells after UVB irradiation (Figure 3.4A and B). Using densitometry, a 2-fold increase in the level of p33 , W G * protein in NHEK cells at 40 mJ/cm 2 and approximately 4-fold increase in HaCaT cells at 100 mJ/cm 2 were observed. It was also noted that the expression of p33ING1 in HaCaT cells is dose-dependent, with a maximum induction at 80 mJ/cm 2 . The observation of similar levels of p33ING1 induction in NHEK and HaCaT cells by UV irradiation further confirms p53-independent expression of p33ING1. To confirm that UV induces p33ING1 expression at the transcriptional level and that this was not due to post-translational modifications, p33ING1 mRNA levels were examined in HaCaT cells over a time-course. Figure 3.4C shows that p33ING1 mRNA 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 p33ING1 in the brain of p53 + / + and p 5 3 " mice. (A) p53 + / + . (B) p53-'\ Arrows indicate p33ING1 expressing neural cells Scale bar, 25 um. IK +/+ -/-'* X * A B 4* :.. 51 Figure 3.4 p33ING1 induction by UVB irradiation in keratinocytes. Proteins were extracted from the cells 24 h after UVB irradiation and subjected to Western blot analysis. (A) NHEK were exposed to UVB at 0 and 40 mJ/cm 2 . (B) HaCaT cells were exposed to UVB at 0, 20, 40, 80, and 100 mJ/cm 2 . p-actin was used as an internal control. (C) Northern blot analysis of p33 , W G * expression levels in HaCaT over a time-course. HaCaT cells were exposed to UVB at 80 mJ/cm 2 and total RNA was collected at 0, 1, 2, 4, 8, 24, and 48 h. The 18S rRNA was used as an internal control. N H E K 40 mJ wmmm P33ING1 A c t i n B HaCaT 0 20 40 80 100 mJ P33ING1 -^"^p^ U P r ^UPr Actin 0 1 2 4 8 24 48 h p33ING1 18s 52 fibroblasts isolated from p53 + / + and p53" /_ mice to 40 mJ/cm 2 of UVB. Our data show that neither p53+/+ nor p53'A fibroblasts exhibit any change in p33ING1 expression (Figure 3.5), suggesting that UV-induction of p33ING1 is cell type-specific. In this study, we found that wt p53 is not required for the expression of p33ING1 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 mammary gland cell lines under normal and stress conditions. Induction of p33ING1 by UV irradiation in human keratinocytes suggests a role of p33ING1 in cellular stress response and skin carcinogenesis. UV induction of p33ING1 seen in keratinocytes suggests two possibilities: 1) p33ING1 is capable of responding to stress in terms of expression independent of p53 but cannot perform its cellular activities without functional p53, and 2) p33 / W G * is capable of responding to stress and carries out its functions independent of p53. Further functional studies are needed to decipher the role of p33ING1 in the cellular stress response. 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 p33ING1 cooperates with p53 in keratinocytes to modulate cell cycle arrest, DNA repair, and apoptosis will further determine the functional significance of p33ING1 in the UV-induced stress response in particular, and in skin carcinogenesis in general. To support the notion that p33ING1 plays a 53 Figure 3.5 Western blot analysis of p33 G expression in UV-irradiated murine fibroblasts. p 5 3 + / + and p53" A dermal fibroblasts were exposed to UVB at 0 and 40 mJ/cm 2 . B-actin was used as an internal control. +/+ Fb -/- Fb 40 0 40 mJ p33ING1 VHJMMP l^iiiiM^ wnf0itt^ A^ctin 54 role in skin carcinogenesis, we have recently found that UV-induced murine squamous carcinomas express significantly higher levels of p33ING1 regardless of p53 status in comparison to normal skin (data not shown). 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 p33ING1 expression is dependent on p53 status in normal and stress conditions. 55 CHAPTER 4. p33 I N G 1 MEDIATES REPAIR OF UV-DAMAGED DNA 4.1 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 NER 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 NER 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, GADD45. The gene was originally cloned on the basis of rapid induction in cells by UV light (Fornace et al., 1998). Later it was shown that GADD45 enhances NER (Smith et al., 2000). Most evidence suggests that the GADD45 response is p53-dependent (Zhan etal., 1996; Kastan etal., 1992). Smith et al. (1994) demonstrated that GADD45 stimulates NER and inhibits entry of cells into S phase. Furthermore, antisense DNA directed against GADD45 mRNA 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 GADD45 to PCNA, a protein involved in cell cycle regulation and DNA repair. Other lines of evidence suggest that p53 may directly participate in NER through a protein-protein interaction with components such as X P B and X P D , 57 subunits of TFIIH involved in NER, 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'A/p53'A (n=14) > Xpc'A/p53+A (n=22) > Xpc'A/p53+/+ (n=19). Hence, inactivation of both p53 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 BRCA1 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 may also participate in cellular stress response to UV irradiation. UV-induction of p33ING1 seems to be a common phenomenon in epidermal cells as we have shown that p33ING1 is upregulated at the transcriptional level in NHEK and a keratinocyte cell line, HaCaT (Cheung et al., 2000). We hypothesized that p33ING1 enhances repair of UV-damaged DNA in the presence of functional p53. 58 4.2 Results and Discussion We first examined if p33ING1 would respond to UVB in a human melanoma cell line, M M R U , which contains wt p53 (Li et al., 1995b). We 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 RNA levels at various time points after UVB irradiation. We found that UVB-induced p33ING1 was indeed a result of transcriptional control (Figure 4.1 C). These results indicate that p33 , W G * was induced in a dose- and time-dependent manner after UVB irradiation. To study if p33ING1 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, p33ING1, or antisense p33ING1 expression vector into MMRU cells. The activity of the reporter gene was used as an indicator of the extent of repair. Our data demonstrated that cells overexpressing the p33ING1 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 CAT plasmids (at 480 mJ/cm 2) were used (p=0.01, t-test) (Figure 4.2A). To confirm the results from the 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 MMRU cells overexpressing p33ING1. The results showed that the repair rate of 59 Figure 4.1 p 3 3 I N G 1 is UV-inducible in a dose- and time-dependent manner. (A) Western analysis of UVB-induced p 3 3 I N G 1 protein expression in M M R U cells. Cells were irradiated with UVB at 0, 10, 20, 40, and 80 mJ/cm 2 and harvested after 24 h incubation. An anti-ING1 antibody was used for primary antibody incubation and p-actin was used as loading control. Lane C represents lysate from MMRU cells overexpressing the pCI -p33 I N G 1 plasmid, confirming that the bands induced by UV irradiation were the p 3 3 ^ G 1 protein. (B) Densitometry of p 3 3 I N G 1 induction in (A). (C) Northern analysis of UVB-induced p 3 3 I N G 1 mRNA in M M R U cells. Cells were irradiated with UVB at 40 mJ/cm 2 and harvested at 0, 2, 4, 8, 12, and 24 h after UV exposure. The p 3 3 I N G 1 probe was first made by amplifying a 577 bp fragment by PCR using primer 1 (5 ' -GATCCTGAAGGAGCTAGACG-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 - 3 2 P[dCTP] (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. 10 20 40 80 mJ Actin 60 Figure 4.2 p33 , / V G 7 enhances UV-damaged DNA repair. (A) Effect of p33 / / V C j 7 on repair of UV-damaged plasmid DNA by host-cell-reactivation assay. Undamaged or UV-damaged pCMVcat plasmids were co-transfected with vector (V), pCI-p33 / A / G 7 (p33ING1) or pCI-antisense p33ING1 (AS) into MMRU cells and incubated at 37°C with 5% CO2 for 40 h. CAT 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 p33ING1 on repair of UV-damaged genomic DNA by radioimmunoassay. MMRU cells transfected with vector or p33 plasmids were irradiated with UVB at 20 mJ/cm 2 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). A 61 C P D was nearly doubled in p33 / / V G 7-transfected cells compared to the vector-transfected control cells 24 h post-UV irradiation (Figure 4.2B). As 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 DNA repair, we disrupted the activity of endogenous wt p53 in MMRU 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 MMRU compared to the vector control, indicating successful transfection (Figure 4.3B). Similar levels of p33ING1 between pED1-transfected and control cells were observed (Figure 4.3B), eliminating the possibility that the overexpressed mutant p53 might block the expression of p33ING1. Using the host-cell-reactivation assay, we noted that the repair enhancement of p33ING1 was dramatically suppressed in pED1 (mut p53)-transfected cells but restored in pECH (wt p53)-transfected cells (Figure 4.3C), suggesting that p33ING1 requires the presence of p53 to repair damaged DNA. To study the pathways involved in p33 / A / G 7-mediated DNA repair, we examined if p33ING1 is the upstream regulator of GADD45, X P A , and X P B , all of which have been shown to have significant involvement in DNA repair (Smith et al., 2000). We found that there was no change in expression in any of the aforementioned proteins in M M R U cells overexpressing p33ING1 (Figure 4.4A), indicating that p33ING1 is not the upstream regulator of them. To test the possibility 62 Figure 4.3 p33 / / V G 7-mediated DNA repair is p53-dependent. (A) Western analysis of p53 protein expression in UVB-irradiated MMRU cells. Cells were irradiated with UVB at 0, 10, 20, 40, and 80 mJ/cm 2 and harvested after 24 h incubation. An anti-p53 antibody was used for primary antibody incubation and B-actin was used as loading control. (B) Western analysis of p53 and p33ING1 proteins in MMRU cells transfected with the dominant-negative mutant p53 (pED1) expression vector. (C) Effect of p53 on p33 / / V G r-mediated DNA repair. Host-cell-reactivation assay was performed on MMRU cells transfected with UV-damaged (40 mJ/cm 2) pCMVcat plasmid and control vector (V), p33ING1, pED1 (mut p53), p33)NG1 + pED1, pECH (wt p53), or p33ING1 + pECH. 40 h later, CAT activity was measured using the undamaged pCMVcat as control. Experiments were performed in triplicates. Shown is a representative of two independent sets of experiments. 63 0 10 20 40 80 mJ p53 Actin V pED1 p53 p33ING1 mm A c t i n ^ & J * & ^ <8> 64 Figure 4.4 ING1 physically interacts with GADD45, but does not transcriptionally upregulate GADD45, X P A , or X P B . (A) Effect of p33ING1 on the expression of GADD45, XPA , and X P B proteins. MMRU cells were transfected with vector alone (V), p33ING1, or antisense p33ING1 (AS) expression vectors. 24 h after transfection, cells were harvested and their lysates were analyzed by Western blotting using anti-GADD45, anti-XPA, and anti-XPB antibodies, p-actin served as loading control. (B) Co-immunoprecipitation of ING1 with XPA, X P B , and GADD45. 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 GADD45 were then used in Western analysis. The physical binding between ING1 and GADD45 was observed in three separate experiments. V p33ING1 A S p33ING1 G A D D 4 5 X P A X P B Actin B 1 2 3 ! G A D D 4 5 X P A X P B 65 that ING1 may physically associate with GADD45, XPA , 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 GADD45 (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 p33ING1 enhances NER of both UV-damaged genomic DNA and exogenous plasmid DNA, further supporting the notion that p33ING1 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 NER, 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 NER 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, ATPase and kinase activity (Wang er al., 1994). However, our results demonstrate that p33ING1 does not transcriptionally regulate or physically bind to X P A and X P B (Figure 4.4). The physical association between ING1 and GADD45 (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 GADD45 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 GADD45 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). It would be of interest to exploit the mechanistic role of p33ING1 in this GADD45-mediated repair process, since recent evidence also suggests that p33ING1 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 p33ING1 enhances NER 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 p33ING1 can potentially contribute to neoplastic development. 67 CHAPTER 5. p33' N G 7 ENHANCES UVB-INDUCED APOPTOSIS IN MELANOMA CELLS 5.1 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. We 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 p33ING1 in cellular stress response to UV irradiation. We previously found that p33ING1 expression was induced in a dose/time dependent manner after UV irradiation in 68 both keratinocyte and melanoma cells (Cheung et al., 2000; 2001b). We also demonstrated that the ability of melanoma cells to repair UV-induced DNA damage could be enhanced by the presence of p33ING1. Studies from other groups further lend credibility to the idea that p33ING1 has a significant role in stress response to UV irradiation. 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. p33ING1 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 p33ING1 have a higher percentage of apoptosis compared to cells receiving vector controls. In order to investigate the molecular pathways of p33ING1 enhancement in UV-induced apoptosis in biologically relevant cells, we hypothesized that p33ING1 and p53 synergistically enhance UV-induced cell death in melanoma cells. 69 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 p33ING1 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 p33ING1 isoform appeared to enhance DNA repair in melanoma cells and apoptosis in fibroblast cells (Cheung er al., 2001b; Scott et al., 2001a; 2001b). The p33ING1 isoform has also been shown to translocate to the nucleolus and bind to P C N A after UV irradiation (Scott et al., 2001a; 2001b). To further investigate the role of p33ING1 in UV-induced apoptosis, we transfected a melanoma cell line, MMRU, with either p33ING1 or antisense p33ING1 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 / A / G 7-overexpressing M M R U cells after UVB irradiation in comparison to cells transfected with vector alone or antisense p33ING1, and found that overexpression of p33ING1 consistently enhanced cell death at various doses of UVB (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 , A / G 7-overexpressing MMRU cells present significantly more condensed apoptotic bodies than the controls (p=0.004, t-test) (Figure 5.1 D). 70 Figure 5.1 Effect of p33 , A / b ' 7 on UVB-induced cell death in MMRU cells. (A) Western blot analysis of p33 expression in MMRU cells transfected with pCI-vector (V), pCI-p33 , / V G 7 (p33ING1), and pCI-antisense p 3 3 / W G 7 (AS). An anti-p33 / A / G 7 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 / A , G 7 (p33ING1), and pCI-antisense p 3 3 / N G 7 (AS). 24 h after transfection, MMRU cells were irradiated with UVB at 0, 40, 80, and 120 mJ/cm 2 . Trypan blue exclusion assay was then performed 24 h after UVB irradiation. Data represent mean + SD from triplicate plates. The experiment was repeated twice with similar results. (C) PI staining images of apoptotic cells after UVB irradiation. MMRU cells were transfected with pCI-p33 / A / G 7 and exposed to 80 mJ/cm 2 as above. 24 h after UVB irradiation, the cells were stained with PI, and images were taken using a fluorescent microscope. Cells without UVB irradiation were used as control. (D) Quantitative data from PI staining of UVB-irradiated M M R U cells transfected with pCI-vector (V), pCI-p33 / W G 7 (p33ING1), and pCI-antisense p33ING1 (AS). Data represent mean + SD from triplicate plates. (E) Microscopic images of UVB-irradiated MMRU cells transfected with vector (V), pCI-p33ING1 (p33ING1), and pCI-antisense p 3 3 / W G 7 (AS). 24 h after transfection, MMRU cells were irradiated with UVB. Photographic images were taken 24 h after UVB irradiation. (F) Quantitation of cell death by flow cytometry. MMRU cells were irradiated with UVB at 0 and 80 mJ/cm 2 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. 71 72 UV P33ING1 73 OmJ 80mJ p33!NG1 4.9% . j L. k 5.7% Lu, _J 0 mJ O 200 400 600 800 1000 0 200 400 600 600 1000 0 200 400 600 800 1000 31.7% 44.8% CO © 25.5% § ^ A o CM L . 80 mJ 0 200 400 600 800 1000 0 200 400 600 S00 1000 0 200 400 600 800 1000 DNA Content 74 Similarly, microscopic images show that there is significantly less spindle-shaped live cells in M M R U cells overexpressing p33ING1 24 h after UVB irradiation compared to vector- and antisense-control cells (Figure 5.1E). To further confirm the role of p33ING1 in UV-induced apoptosis, we performed flow cytometry analysis, and the results indicate that cells overexpressing p33ING1 displayed more sub-G1 population compared to the controls (Figure 5.1 F), which is consistent with the results from the trypan blue assay and PI counts. We 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 p33ING1 with p53 synergistically induced apoptosis in glioma cells (Shinoura et al., 1999) led us to hypothesize that p33ING1 and p53 may work together in the enhancement of UV-induced apoptosis in melanoma cells. We transfected MMRU cells with vector, p33ING1, pECH (wt p53), or p33ING1 + pECH, and exposed to UVB irradiation at 80 mJ/cm 2 . Using flow cytometry analysis, we found that overexpression of wt p53 alone had no effect on UV-induced cell death in MMRU 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 p33ING1 and p53 shows synergistic enhancement (0.001, t-test) (Figure 5.2A). This cooperation between p33ING1 and p53 has also been observed in other cellular responses such as nucleotide excision repair of UV-damaged DNA (Cheung et al., 2001b). To confirm that p53 is required for p33ING1-75 Figure 5.2 Synergistic effect of p33 , A / G 7 and p53 on UVB-induced cell death in M M R U cells. (A) Cell death assessment using flow cytometry on UVB-irradiated MMRU cells transfected with pCI-vector (V), pCI-p33 , / V G 7 (p33 / A f e 7 ) , pECH (wt p53), and pCI-p33 / A / G 7 + pECH. MMRU cells were irradiated with UVB at 0 and 80 mJ/cm 2 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 , A / G 7 , p E C H , pCI -p33 / W G 7 + pECH, and pCI-p33 , A / G 7 + pED1 (mut p53). The procedures for transfection and UVB irradiation were performed as in (A). Percentages of dead cells were determined 24 h post UVB irradiation. (C) Western analysis of p53 protein expression in MMRU cells transfected with pCI-vector (V), pCI-p33 / A / G 7 and pCI-antisense p33ING1 (AS). Western analysis was performed 24 h after transfection. p-actin was used as a loading control. 76 70 -60 50 40 -30 20 10 -0 • v • P33ING1 H wt p53 • p33ING1 + wtp53 • p33ING1+ mut p53 0 80 UVB dose (mJ/cm 2) V p33ING1 AS p33ING1 mamumm p53 Actin 77 induced apoptosis, we transfected MMRU cells with vector, p33INU\ pECH (wt p53), p33ING1 + p E C H , or p33ING1 + pED1 (mut p53). Trypan blue exclusion assay was performed after exposure to UVB irradiation at 80 mJ/cm 2 . The results show that, similar to those of flow cytometry analysis, significant enhancement in cell death was observed in cells transfected with p33ING1 + pECH (p=0.002, t-test) (Figure 5.2B). 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 / N G 7 and p53 in UV-induced apoptosis is not due to the ability of p33ING1 to elevate p53 expression, p53 protein levels were determined in MMRU cells transfected with vector, p33ING1, and antisense p33ING1 by Western blotting. The results indicate that p33ING1 does not induce the expression of p53 protein (Figure 5.2C). To further provide evidence of p53-dependence of p33ING1 in UV-induced apoptosis, we transfected a melanoma cell line M E W O that contains mutant p53, with vector, p33ING1, or antisense p33ING1 and irradiated the cells with UVB. Twenty-four hours later, the percentage of cell death was determined by trypan blue exclusion. Our results indicate that overexpression of p33ING1 did not enhance UVB-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, p33ING1, and antisense p33ING1 24 h after UVB irradiation (Figure 5.3C). To eliminate the possibility that this observed lack of enhancement by p33ING1 78 Figure 5 . 3 Effect of p33ING1 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\-p33ING1 (p33ING1), and pCI-antisense p33ING1 (AS). 24 h post-transfection, cells were irradiated with UVB at 0, 20, 40, 80, and 120 mJ/cm 2 . Trypan blue exclusion assay was then performed 24 h after UVB irradiation. Experiments were performed in triplicate. (B) Twenty-four hours after transfection, cells were exposed to 120 mJ/cm 2 of UVB and sub-G1 population was determined by flow cytometry 24 h post UVB irradiation. (C) Selected microscopic images of UVB-irradiated M E W O cells transfected with vector (V), pCI-p33 / A / G 7 (p33ING1), and pCI-antisense p33ING1 (AS). (D) Western analysis of p33ING1 protein expression in MEWO and MMRU cells. (3-actin was used as a loading control. B c 3 O o 0 mJ 120 mJ 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 DNA Content 79 0 mJ 120 mJ V \ • •*•'- A •, ; .,\ p33ING1 A S D MEWO MMRU • ^Ml^^^B wrfb^HW p33ING1 ^^^^ Actin 80 overexpression was not due to the presence of lower endogenous p33INU1 expression in the M E W O cell line compared to MMRU, we assessed the levels of p33ING1 protein in both cell lines using western analysis. Figure 5.3D shows that there is no obvious difference in p33ING1 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 p33ING1 activates the mitochondrial apoptosis pathway after UVB irradiation, cells transfected with p 3 3 , W G 7 or vector alone were irradiated with 80 mJ/cm 2 of UVB and stained with a cationic dye using the MitoCapture™ Apoptosis Detection kit 24 h after UVB irradiation. The cationic dye accumulates and aggregates in the mitochondria in 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 UVB. There is a significantly higher percentage of green-stained cells in p33 , A / G 7-transfected cells compared to vector controls (25.1% vs 12.7%) (Figure 5.4B). These data suggest that p33ING1 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 p33ING1 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 MMRU cells transfected with pCI-vector (V) and pCI-p33 / A / G 7 , and irradiated with UVB. 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), pCI -p33 / N G ) , and pCI-antisense p33ING1 (AS) were irradiated with UVB at 0 and 80 mJ/cm 2 . 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). 82 83 0 mJ 80 mJ V p33ING1 AS V p33ING1 AS B a x A c t i n 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). Recently, Nagashima et al. (2001) reported that p33ING1 can upregulate Bax promoter in colorectal carcinoma RKO cells. To investigate if p33ING1 activates endogenous Bax and to confirm the role of p33ING1 in the mitochondrial apoptotic pathway, we compared the levels of Bax protein in cells transfected with vector, p33ING1, and antisense p33ING1 before and after UVB irradiation. Our results demonstrate that cells overexpressing p33ING1 have higher Bax expression compared to vector and antisense controls, while UVB 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, p33ING1, enhances UVB-induced apoptosis in melanoma cells and that this enhancement requires the participation of p53. It is interesting to note that p33ING1 is also capable of enhancing repair of UV-damaged DNA. So under what different circumstances does p33ING1 participate in DNA repair or induce cell death? Presumably, p 3 3 / W G 7 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. With the ever increasing 85 evidence that p33ING1 plays an important role in cellular stress response, such as DNA repair and apoptosis, to UV irradiation, mutation and/or abnormal expression of the p33ING1 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 the mutation rate in these culture cells was relatively low but their pattern of p33ING1 expression and mutation follow that of p53. 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; Essnere fa / . , 1998; Whiteman etal., 1998). Our novel finding that p33ING1 cooperates with p53 to activate the mitochondrial pathway not only provides a better understanding of the p33ING1 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 disease -melanoma. 86 CHAPTER 6. p33 / N G t 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). We selected CPT 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. As 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 p24ING1 isoform is capable of enhancing chemosensitivity in human fibroblasts containing wt p53 after exposing to etoposide. To determine if the p33ING1 isoform also had a role in chemosensitivity, we hypothesized that p33ING1 overexpression enhances CPT-induced cell death in melanoma cells. 88 6.2 Results and Discussion In this study, we investigated whether p 3 3 / N G 7 enhances CPT-induced cell death in melanoma cells. We first confirmed that the melanoma cell line, R P E P , was transfectable and that the p33ING1 construct could be expressed (Figure 6.1A). Next, we compared the cell survival rate among cells overexpressing p33ING1 and controls (vector and antisense p33ING1) after treatment with various doses of CPT. 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 / W G 7-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, p33ING1, or antisense p33ING1. Results from flow cytometry analysis were consistent with those of S R B staining (Figure 6.3), indicating that overexpression of the isoform p33ING1 or suppression of p33ING1 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, MMRU (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 p33ING1 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 p33INU1 and antisense p33ING1. (A) Western blot analysis of p33ING1 expression in R P E P cells transfected with pCI-vector (V), pCI -p33^ G 7 (p33ING1), and pCI-antisense p33ING1 (AS). An anti-p33 , A / G J 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. 90 V p33ING1 AS p33ING1 ^ Actin 91 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 , A / G ' (p33ING1), and pCI-antisense p33ING1 (AS) for 24 h and then treated with 0 or 400 nM CPT. Cells were viewed and photographed using a Nikon microscope with a 10X objective. Control 400 nM 92 Figure 6.3 Quantitation of cell death by flow cytometry. Sub-G1 population represents dead cells. V, vector; p33ING1, pCI-p33 , A / G I ; A S , pCI-antisense p33 , A / G ) ; Ctl, no C P T treatment; CPT , 400 nM treatment for 24 h. V p33ING1 AS DNA Content 93 Figure 6.4 Effect of p 3 3 / W G ) on MMRU cell survival after CPT treatment. Cells were transfected with vector (V), p33ING1 (p33ING1), or antisense p 3 3 , N G 7 (AS) for 24 h and then treated with 0, 50, 200, or 400 nM of CPT. Cell survival was determined by S R B assay. Data represent mean + SD from three independent experiments. 94 6.5 shows that overexpression of both constructs, p33 , A / b 7 and pECH (containing human wt p53), had no enhancement on CPT-induced cell death compared to either one alone. Taken together, our data indicate that the alternatively spliced form, p33ING1, 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 its relationship with p53. Numerous tumor suppressive functions of ING1 have been observed, including G1 cell cycle arrest (Garkavtsev et al., 1996), anchorage-dependent 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). The interesting fact that ING1 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. We 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 p33ING1 and p53 co-expression on melanoma chemosensitivity. R P E P cells were transfected with vector (V), pCI-p33 / A / G 7 (p33ING1), p E C H (p53), or pCI-p33 , A / G ' + pECH 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. V P33ING1 p53 p33ING1+p53 DNA Content 96 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 MCF7 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 p33ING1 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. Four alternatively spliced forms of ING1, named p47ING1, p33ING1, p27ING1, and p24ING1, 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 p33ING1 isoform. W e first investigated how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model, we examined if the expression of p33ING1 is dependent on p53. We found that there was no difference in p33ING1 mRNA and protein levels between p53+/+ and p53"7~ murine organs. In addition, when normal human epithelial keratinocytes and a keratinocyte cell line, HaCaT, which lacks wt p53 function, were exposed to UVB irradiation, the expression levels of p33ING1 were elevated in both normal human epithelial keratinocytes and HaCaT cells. It is interesting, however, that UVB irradiation did not induce p33ING1 expression in dermal fibroblasts isolated from p53+ / + and p53"A mice. Based on our findings, we therefore concluded that the expression of p33ING1 is independent of p53 status. UV induction of p33ING1 in keratinocytes suggests that 98 p33ING1 may play a role in cellular stress response and skin carcinogenesis. The 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 p 3 3 / W G 7 plays a role in UV-stress response, such as repair of UV-damaged DNA. We found that overexpression of p33ING1 enhances repair of UV-damaged DNA and that p53 is required for the repair process in melanoma cells. Furthermore, physical binding between ING1 and GADD45 was detected by immunoprecipitation. These observations suggest that p33ING1 cooperates with p53 in nucleotide excision repair and that GADD45 may be one of its components. Next we investigated the molecular pathways of p33ING1 enhancement in UV-induced apoptosis in biologically relevant cells using melanoma cell lines. We found that overexpression of p33ING1 increased while the introduction of an antisense p33ING1 plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33ING1 again required the presence of p53. Moreover, we found that p33ING1 enhanced the expression of endogenous Bax and altered mitochondrial membrane potential. These observations strongly suggest that p33ING1 cooperates with p53 in UV-induced apoptosis via the mitochondrial cell death pathway in melanoma cells. Previous findings indicate that the isoform p24ING1 is capable of enhancing chemosensitivity in human fibroblasts. To investigate if the p33ING1 isoform is also involved in chemosensitivity, we overexpressed p33ING1 in melanoma cells and assessed for cell death after treatment with camptothecin. Results from cell survival assay and flow cytometry analysis show no significant difference among cells transfected with vector, p33ING1, 99 and antisense p33ING1. 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 p33ING1 and p53 in DNA repair and apoptosis. We therefore demonstrate that p33ING1 does not enhance camptothecin-induced cell death in melanoma cells. Taken altogether, we have elucidated in this thesis some of the novel functions of p33ING1 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, better scientific tools are needed. For instance, although we were able to consistently demonstrate relatively high expression of the wt p33ING1 plasmid in our cell lines, the 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. We are also interested in establishing cell lines expressing wt p33ING1 and antisense p33ING1 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 p33ING1 may not represent physiologically normal conditions in the cell and that the antisense p33ING1 construct consistently appears weak in suppressing the levels of endogenous p33ING1 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 gene has any effects on normal development of mammals. An alternative and recently developed technique for disrupting endogenous genes in mammalian cells is the RNA interference (RNA i) method. Initially used in the nematode Caenorhabditis elegans in the early 1990s (Fire et al., 1991), this technique employs double-stranded RNA with sequence specific against the target gene mRNA 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 p33ING1 isoform, one may be able to correct such defect by gene therapy. In the case of skin cancer, our laboratory has recently demonstrated that p33ING1 is overexpressed in 100% of melanoma cell lines (Campos et al., 2002) and 101 96% of melanoma primaries (data not shown) compared to their normal counterparts. 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 p33ING1 transcript, may account for the overexpression of the dysfunctional p33ING1 protein. Gene therapy using wt p33ING1 may be one option to eliminate the maligancy by apoptosis. Melanoma-specific expression can be achieved by constructing a vector containing the wt p33ING1 DNA attached to the tyrosinase promoter, which is only activated in melanin-producing cells (Bertolotto et al., 1996). To capitalize our finding that overexpression of p33ING1 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 p33ING1 delivered into the area of target for UV light application. 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Science 1991, 254: 1167-73. 122 2001 UBC Division of Dermatology Annual Research conference, Vancouver, BC (Topic: p33ING1 role in DNA repair) 2000 UBC Experimental Medicine Annual Student Research Day, Vancouver, BC (Topic: ING1 role in DNA repair) 1999 UBC Experimental Medicine Annual Student Research Day, Vancouver, BC (Topic: p33ING1 involvement in UV-induced stress response and its in vivo relationship with p53) 1999 77 t h International Association for Dental Research (IADR), Vancouver, BC (Topic: Increased allelic loss in early oral dysplasia from high-risk sites) 1999 Frost Road Elementary School, Surrey, BC (Topics: Genetics: Applications, Career, and Training) 1998 British Columbia Cancer Research Center, Vancouver, BC (Topic: LOH profile of oral premalignant lesions in low-/high-risk sites) 1998 Burnaby North Secondary School, Burnaby, BC (Topic: Applications of Genetics) 1997 British Columbia Cancer Research Center, Vancouver, BC (Topic: Genetic susceptibility and human chromosome 11) ABSTRACTS 1. Chin M, Cheung K-J, Ho V, Li G . Induction of apoptosis p33INh1 via bcl-2 and caspase 3. Annual meeting for Society of Investigative Dermatology, Florida, USA. April 30-May4, 2003. 2. Cheung K-J, Ho V, Li G . Role of p 3 3 / N G ' in UV-induced apoptosis. Annual meeting for Society of Investigative Dermatology, Los Angeles, USA. May 15-18, 2002. 3. Cheung K-J, Mitchell D, Lin P, Ho V, Li G. p33ING1 role in DNA repair. Annual meeting for Society of Investigative Dermatology, Washington, DC, USA. 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 Dermatology, Washington DC, May 9-12, 2001. 5. Campos E l , Cheung K-J, Li G. Mutational analysis of the p 3 3 / N G ? gene in human melanoma cell lines. Annual Meeting of the Society for Investigative Dermatology, Washington DC, May 9-12, 2001. 6. Cheung K-J, Lin P, Ho V, Li G. Role of p 3 3 , W G ' in DNA repair. Annual Meeting of the Canadian Society for Investigative Dermatology, Halifax, NS, June 28-30, 2001. 7. Cheung K-J, Bush JA , Ho V, Li G. The Role of p 3 3 m G ' in Cellular Stress Response and its In Vivo Relationship. Annual Meeting of Canadian Society for Investigative Dermatology, Montreal, June 29-July 2, 2000. 8. Cheung K-J, Ho V, Li G . p33ING1 involvement in UV-induced stress response and its in vivo relationship with p53. Journal of Investigative Dermatology, 114(4), p854. 2000. 9. Cheung K-J, Zhang L, Rosin MP. Increased allelic loss in early oral dysplasia from high-risk sites. 77th International Association for Dental Research (IADR), Vancouver, B C , Canada. 1999. 124 PUBLICATIONS 1. Cheung K-J and Li G. p53 and p33 INU1: Role in nucleotide excision repair of UV-damaged DNA. Book chapter in Comprehensive Series in Photosciences. In press. 2003. 2. Cheung K-J and Li G. The tumour suppressor p33 I N G 1 does not regulate migration and angiogenesis in melanoma cells. International Journal of Oncology. 21(6): 1361-5,2002. 3. Cheung K-J and Li G. p33 I N G 1 enhances UVB-induced apoptosis in melanoma cells. Experimental Cell Research. 279(2): 291-8, 2002. 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 I N G 1 mediates repair of UV-damaged DNA. Cancer Research. 61(13): 4974-77, 2001. 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: 305-14,2001. 13. Cheung K-J, Bush J, Jia W, Li G. Expression of the novel tumor suppressor p33 I N G 1 Is 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 • 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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