INITIAL CHARACTERIZATION AND DETE RMINATION OF THE MOLECULAR MECHANISM(S) THAT CONTROL TR ANSCRIPTION OF THE HUMAN PKC EPSILON GENE IN LUNG CANCER CELLS By LINNET AKINYI A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2004
Copyright 2004 by Linnet Akinyi
iii ACKNOWLEDGMENTS I thank the University of Florida Anat omy and Cell Biology department and the Graduate School for giving me the opportunity to further her studies. Next, I thank my mentor, Dr. Lei Xiao, was most in strumental in my growth as a scientist. I also thank the other members of my committee, Dr. Kilber g and Dr. Wallace for assistance, support, and advice they provided during th e course of this thesis stud y. Special thanks go to the my colleagues in the Xiao lab, (Dr. Kyung-Mi Bae, Benjamin Sutter, Heiman Wang, and Dr. Guoha Jiang). Finally, but most importan tly, I thank my family and friends for their faith in me, their love, and their unfailing support for my e ducational endeavors.
iv TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iii LIST OF TABLES...............................................................................................................v LIST OF FIGURES...........................................................................................................vi ABSTRACT......................................................................................................................v ii CHAPTER 1 INTRODUCTION........................................................................................................1 Protein Kinase C...........................................................................................................1 Isotype PKC Epsilon.....................................................................................................2 Lung Cancer..................................................................................................................6 DNA Methylation.........................................................................................................8 2 MATERIALS AND METHODS...............................................................................14 Primer Extension........................................................................................................14 RT-PCR......................................................................................................................15 Transient Transfection Assays....................................................................................17 Bisulfite Genomic Modification.................................................................................20 3 RESULTS...................................................................................................................23 The Transcription Start Site for the PKCGene is Located 132 Nucleotides Upstream From the Translation Start Site.............................................................23 Promoter Activity in the 5 Regulatory Region of the PKCGene..........................25 Synergistic Effects of 5-Aza-CdR a nd TSA in The Reactivation of PKCGene.....26 Methylation Status f the PKCPromoter..................................................................29 4 DISCUSSION.............................................................................................................31 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................44
v LIST OF TABLES Table page 2-1 PKCexpression in lung cancer cell lines..............................................................15
vi LIST OF FIGURES Figure page 1-1 Schematic of primary structures of protein kinase C family members showing domain composition and activators............................................................................2 1-2 The mechanism whereby DNA methylation and histone deacetyl ation cooperate to repress transcription.............................................................................................12 2-1 A physical map of the 5 regulatory region of the human PKCgene depicting deletion constructs....................................................................................................20 2-2 Schematic diagram of the CpG island lo cated in the 5' regulatory region of the PKCgene (-1930 to -220) relative to the transcription start site..........................22 3-1 Determination of the transc ription start site of the PKCgene..............................24 3-2 A schematic representation of th e 5' regulatory region of the PKCgene showing the transcriptio n start site (+1)...................................................................25 3-3 Induction of luciferase activity by the PKCpromoter in H157 cell line...............26 3-4 Synergistic effects of 5-Aza-CdR and TSA in the reactivation of PKCgene.......28 3-5 Restriction enzyme digestion profile of bisulfite-converted DNA..........................30
vii Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science INITIAL CHARACTERIZATION AND DETE RMINATION OF THE MOLECULAR MECHANISM(S) THAT CONTROL TR ANSCRIPTION OF THE HUMAN PKC EPSILON GENE IN LUNG CANCER CELLS By Linnet Akinyi December 2004 Chair: Lei Xiao Major Department: Anatomy and Cell Biology Protein kinase C has been implicated in the regulation of diversified biological functions in both normal and tumor cells. We recently learned that expression of PKC, a novel PKC isoform, is specifically linked to the chemo-resistant phenotype of nonsmall cell lung cancer (NSCLC). Our studies suggest that the expr ession level of PKCin a cell is a key determinant of cellular suscepti bility to chemothe rapy. The cell typespecific regulatory mechanisms that control PKCexpression are currently unknown. We investigated this transcriptional regula tion by cloning and characterizing the promoter region of the human PKCgene. A 6-kb genomic fragment containing the 5' regulatory region was isolated, cloned by high fidelity PCR, and sequenced. The transcriptional start site, which is located 132 bases upstream of the translational in itiation codon was determined by primer extension analysis. The putative PKCpromoter lacks the TATA and CAAT boxes but contains several GC-ri ch regions as well as Sp1, Ap1, and CRE
viii binding sites. Promoter func tion was analyzed in the PKCexpressing cell line H157. A deletion analysis and lucifera se reporter gene assays of th e promoter demonstrated that a positive cis-acting regulatory region was pres ent in this TATA-less promoter. Results of the deletion analysis revealed that the region from -5463 to +49 relative to the transcription start site is important for transcriptional regulation of the PKCgene. These observations suggest the presence of cis-acting elements that confer the phenotype specific transcription of PKCin the distal promot er region. As sequence analysis revealed a CpG-rich island in th e 5' regulatory region of the gene, we investigated DNA methylation as a plausible mechanism in the transcriptional repression of the PKCgene. The NCI-H82 SCLC cells, (which do not express PKCowing to the transcriptional inactivati on of the gene) were treated with the inhibitor of DNA methylation, 5-Aza-2'-deoxycy tidine (5-Aza-CdR). Results demonstrate that 5-Aza-CdR induces PKCexpression suggesting that DNA methyla tion plays a poten tial role in the PKCgene silencing in SCLC cells. As aberrant DNA methylation at the CpG-rich islands has been shown to play a key role in the transcriptional repression, these results provide a necessary basis for further studies of the methylation patte rns at the promoter region, in particular the CpG island, in PKCexpressing NSCLC cells and non-expre ssing SCLC cells. Understanding these methylation patterns could be the key to elucidating the molecular mechanism(s) that control differential tr anscription of PKCin human lung cancer cell lines.
1 CHAPTER 1 INTRODUCTION Protein Kinase C Protein kinase C comprises a family of seri ne/threonine protein kinases that play a critical role in many signal-tr ansduction pathways in the cell. Protein kinase C is a ubiquitous enzyme that was originally described as a Ca2+-activated, phospholipiddependent protein kinase (Inoue et al.1977). Molecular cloni ng and biochemical analysis revealed that the enzyme exists as a family of multiple subspecies having closely related structures. Up to 12 distinct family memb ers have been discovered in mammalian cells, and several non-mammalian PKCs have been described. The mammalian protein kinase C family comprises 10 isozymes grouped into 3 classes: conventional PKCs (cPKC) includi ng alpha, betaI and the sp lice variant betaII, and gamma; novel PKCs (nPKC), delta, epsil on, eta, and theta; and atypical PKCs (aPKC) comprising zeta and lambda (also know n as iota) (Nishizuka et al.1995; Mellor and Parker 1998; Toker 1998). An additional family member may be considered by the more recently discovered PKCmu. This PKC was independently discovered by two laboratories,( Valverde et al.1994 and is also known as protein kinase D (PKD) (Johannes et al.1994). All members have in common a conserved kinase core carboxyl terminal to a regulatory moiety. This re gulatory moiety contains tw o key functionalities: an autoinhibitory sequence (pseudosubstrate) and one or two membrane targeting modules (C1 and C2 domains and, in the case of protein kinase D, PH domain) (Newton 2001) (Figure 1). The cPKCs alpha , beta I/II and gamma were the first
2 described and are activated in vitro and in vivo by phosphatidylserine (PS) in a calciumdependent manner. In addition, they bind to and are activated by diacylglycerol (DAG), which increases the specificity of the enzyme for PS and also increases the affinity of the enzyme for calcium (Newton 1995). Novel PK Cs are also activated by DAG and require PS as a co-factor, but have lost the requireme nt for calcium. This loss was found to be partly due to the absence of a classical C2 domain. Neither the atypical PKCs zeta and lambda/iota nor PKCmu/PKD respond to DAG or calcium, but still require PS as a cofactor (Newton 2001) (Figure 1). Figure 1-1. Schematic of primary structur es of protein kinase C family members showing domain composition and activators . The N-terminal moiety contains the regulatory modules: the pseudosubstrate (green); the C1 A and B domains which bind phosphatidylserine and, for all atypical protein kinase CÂ’s, diacylglycerol/phorbal esters (orange); the C2 domain which binds anionic lipids and, for conventional protein kinase CÂ’s, Ca2+ (yellow); and the PH domain which binds phosphoinositides (purple). The C-terminal moiety contains the kinase domain (cyan). The requirements for the classical cofactors for protein kinase C subc lasses are shown on the right: PS, phosphatidylserine; DG, diacylglycerol; Ca2+. (Newton, A. C. (2001) Structural and spatial regulati on by phosphorylation, cofactors, and macromolecular interactions. Chem. Rev. 101, 2353-2364). Isotype PKC Epsilon PKC epsilon is a novel PKC isotype that is characterized as a calcium-independent and phorbol ester/diacylglycerol-s ensitive serine/threonine kina se (Ono et al., 1988). It is
3 expressed in many tissues and cells, but a bundantly in neuronal, hormonal and immune cells (Ohno et al.1988). PKCplays essential roles in many signaling systems including proliferation (Balciunaite et al. 2000), diffe rentiation (Racke et al. 2001), gene expression (Jobbagy et al. 1999), muscle contraction (Ali et al. 2002), mechanical force adaptation (Traulo et al.1997), metabolism (Mehta et al. 2002), transport (DeCoy et al. 1995), exocytosis (Akita et al. 1994), and endocytosis (Song et al. 1999) systems. PKCalso has roles in the nervous, inflammatory, imm une, and circulatory systems. Castrillo et al (2000) showed that PKCis required for macrophage activation and defense against bacterial infection in the immune system. The macrophages from PKCknockout mice have severe deficiencies and, in the absence of PKC, host defense against bacterial infection is severely compromised, resulting in increased mortality (Lar sen et al. 2000). PKCalso participates in nerve gr owth factor (NGF) signaling. PKCis activated after NGG-stimulation of the PC12 ra t pheochromocytoma cell line (Ohmichi et al. 1993). Overexpression of PKCin the same cells induces both neurite extension and activation of MAPK in an NGF-dependent manner (Hundle et al. 1995). Similar observations were made in studies with hum an neuroblastoma cells (Fagerstrom et al. 1996). PKChas been found to be enriched at the growth cones of extending neurites in differentiating neural cells (Parrow et al. 1995). It therefore appears that PKCregulates certain aspects of neural function, and this is consistent with the fact that this PKC isoform is abundant in brain and other neural tissues. PKChas also been linked to the expression of certain transcripti on factors, and to the induction of immediate-early genes. This may account, at least in part, for its effects on cell growth (Toker 1998). Studies have shown that PKCcan induce the accumulation of both c-fos and c-Jun mRNAs,
4 and this is dependent on the ac tivation of high-affin ity IgE receptors (Razin et al. 1994). PKChas also been shown to regulate the tr anscription factors NF-AT-1 and AP-1 in activated T cells, similar to the stimulatory e ffects of activated Ras in the same system (Genot et al. 1995). PKCs are known to be important regulator s of the cytoskeleton in cells. Recent studies indicate that PKCplays an important role in cyto skeletal organization. Prekeris et al (1996) identified an actin-bin ding motif that is unique to PKCand proposed that this serves to localize the act ive PKC within intact nerve terminals. The actin-binding motif is located between the fi rst and second cysteine-rich re gions of the C1 domain, and associates with actin filaments in response to extra stimuli in a manner independent of phosphatidylserine (Prekeris et al. 1996; Ze idman et al. 2002). Arachidonic (AA) and diacylglycerol (DAG) syne rgistically stimulate th e association of PKCand actin. Filamentous actin serves as the unique anchoring protein of PKC, and also activates the kinase by maintaining it in a catalytically active conformation. The C1 and C2 domains of PKCultimately function as subcellular loca lization signals (Leh el et al. 1995). PKCseems to be involved in tumor development and tumor cell metastasis in several tissues. Cacace et al (1993) and Misc hak et al (1993) demonstrated that PKCcan function as an oncogene when overexpr essed in fibroblasts . Increased PKCactivity in these cells correlated with formation of de nse foci in monolayer cultures, decreased doubling times, increased cell sa turation densities, a decrease in serum requirements, growth in soft agar and tumor formation in nude mice (all characteristics of neoplastic transformation). In further studies Cacace et al (1996) indicated that PKCmay function as an oncogene by enhancing the activity of the Raf-1 kinase, thus
5 modulating the MAPK pathway. Evidence ha s also been presented that oncogenic potential of PKCis due in part to the production of autocrine growth factors, in particular transforming growth factor beta 2 and 3 (TGF-beta) (Ueffing et al. 1997; Cacace et al. 1998). Transcriptional activa tion of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcription factors that interact with distinct elements of the cyclin D1 promoter. Investigation of the role of PKCin the Ras-Rac-mediated transcriptional regulation of cyclin D1 showed that PKCmediates cyclin DI induc tion in Ha-ras-transformed fibroblasts (Kampfer et al. 2001). Squamous cell carcinoma (SCC) and basa l cell carcinoma (BCC) are the most common forms of human skin cancer. In murine skin carcinogenesis models for SCC, the incidence of metastasis is very low. Jansen et al (2001) showed that epidermisspecific transgenic overexpression of PKCcauses mice to develop highly malignant/metastatic carcinomas. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote ce ll spreading. Studies have shown that PKCis required for the cell spreading mediated by integrin 1; and that Rac1 acts downstream of PI3-kinase and PKC(Berrier et al. 2000; Chun et al. 1996). PKCis reported to be linked to integrin 1 through interaction with RACK1, a nd to associate with F-actin via its actin-binding site, thereby mediating increased adhesion and mobility (Besson et al. 2002; Tachado et al. 2002). Ivaska et al (2002) reported that PKCappears to contribute to motility by regulating 1 integrin traffic that permits its recycling in cells. In human breast carcinoma cells, cis-polyunsat urated fatty acids stimulate 1 integrin-mediated
6 adhesion to type IV co llagen by activating PKCand PKC(Palmantier et al. 2001). PKChas been implicated in apoptosis in a variety of cells. In thyroid tumors, rearranged amplification of the PKCgene or post-transcrip tional changes have been reported (Knauf et al. 2002). In addition, PKChas been implicated in ultravioletinduced apoptosis and tumor promotion (Che n et al. 1999). Koriyama et al (1999) showed that PKCappears to be subjected to restric tive proteolysis by caspase (3 and 7). Studies have also de monstrated that PKCcan be selectively cleav ed by calpain (Eto et al.1995; Hiwasa et al. 2002). All these studies s uggest that PKCplays an important role in the signaling cascade that accom panies apoptosis and tumorigenesis. Lung Cancer Lung cancer is the most prevalent and leth al cancer in the world. In the United States 160, 000 people died of l ung cancer last year, which repr esents 28% of all cancer deaths (Jemal et al. 2001). Although the rate of lung cancer d eaths for males is decreasing in the United States, the mortal ity associated with lung cancer among women continues to increase (Greenl ee et al. 2000). The main risk factor for lung cancer is cigarette smoking, accounting for about 90% of th e cases in men and 70% of the cases in women (Doll et al. 1981; Shopland et al. 1995). The incidence of lung cancer can also be influenced by exposures to other environm ental and occupational respiratory carcinogens that interact with cigarette smoking. At this time, preventing smoking initiation and increased smoking cessation remain the best long-term methods to prevent lung cancer development. Lung cancer is divided into f our major histological subtypes as proposed by the World Health Organization (Whole H ealth Organization, 1982): Adenocarcinoma (AD), Squamous Cell Carcinoma (SCC), Large Cell Lung Carcinoma (LC), and Small
7 Cell Lung Carcinoma (SCLC). Since the progno sis and treatment of SCLC is markedly different from non-SCLC (NSCLC), the four histological subtypes are clinically divided into SCLC and NSCLC (comprising AD, SCC, LC and other minor forms) (Travis et al. 1996). NSCLC represents about 80% of all lung cancers. SCLC represents about 20% of all lung cancers and is characterized by early metastasis and initial responsiveness to chemotherapy and radiation. However, near ly all patients with SCLC relapse and develop resistance to cytotoxic therapy and ev entually die from the disease (Smit et al. 1995). The underlying mechanism of developi ng drug resistance in relapsed SCLC patients is unclear. This resistance may be due, in part, to a transition of the SCLC toward the NSCLC histology (Abeloff et al. 1979; Mabry et al. 1991). Development of metastases when the primar y tumors are still small, coupled with lack of methods for early diagnos is and of systemic therapies with great efficacy to deal with micrometastatic disease are the main reasons why the prognosis of lung cancer patients is still poor. Nevertheless, some improvement in survival has been shown over the last two decades (Chute et al. 1999; Lebitasy et al. 2001), bringing the overall 5-year survival rate to approximat ely 14% (Travis et al. 1995). Thus new methods for early detection and identification of smokers at grea test risk for developi ng lung cancer, such as spiral-computed tomography screening for early lung cancers, biomarkers for lung cancer risk assessment, new approaches fo r lung cancer prevention (chemoprevention), and new drugs based on rational targets, ar e necessary and need to be developed (Zochbauer Muller et al. 2002). Human lung cancer cells express multiple PKC isoforms. It has been reported that the drug-resistant phenotype is associated with expression and/or activity of PKCs in lung cancer cell lines and lung carcinomas (Basu et
8 al. 1996; Volm 1995). Ding et al (2002) reported that the expression of PKC, a novel isoform, is specifically linked to the chem o-resistant NSCLC cells, but are absent in SCLC cells tested. They observe d that forced expression of PKCconferred resistance to chemotherapy in SCLC cells, while down-regulation of PKCexpression sensitized NSCLC cells. This suggests th at the expression level of PKCin a cell is a key determinant of cellular sus ceptibility to chemotherapy. Currently, the molecular mechanism(s) that control the differential expression of PKCin human lung cancer cells are unknown. DNA Methylation It is well known that a variety of gene tic changes influence the development and progression of cancer. These changes may result from inherited or spontaneous mutations that are not correct ed by repair mechanisms prior to DNA replication. It is increasingly clear that so called epigenetic effects that do not affect the primary sequence of the genome also play an important role in tumorigenesis. One form of epigenetic information in mammalian cells is DNA methyla tion, or the covalent addition of a methyl group to the 5-position of cytosine predomin ately within the CpG dinucleotide (Plass et al. 2002). DNA methylation ha s profound effects on the mammalian genome. Some of these effects include transc riptional repression, chromatin structure modulation, X chromosome inactivation, genome imprinting, and the suppression of the detrimental effects of repetitive and parasitic DNA seque nces on genome integrity (Baylin et al. 2000; Jones et al. 1999; Robertson et al . 2000). CpG dinucleotides (CpGs) are underrepresented in vertebrate DNA; they occu r at 0.2 to 0.25 of the frequency expected from the overall genomeÂ’s nucleotide compos ition (Swartz et al. 1962). Despite the low
9 average abundance of CpGs, the human ge nome contains approximately 45,000 limited regions of high-density CpGs known as CpG islands (Antequera et al. 1994). These regions are mostly associated with promoters, the 5' end of genes, or both (Cross et al. 1995). Housekeeping genes regularly contain CpG islands, as do approximately 40% of genes with tissue-specific expression patte rns (Larsen et al. 1992). Approximately 6090% of the CpGs in the genome of the adu lt mammal are methylated by the covalent addition of a methyl group to the 5-position during embryogenesis (Y eivin et al. 1993). In contrast, cytosines in CpG islands in the promoters of active genes are largely unmethylated (Naveh-Many et al. 1981). Cellular DNA methylation patterns seem to be established by a complex interplay of at least three independent DNA me thyltransferases: DNMT1, DNMT3A, and DNMT3B. Bestor et al (1988) discovered the first methyltransferase, DNMT1 in 1988. Studies have shown that DNMT1 has a 10 to 40 fold preference for hemimethylated DNA (Pradhen et al. 1997; Pradhen et al . 1999). DNMT1 is the most abundant methyltransferase in somatic cells (Robertson et al. 1999). It localizes to the replication foci via several independent domains and inte racts with the proliferating cell nuclear antigen (PCNA) (Chuang et al. 1997). This set of features is why DNMT1 is often referred to as the maintenance methyltransfer ase since it is believed to be the primary enzyme responsible for copying methylati on patterns after DNA methylation. The generation of DNMT1-knockout mice has revealed that DNMT1 is required for proper embryonic development, imprinting, and X-inacti vation (Li et al. 1993; Beard et al. 1995; Xie et al. 1999). In 1998, Okano et al (1998) reported the cloning and initial characterization of the DNMT3 family of methyltransferases. The mouse and human
10 enzymes are highly conserved (approximately 95% identical at the amino acid level). Homologous genes have been identified in zebra-fish, Arabidopsis thaliana and maize (Okano et al. 1998; Cao et al. 2000). These enzymes are required for the de novo methylation that occurs in the genome following embryo implantation and for the de novo methylation of newly integrated retroviral sequences in mouse ES cells. Studies by Okano et al (1999) showed that DNMT3a knoc kout mice are born live but become runted and die at about four weeks of age. DNMT3b knockout mice on the other hand are not viable, and mutant embryos show numer ous developmental defects and growth impairment after embryonic day (E) 9.5, close to the time at which the DNMT1 knockout mice begin to show growth defects (Li et al . 1992). These observations, coupled with in vitro data indicating that the DNMT3 enzyme s have an equal preference for hemi-and unmethylated DNA substrates, have led to them being termed the Â‘ de novo methyltransferasesÂ’ (O kano et al. 1998). It is now well established that DNA met hylation is involved in regulating gene transcription. The methylat ion state of promoter CpG isla nds confers information about the transcriptional activity at these loci. It has been know n for years that, in general terms, there is an inverse relationship betw een the density of promoter methylation and the transcriptional activity of a gene. H ypermethylated promoters are almost always transcriptionally silent, packaged into a chromatin structure resistant to nucleases, and enriched in hypoacetylated core histones (Eden et al. 1998). However, the actual mechanisms by which DNA methylation modulates gene expression are still not entirely clear.
11 Early studies by Tate et al (1993) showed that DNA met hylation appears capable of directly preventing the binding of some transcription factors to their DNA binding sites. This is achieved when methyl groups proj ect into the major groove of DNA, thus interfering with the binding of specific transc ription factors that have methylated CpG(s) within their response elements (Tate et al. 1993). In 1992, a transcri ption repressor that selectively recognizes methylated DNA, me thyl-CpG binding protein 2 (MECP2), was characterized (Lewis et al. 1992). MECP2 can be divided in to two structural domains: a methyl-CpG binding domain (MBD) which re cognizes a symmetrically methylated CpG dinucleotide through contacts in the major gr oove of the double helix (Wakefield et al. 1999), and a transcriptional repression domain (TRD) which interacts with several other regulatory proteins (Nan et al. 1997). In 1998, two independent groups not only linked DNA methylation and transcriptional si lencing, DNA methylat ion and histone hypoacetylation, but also showed that MECP2 could recruit histone deacetylase (HDAC) (Nan et al. 1998; Jones et al. 1998). This led to proposal off a rational mechanism for explaining DNA methylati on could repress transcription and result in chromatin structure change: recruitment of MBDÂ’ s and their associated HDAC s to methylated DNA would result in local deacetylation of core histone tails, which would result in tighter packaging of DNA and reduced access to transcription factors to their binding sites (Fig. 1-2 )
12 Figure 1-2. The mechanism whereby DNA me thylation and histone deacetylation cooperate to repr ess transcription . A transcriptionally active region targeted for silencing is proposed to acquire DNA methylation first, which then recruits the methyl-CpG binding proteins and their associated co-repressors and histone deacetylases (HDAC). As DNA methyltransferase 1 (DNMT1) can interact directly with hi stone deacetylase, it is po ssible that transcription is first silenced by deacetylation by other tethering factors, after which the methylation machinery and the methyl-C pG binding proteins are recruited to Â‘cementÂ’ the promoter in the silent st ate. In either case, the deacetylated nucleosomes adopt a more tightly packed structure that inhibits the access of transcription factors to their binding sites. (Gul dberg, J.W. (2002) DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy. J. Oral. Pathol. Med. 31, 443-449. Early studies by Jemal et al (2001) showed a mechanistic link be tween DNA methylation and histone deacetylation. They demonstrat ed this by reactivating the aberrantly methylated genes p16 , TIMP3 , and MLH1 in a colon cancer cell lin e by treating the cells with a combination of the DNA methyltransf erase inhibitor 5-Aza-CdR and the histone deacetylase inhibitor trichostatin A (TSA). Low doses of 5-Aza-CdR resulted in lowlevel re-expression and minimal demet hylation of hypermethylated CpG-islandassociated genes, however a combination of 5-Aza-CdR and TSA resulted in robust
13 activation of these same genes. Treatment w ith TSA alone had no effect. This revealed not only that DNA methylation and histone deacetylation wo rked together to silence transcription, but also that DNA methylati on was dominant over histone acetylation status. The genes APC, CDKN2A, CHD 13, RARB, and RASSF1A are a few of the genes that have been found to be hypermet hylated in over 30% of lung cancer tumors (Tsou et al. 2002). Each of these five genes ha s been demonstrated to be transcriptionally silenced in cell lines/tissues showing methylat ion. Re-expression of these genes was seen in lung cancer cell lines following treatment with 5-Aza-CdR, further supporting the notion that methylation caused their inactivation (Brabender et al. 2001; Dammann et al. 2000; Zhu et al. 2001). Their silencing argues that DNA methylation as a common mechanism for gene inactivation in lung cancer. This leads to the hypothesis of my stu dy: that DNA methylation is one of the molecular mechanism(s) that control the transcription of the PKCgene in human lung cancer cell lines. Previous studies from this laboratory (Ding et al., 2002) reported that the expression of PKC, a novel isoform, is specifically linked to the chemo-resistant NSCLC cells, but is transcriptionally silent in SCLC cells tested. It was observed that forced expression of PKCconferred resistance to chemotherapy in SCLC cells, while down-regulation of PKCexpression sensitized NSCLC cel ls. This suggested that the expression level of PKCis a key determinant of cellula r susceptibility to chemotherapy. Results from this study, though not conclusi ve, suggest that DNA methylation plays a role in the transcriptional inactivation of PKCin human lung cancer cell lines.
14 CHAPTER 2 MATERIALS AND METHODS Primer Extension Primer extension assays were performe d using a 20-bp anti-sense primer, PKCreverse 2 (5 -GAAGGCCATTGAACACTACC-3 ) complementary to the PKCcDNA sequence spanning positions (+135/+152) relative to the transcription start site. The primer was end-labeled with [ 32P] ATP (ICN, Costa Mesa, CA) by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). We hybridized 40 g of total RNA form H157 NSCLC cells was hybrid ized with 1 pmol of 32P-PKC reverse 2 primer in aqueous hybridization buffer (5M NaCl, 1M Tris-Cl, pH7.6, 0.5M EDTA, pH 8) and denatured at 84oC for 10 min. The primer extension reaction was then gradually cooled to 50oC followed by precipitation with 3 M NaOAc pH 5.2 and 2 volumes of ethanol in dry ice for 30 min. The reaction was washed with 75% ethanol/25% 0.1 M NaOAc pH 5.2, air dried and resuspended in DEPC-ddH2O. First Strand buffer (Invitrogen Life technologies, Carlsbad, CA), 0.1 M DTT, 10 mM 4X dNTPs (Boehringer Mannheim, Germany), RNaseOut (40U/ l), Superscript II reverse transcriptase (200U/ l) was added to the reaction and incubated at 42 C for 60-90 min. This was followed by the addition of 0.5 M EDTA, RNase A (1 mg/mL) and incubation at 37 C for 30 min. The primer extension reaction was then precipitated with 10 M NH4OAc at -20 C for 1 hr, washed with 80% ETOH and resuspended in LTE. The cDNA product was denatured at 100 C and analyzed by electrophoresis using an 8% denaturing Polyacrylamide gel (8 M urea,
15 30% Bis/acrylamide) run at 200V for 3 hr. The size of the extension products was determined using a 32P-end-labeled molecular weight marker pBR322 DNAMspI Digest (Invitrogen Life Technologies , Carlsbad, CA) and DNA sequencing. The gel was fixed with 25% methanol/ 25% acetic acid for 10 mi nutes, dried using a gel dryer and exposed to Kodak XAR film (Eastman K odak Company, Rochester, NY). Table 2-1. PKCexpression in lung cancer cell lines. negative SCLC H249 negative SCLC H82 negative SCLC H69 positive NSCLC H1299 positive NSCLC A549 positive NSCLC H157 positive NSCLC H23 PKCHistological type Cell line negative SCLC H249 negative SCLC H82 negative SCLC H69 positive NSCLC H1299 positive NSCLC A549 positive NSCLC H157 positive NSCLC H23 PKCHistological type Cell line RT-PCR The human lung cancer cell line H82 was grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin-streptomycin in an incubator (37 C, 5% CO2). Cells were seeded at a density of 3 X 106 cells/30ml medium for control cells and 6 X 106 cells/60mL medium for drug trea tment. Control cells were treated with dimethyl sulphoxide (DMSO) (S igma, St Louis, MO). The drug treatment cells were incubated with 10 M 5-Aza 2 -deoxycytidine (5-Aza-CdR) (Sigma, St Louis, MO) for 24 hr. At the 24 hr time-point, drug treated cells were split into two sets and reseeded at a density of 3 X106 cells/30mL medium with one set receiving the vehicle control DMSO while the other received a dose of 100 nM of trichostatin A (TSA) (Sigma, St Louis, MO). The rest of th e cells were harvested for mRNA expression analysis. Drug treated cells were incubated for an additi onal 24 hr then harvested.
16 Isolation of total cellula r RNA was done with TRIZOL reagent (Invitrogen Life Technologies, Carlsbad, CA ) as sp ecified by the manufacturer. Isolated RNA was resolved on a 1% fo rmaldehyde/agarose gel to verify RNA integrity prior to cDNA synthesis. RNA concentration was determined by spectrophotometric analysis using the DU 640 spectrophoto meter (Beckman Coulter, Fullerton, CA). RT-PCR was then perfor med using a two step RT-PCR methodology. Briefly, 1 g of RNA was incubated with 20 M Oligo dT primers (Promega, Madison, WI), 10 mM 4X dNTPs (Roche, I ndianapolis, IN), and DEPC ddH2O. The mixture was heated at 65 C for 5 minutes followed by a quick chil l on ice. After a brief spin down, First Strand buffer, 0.1 M DTT, RNaseO ut Recombinat Ribonuclease (40U/ l) (Invitrogen Life Technologies, Carlsbad, CA) was added to the mixture. The mixture was incubated at 42 C for 2 minutes after which I unit of Superscr ipt II reverse transcriptase (Invitrogen Li fe Technologies, Carlsbad, CA) was added. Priming conditions were as follows: 1 cycle of 42 C for 50 min and 70 C for 15 min. The cDNA obtained was then amplified by PCR. Th e PCR conditions were as follows: 94 C for 2 min, 40 cycles of 94 C for 30 sec, 58 C for 30 sec, 68 C for 1 min; and finally 2 min at 68 C. The PCR mixture contai ned PCR buffer (Roche, Indi anapolis, IN), 8% DMSO (Indianapolis, IN), 5 mM dNTPs (Boehringer Mannheim, Germany), 3 l cDNA, 45 M each of PKCor glyceraldehyde-3-phos phate-dehydrogenase (GAPDH) primers in a 25 l total reaction. PKCprimers were: sense primer, 5 AGCTTGAAGCCCACAGCCTG-3 and anti-sense primer 5 CTTGTGGCCGTTGACCTGATG-3 . The GAPDH primers were: sense primer, 5 TCTAGACGGCAGGTCAGGTCCACC-3 and the anti-sense primer 5 -
17 CCACCCATGGCAAATTCCATGGCA-3 . The PCR products (10 l) were directly loaded onto a 1.2% agarose gel, stained with ethidium bromide (0.5 g/mL), and visualized under UV illumination using the FluoChem 9900 DNA imaging system (Alpha Innotech Corp, San Leandro, CA). Transient Transfection Assays The NCI-H157 NSCLC cell line obtained from ATCC (Rockville, MD) was maintained in RPMI 1640 medium supplemented with 9% (v/v) calf serum, 100 units/mL penicillin, and 100 units/mL streptomycin at 37 C in a 5% CO2 atmosphere. Cells were seeded at 3 X 105 cells/ well plate containing 3 mL of normal growth medium. The cells were incubated for 18-24 hr to approximately 40-50% confluency. The vectors used for transfection experiments included pGL3-Basic promoter-less vector, pGL3-promoter, and pGL3-1.2 kb NcoI construct. Transient transf ections were carried out using Lipofectamine, according to the procedures recommended by the manufacturer (Invitrogen Life Technologies, Ca rlsbad, CA). To monitor fo r transfection efficiency, the plasmid pSV-gal (Promega, Madison, WI) was co-t ransfected with the luciferase constructs. 48 hr post-transfection, cells were harvested by scraping using a rubber policeman, washed twice with ice cold PB S, and lysed in 1X Reporter Lysis Buffer (Promega, Madison, WI). Assays for luciferase and -galactosidase activities were performed using colormetric assa ys. All of the luciferase ex periments were carried out in triplicate and repeated at least four times. Western Blot Analysis Protein expression was detected by West ern immunoblotting. Briefly, cells were harvested by centrifugation at 900 rpm. Cells were washed twice in ice cold phosphate
18 buffered saline (PBS) and lysed in a modi fied radioimmuno-precipitation assay buffer (RIPA) (50 mM HEPES pH 7.5, 150 mM NaCl , 2 mM EDTA pH 8.0, 2 mM EGTA pH 8.0, 1% Triton X-100, 50 mM Sodium fluoride, 50 mM sodium -glycerophosphate) containing protease and phosphata se inhibitors (1 mM sodium orthovanadate, 1 mM DTT, 1 mM PMSF, 10 g/mL leupeptin, 10 g/mL aprotinin) in a rotating apparatus for 1 hr at 4 C. The cell lysates we re centrifuged at 4 C for 10 min. Protein concentrations were determined using the BCA protein a ssay reagent (Pierce, Rockford, IL). 100 g each of whole cells lysates were reso lved on a 7.5% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE) at 40 mA in 1X running buffer (25 mM Tris base, 192 mM glycine, 0.1% SDS). Proteins were tran sferred to a nitrocellu lose membrane (BioRad Labs, Hercules, CA) in 1X transfer bu ffer (25 mM Tris base, 192 mM glycine, 10% MeOH) using BIO-RAD Trans-Blot system (Bio-Rad Labs, Hercules, CA). The nitrocellulose membrane was blocked in 5% nonfat milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20) with agitati on at room temperature for 2 hr. The blot was rinsed four times with TBST for five minutes each, followed by incubation with the primary antibody (1:1000 for PKC) (Transduction Labs, ),(1:1000 for PKCII) (Santa Cruz, CA) in 1% BSA or 5% BSA respectively. Incubation was done at room temperature for 2 hr with agitation. The blot was washed briefly with fresh TBST and incubated with secondary anti-body (1:3000 fo r mouse HRP or 1:5000 for rabbit HRP) in 5% nonfat milk in TBST. Incubation was done at room temperature for 2 hr with agitation followed by four washes with TBST . Proteins were visualized using the enhanced chemiluminescence (ECL) detec tion system (Amersham Biosciences, Piscataway, NJ).
19 Deletion Constructs For Luciferase and -galactosidase assays, dele tion constructs of the 5 regulatory region of the PKCgene were generated by restricti on enzyme digestion and cloned into the PGL3-Basic vector. Briefly, a mutati on was introduced immediat ely upstream of the transcription start site of the PKC6-kb 5 -genomic clone using the QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jo lla, CA). The mutation changed two bases at positions 5650 and 5652 (G G C T CG G C T A GC) generating a NheI (GCTAG) restriction site at the 3 end of the cloned 6-kb PKCconstruct. The PKCprimers utilized for this mutagenesis were: sense primer, 5 GAGTCCCTGTGGCTACCAGTGCCGGGCCGTC-3 and the anti-sense primer, 5 GACGGCCCGGCACTGCTA GCCACAGGGACTC-3 . The presence of the mutation was verified by sequencing (Interdiscip linary Center for Biotechnology Research, Gainesville, FL). The cloned 6-kb 5 regulatory region containing the mutation was digested with the enzymes NheI, ScaI, PstI, BspHI, and NcoI (New England Biolabs, Beverly, MA) to generate a series of 1-kb deletion fragments (Fig. 3). The fragments were resolved on a 1% agarose gel, excised from the gel, and eluted from the gel using an Electro-Eluter (BioRad Labs, Hercules, CA ). The DNA obtained was extracted with phenol/chloroform/isoamyl alcohol (25:24:1) (PC9), followed by precipitation with 2.5 M ammonium acetate and 2 volumes of 100% ethanol. The DNA frag ments were then sub-cloned upstream of the Luciferase reporte r gene in the pGL3-Basic vector. The resulting constructs PKC-promoter-Luc were transfected into H157 NSCLC cells and assayed for Luciferase and -galactosidase activity.
20 6kb 5.3kb ScaI 4.1kb PstI 2.6kb BspHI 1.2kb NcoI NheI Exon I5 3 ScaI PstI BspHI NcoI 6kb 5.3kb ScaI 4.1kb PstI 2.6kb BspHI 1.2kb NcoI NheI Exon I5 3 6kb 5.3kb ScaI 4.1kb PstI 2.6kb BspHI 1.2kb NcoI NheI Exon I5 3 ScaI PstI BspHI NcoI Figure 2-1. A physical map of the 5 regulatory region of the human PKCgene depicting deletion constructs. The four deletion cons tructs are shown from largest to smallest. The NheI restriction site is observed at the 3 end of the 6 kb cloned genomic fragment while the ScaI, PstI, BspHI , and NcoI 5 end restriction sites represent the 5 end of the deletion constructs. Bisulfite Genomic Modification Genomic DNA was isolated from H82, H69, H249 SCLC cell lines and H157, H23 NSCLC cell lines. Adherent cell lines H157, and H23 were trypsinized and collected, while suspension cell lines H82, H249, and H 69 were collected by centrifugation at 900 rpm for 5 min at room temperature. Cells were washed twice with ice cold PBS and resuspended in Tris-EDTA buffer (TE9) ( 500 mM Tris-base, 20 mM EDTA pH 8.0, 10 mM NaCl). 10mg/mL Proteinase K (Roche, I ndianapolis, IN) in 10% SDS was added and the samples incubated at 50 C overnight. The samples were extracted with phenol/chloroform/isoamyl alcohol (25:24:1) (PC9), followed by precipitation with 2.5 M ammonium acetate and 2 volumes of 100% ethanol. DNA was recovered by
21 centrifugation at 1700 X g for 2 min, washed w ith ice cold 80% etha nol, air-dried and resuspended to a final concentration of approximately 1mg/ml. For bisulfite modification, 5 g of genomic DNA was digested with the enzyme BspHI (New England Biolabs, Beverly, MA) at 37 C overnight. BspHI cleaves the genomic DNA on both sides of the 2kb CpG Island. The digest ed DNA was extracted with PC9, ethanol precipitated and resuspended in LTE. Th e genomic DNA was then denatured with freshly prepared 0.3 M NaOH at 37 C for 30 min. The denatured DNA was treated with freshly prepared 1.55 M sodium metabisulfit e (Sigma, St Louis, MO) containing 0.05 mM hydroquinone and incubated at 55 C in the dark for 16-20 hr . The bisulfite treated DNA was desalted using the Wizard DNA Cl ean-up system (Promega, Madison, WI) according to the protocol provided by the manufacturer, eluted in 50 L of water, and treated with 0.3 M NaOH at 37 C for 15 min for desulfonation. The DNA was then precipitated with 7.5 M ammonium acetate and three volumes of 100% ethanol with 1 g of glycogen (Roche, Indianapolis, IN) as carrier at -80 C overnight. The DNA pellet was washed with 75% ethanol, dried in a speed-vacuum, dissolved in 100 L of ddH2O, and used for PCR. 10 L of the bisulfite treated DNA ( 300 to 500 ng) was PCR amplified in a 50 l reaction volume containing 10X Herculase buffer (Stratagene, La Jolla, CA), 5 mM dNTPs (Boehringer Mannheim, Germany), 8% DMSO, (Stratagene, La Jolla, CA), 10 M forward and reverse primers overlaid with mineral oil (Promega, Madison, WI). Cycling conditions were: 95 C for 15 min, 10 cycles of 94 C for 1 min, 54 for 2 min, 72 C for 3 min, 30 cycles of 94 C for 1 min, 54 C for 2 min, 72 C for 1 min, and finally 30 min at 72 C. The primers utilized for bisulfite PCR were as follows: For region A
22 (Fig 4 ): forward primer, 5 -TTTTGTTTTTTTAGG TTTTTAGTTT-3 ; reverse primer, 5 -AACATTCTCCCCTTTA AAATCTATC-3 . For region B: forward primer, 5 GGTTTTAAAAGATAG TGTTGGT-3 ; reverse primer, 5 ATTTAAAAAAATAA AAAATTTTACC-3 . For region #3: forward primer, 5 TTTTTGTATTAGAGGGAG GGAGATT-3 ; reverse primer, 5 AACTACACCTCCAAAAAAAAAAAC-3 . Figure 2-2. Schematic diagram of the CpG isla nd located in the 5' regulatory region of the PKCgene (-1930 to -220) relative to the transcription start site. Overlapping primers were designed to amplify regions A (422bp), B (238bp), and C (502bp), respectively. With in this region there are two TaqI (T) recognition sites (TCCA), two EagI (E) (CGGCCG) recognition sites, and four SmaI (S) (CCCGGG) recognition sites.
23 CHAPTER 3 RESULTS The Transcription Start Site for the PKCGene is Located 132 Nucleotides Upstream From the Translation Start Site. To investigate the molecular mechanisms i nvolved in the differen tial transcription of the PKCgene in human lung cancer cells, the 5' regulatory sequences of the PKCgene were cloned by high fidelity PCR. Seque ncing analysis revealed that the cloned sequence spans a distant of approximately 6kb upstream of the tran slation start codon. As a first step in the ch aracterization of the PKCgene, primer extension analysis was performed to identify the transcription start site using total cellular RNA isolated from the PKCexpressing cell line H157. As shown in Figure 5, a single transcription start site was identified which is located at 132 bp upstream from the tran scription initiation site. Identical results were obtained by pr imer extension analysis using other PKCexpressing cell lines H358 and H460. Sequen ce analysis showed that the putative PKCpromoter lacks the typical TATA and CAAT boxes commonly found within 100 bp upstream of transcriptional initiation site of a gene. However, the PKC5' regulatory sequence was found to have a high G/C conten t (55%) within a 2-kb region immediately upstream of the ATG, resembling a CpG-rich island (Figure 6). There are a total of 148 CpG dinucleotides in the 1.2-kb NcoI fragment (-1133/+130). Ar eas of high G/C content have been previously described in TATA -less promoter regions of other genes (Robertson et al. 2000; S hopland et al. 1995).
24 * *A B. * *A B * *A B. * *A B Figure 3-1. Determination of the tr anscription start site of the PKCgene. (A) Primer extension assay. Total cellular RNA (40 g) was isolated from the PKCexpressing cell H157. The RNA was hybridized to a 32P-end-labeled primer and extended using Superscrip t II reverse transcriptase. The primer extension product was denatured and analyzed by sequencing gel electrophoresis followed by autoradiography. The asterisk indicates the transcription start site. (B) Nucleotide sequence of the transcription start site. Bent arrow indicates the transcription start site identified by primer extension analysis (panel A). The translation initiation site ( ATG) is indicated in bold letters. The PKCreverse 2 primer utilized in the primer extension assays is also shown.
25 Exon 1 Transcriptional start site (+1)Nco1(-1133)Nco1 (+130) 1.2kb 2kb CpG island5'3' (-5597) Exon 1 Transcriptional start site (+1)Nco1(-1133)Nco1 (+130) 1.2kb 2kb CpG island5'3' (-5597) Exon 1 Transcriptional start site (+1)Nco1(-1133)Nco1 (+130) 1.2kb 2kb CpG island5'3' (-5597) Exon 1 Transcriptional start site (+1)Nco1(-1133)Nco1 (+130) 1.2kb 2kb CpG island5'3' (-5597) Figure 3-2. A schematic representation of the 5' regulatory region of the PKCgene showing the transcription start site (+1) . The closed circles represent the 2-kb CpG-rich island located immedi ately upstream of the transc riptional start site. Promoter Activity in the 5 Regulatory Region of the PKCGene To define the promoter regi ons responsible for the PKCgene transcription, 5 deletion analyses were performed using rest riction enzyme digestion. The cloned 6-kb 5 regulatory region of the PKCgene was digested with the enzymes NheI, ScaI, PstI, BspHI, and NcoI (New England Biolabs, Beverly, MA) generating 5.3kb ScaI , 4.1kb PstI, 2.6kb BspHI , and a 1.2kb NcoI fragments. The deleted fragments were then subcloned upstream of the luciferase reporter gene in the pGL3-Basic vector. Transient transfection experiments were conducted using the PKCexpressing cell line H157. Transfection efficiency was normalized by co -transfection with the internal control plasmid pSV-gal (Promega, Madison, WI). Lucife rase reporter assay was performed as recommended by the manufacturer (Promega, Madison, WI). -galactosidase activity was detected using a colorometric assay. The smallest fragment tested, (1.2-kb NcoI ), produced a high level of luci ferase activity in the PKCpositive cell line H157 as compared to the promoterless pGL3-Basic. E qual or slightly less promoter activity was observed with the other two constructs (2.6-kb BspHI and 4.1-kb PstI ). Transfection of
26 the largest cons truct (5.3-kb ScaI ) of the 5 regulatory region of the PKCgene produced the highest promoter activity (Figure 7). pGL3-Basic1.2kb2.6kb4.1kb5.3kb LifAtiit 0 20 40 60 80 100 120 Relative luciferase activity pGL3-Basic1.2kb2.6kb4.1kb5.3kb LifAtiit 0 20 40 60 80 100 120 Relative luciferase activity Figure 3-3. Induction of luci ferase activity by the PKCpromoter in H157 cell line. Deletion constructs of the 5' regulatory region of the PKCgene (See Fig. 3) were cloned into the pGL3-B asic vector. The resulti ng constructs were then transiently transfected into NSCLC cell li ne H157. Lucifera se activity assays were performed in triplicate. Data presented are normalized luciferase activity relative to the pSV-galactosidase activity. Synergistic Effects of 5-Aza-CdR a nd TSA in The Reactivation of PKCGene Previous studies from this laboratory show ed that a differentia l expression pattern for the PKCisoform exists between the NSCL C and SCLC phenotypes (Ding et al. 2002). Northern blot analysis showed there to be a deficiency in the expression of steady-state PKCin tested SCLC cells. NCI-H82 cell line which does not express PKCwas used to test whether DNA methylati on activity and/or histone deacetylation contributes to re pression of PKCexpression in SCLC cells . H82 SCLC cells were pretreated with the demet hylating agent 5-aza-2'-deoxyc ytidine (5-Aza-CdR) for 24hr followed by treatment with histone deacetylas e (HDAC) inhibitor trichostatin A (TSA)
27 for 12hr. At this point the drug was washed out and cells were incubated in normal media for an additional 12hr. Total cellula r RNA was isolated from one set of drug treated cells while another set was prepared fo r western analysis. Treatment of cells with inhibitors of HDAC increases acetylation of hi stones, which in turn could alter chromatin structure and induce gene expressi on. A detectable level of PKCmRNA, as demonstrated by a RT-PCR product with predicted 464-bp size, was observed after treatment with 10 M 5-Aza-CdR for 24 hr (Figure 3-4, lane 2). As shown in Figure 9, lane 5, a combination treatment with both ag ents led to a more pronounced re-expression of PKCmRNA. Western blot analys is was used to detect PKCexpression. PKCgene expression was observed after treatment with 5-Aza-CdR alone for 48hr (Figure 34, lane 4). Interestingly, when the cells we re exposed to 5-Aza-CdR for 24 hr and then TSA for 12 hr, PKCinduction rose to an even higher level (Figure 3-4, lane 5). In contrast, treatment with 5-Aza-CdR did not a ffect the levels of other PKC isoforms (e.g. PKCII) (Figure 3-4). Therefore, it appears that 5-Aza-CdR and TSA treatment can induced PKCre-expression in a PKCnegative cell line. Thes e results suggest a role for DNA methylation and HDAC in PKCgene silencing.
28 Control Aza Control Aza Aza + TSA 24hr 48hr PKCPKCII GAPDH PKCControl 5-Aza Control 5-Aza Aza + TSA24hr 48hrA B 1 2 3 4 5 1 2 3 4 5 Control Aza Control Aza Aza + TSA 24hr 48hr PKCPKCII GAPDH PKCControl 5-Aza Control 5-Aza Aza + TSA24hr 48hrA B 1 2 3 4 5 1 2 3 4 5 Figure 3-4. Synergistic effects of 5-AzaCdR and TSA in the reactivation of PKCgene. (A) H82 SCLC cells we re treated with 10 M 5-Aza-CdR alone or a combination of 5-Aza-CdR + 100 nM TSA. Whole cell lysates (100 g) were subjected to Western blot an alysis using an anti-PKCantibody. PKCexpression was observed with 48hr trea tment with 5-Aza-CdR alone and a more pronounced induction was seen at 48hr with a combination treatment (lane5). (B) Re-expression of PKCmRNA using RT-PCR was observed at 24 hr with 5-Aza-CdR alone (lane 2) , but a higher induction was observed with a combination treatment using 5-Aza-CdR and TSA (lane 5).
29 Methylation Status f the PKCPromoter To examine the overall levels of methylat ion in the 2-kb CpG-rich island of the human PKCgene, the bisulfite genomic se quencing methodology was utilized. Primers were designed to amplify 3 fragment s within the 2-kb CpG island of the PKCpromoter region (Figure 4). Primers were de signed to avoid potenti al methylation sites (CpG) so that both methylated and unmet hylated DNA could be amplified equally. Genomic DNA from one PKCnon-expressing SCLC (H249) and a PKCexpressing NSCLC (H23) was treated with bisulfite. The treated DNA was amplified by PCR using oligonucleotide primers flanking the 5' and 3' target seque nce, followed by restriction enzyme digestion with the endonucleases SmaI (CCC GGG) or SacII (CCGC GG). In this assay, bisulfite modification results in a change from C to T when the C is unmethylated in the context of a CG dinucle otide. However, when the cytosine is methylated, the bisulfite modification leaves the methylated cytosine intact. Therefore, after bisulfite modification and PCR amplifi cation, methylated CG dinucleotides will be intact, and restriction digestion with th e endonucleases can occur. In contrast, unmethylated CG dinucleotides are alte red to TG, and CG-containing restriction enzymes, such as SmaI , or SacII are unable to digest the fr agment. Analysis of the digested genomic DNA will provide us with a cl ue as to the methylation status of the PKCpromoter region. Digestion pattern is not expected to be uniform across the board, as DNA from different cell lines ma y be methylated at certain restriction endonuclease sites and not at others . As seen in Figure 9, neither SmaI nor SacII was able to digest the bisulfite treated genomic. This demonstrates that the PKCgene
30 promoter is not methylated at these restriction sites. Alternatively, lack of cleavage by these enzymes could indicate incomplete bisulfite conversion of genomic DNA. H249 H23422-bp SmaIA. H249 H23422-bp SacIIB. H249 H23422-bp SmaIA. H249 H23422-bp SacIIB. Figure 3-5. Restriction enzyme diges tion profile of bisulfite-converted DNA. PCRamplified DNA from bisulfite-treated H249 and H23 lung cancer cells was digested with SmaI and SacII . The products were then resolved on a 1.2% agarose gel, stained with ethidium bromide and visualized under UV illumination.
31 CHAPTER 4 DISCUSSION One of the aims of this thesis was to de termine the transcription start site for the human PKCgene. The promoter region of the PKCgene lacks a defined TATA box or CAAT box commonly found within 100bp upstream of transcriptiona l initiation site. However, the PKC5' regulatory sequence was found to have a high G/C content (55%) within a 2-kb region immediately upstream of the ATG, resembling a CpG-rich island (Figure 6). We first characterized the gene w ith regard to promoter structure. Primer extension analysis was performed to identify th e transcription start site using total cellular RNA isolated from the PKCexpressing cell line H157. As shown in Figure 5, a single predominant transcriptio n start site was identified at 132bp relative to the translation start site (ATG). Identical results were obtain ed by primer extension analysis using other PKCexpressing cell lines H358 and H460. Gene expression is controlled primarily at the level of initiati on of transcription. In most cases transcription is initiated at a specific base pair bi nding in the template DNA or at alternative sites within a few base pairs. Trans-acting f actors regulate gene transcription by binding directly or through an intermediate protein to the gene at a particular DNA sequence, called a cis-regulato ry region. This cisregulatory region is usually located in the 5' flanking promoter region of the gene and is composed of a specific nucleotide sequence. There are two cl asses of cis-acting elements that can exert their effects at considerable distance from the promoter, enhancers and silencers. To
32 examine the functionality of the PKCpromoter, a series of deletion constructs were introduced upstream of the luciferase reporte r gene in the promoterless vector pGL3Basic. The smallest fragment tested, (1.2-kb NcoI ), produced promoter activity in the PKCpositive cell line H157. Equal or sli ghtly less promoter activity was observed with the other two constructs (2.6-kb BspHI and 4.1-kb PstI ), suggesting that the 1.2-kb fragment contains essent ial cis-elements for PKCtranscription. Several putative transcription factor consensus sequences were found in the 5 regulatory region of the PKCgene, such Sp-1, E-boxes, AP-1, and CRE . These elements may be involved in the regulation of PKCgene in cancer cell lines. Tran sfection of the largest construct (5.3-kb ScaI) of the 5 regulatory region of the PKCgene, brought about the highest promoter activity as seen in Fi gure 7, indicating that there ma y be an enhancer element in this region. Further functional studies of these promoter regi ons are required to confirm which of the transcription factors regulate PKCexpression in SCLC cell lines. A growing body of data demonstrates the importance of histone acetylation and deacetylation, DNA methylation, an d corresponding structural al teration of chromatin in gene transcriptional regulation (Ohmchi et al.199 3; Traulo et al. 1997; Travis et al. 1995). Sequencing analysis revealed that the cloned 5' regulatory sequence was found to have a high G/C content (55%) within a 2-kb re gion immediately upstream of the ATG, resembling a CpG-rich island. We therefore, proposed to determine whether DNA methylation plays a role is in the transcriptional silencing to the PKCgene in SCLC cells. A DNA demethylating agent was utilized as a pharmacological tool. 5-Aza-CdR is incorporated into DNA during replica tion followed by covalent binding of DNA methyltransferase to the analog causing irre versible inactivation of the enzyme and
33 formation of demethylated DNA. H82 SCLC cells were pretreated with the demethylating agent 5-Aza-CdR for 24hr follo wed by treatment with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) fo r 12 hr. A detectable level of PKCmRNA, as demonstrated by a RT-PCR product with predicted 464-bp size, was observed after treatment with 10 M 5-Aza-CdR for 24 hr. As s hown in Figure 8, a combination treatment with both agents led to a more pronounced re-expression of PKCmRNA. Therefore, it appears that 5-Aza-Cd R and TSA treatment can induce PKCmRNA expression in a PKCnegative cell line. These re sults suggest a role for DNA methylation and HDAC in PKCgene silencing in SCLC cells. Western blot analysis was used to detect PKCexpression in another set of similarly treated H82 cells. PKCprotein expression was obse rved at 48hr with 5-AzaCdR alone (Figure 8, lane 4) and a higher induction was observed with a combination treatment of 5-Aza-CdR and TSA (Figure 8, lane 5). Methylation pr ovides two levels of control, both dependent on DNA/protein inte ractions. Methylation of CpGs in the proximal promoter may be what is blocking th e binding of essential trans-acting proteins, thereby indirectly repressi ng transcription of the PKCin SCLC cells. On the other hand, we observed that the 5 regulatory region of the PKCis has a high density of CpGs which may be methylated permitting th e binding of a methyl-CpG binding protein that either directly represses transcription or recruits corepressors, histone deacetylases, or both. Additional studies using other SCLC cell lines will be necessary to verify that methylation is the cause of the phenotype-spe cific transcriptional silencing of the PKCgene. Further analysis will be performed to identify the factors and control elements necessary for the silencing of PKCgene in SCLC cells.
34 To detect the presence of Cp G sites, if any, after bisulf ite treatment, we digested the amplified DNA with the endonucleases SmaI (CCC GGG) or SacII (CCGC GG). As seen in Figure 9, lack of digestion of the SCLC cell line H249 and the NSCLC H23 with both enzymes reveals that these sites c ontain unmethylated CG dinucleotides. We expected to observe digestion of the SCLC amplified DNA if methylation plays a role in the silencing of the PKCgene. It has been shown that methylation of just one CpG sequence is sufficient to silence the Epstein Barr virus latency C promoter that codes for six viral proteins (Zochbauer-M ulle et al. 2002). Sequencing analysis showed that there are over 180 CpG dinucleotides in the 2-kb CpG island of the PKCgene. Lack of digestion at the SmaI and SacII restriction sites may be due unmethylated CG dinucleotides in both SCLC and NSCLC cell lin es. We only have only tested one region at two CpG dinucleotides. Further analysis of the regions could reve al other methylated CpGs. Fine methylation mapping using bisulf ite genomic sequencing analysis will be performed to further reveal cell-specific met hylation patterns. This assay allows one to detect the methylation at the sp ecific CpG dinucleotide. In summary, our data suggest DNA methyla tion is a potential mechanism(s) for the transcriptional inactivation of the PKCgene in SCLC cells. Our work which utilized a pharmacological mode to re-express the PKCin the transcriptionally inactive cell line, H82, supports the notion that DNA methylation may control the expression of this gene in lung cancer cells. Further studies invol ving fine methylation mapping of the CpG island found in the 5 regulatory region of the PKCgene should provide a better understanding of how PKCsilencing occurs in SCLC cells.
35 LIST OF REFERENCES Abeloff, M.D., Eggleston, J.C., Mendelsohn, G ., Ettinger, G., Baylin, S.B. (1979) Changes in morphologic and biochemical char acteristics of small cell carcinoma of the lung. A clinicopathologic study. Am. J. Med. 66, 757-764. Akita, Y., Ohno, S., Yajima, Y., Konno, Y., Sa ido, T.C., Mizuno, K., Chida, K., Osada, S., Kuroki, T., Kuroki, T., Kawashima, S., Suzuki, K. (1994) Overproduction of a Ca2+-independent protein kinase C isozymes , nPKC-epsilon, increases the secretion of prolactin from thyrotropin-releasi ng hormone-stimulated rat pituitary GH4C1 cells. J. Biol. Chem . 269, 4653-4660. Ali, I and Sarna, S.K. (2002) Selective modulation of PKC isozymes by inflammation in canine colonic circular muscle cells. Gastroenterology. 122, 483-494. Antequera, F., Bird, A. (1994) Predicting the total number of human genes [letter; comment] Nat. Genet. 8:114. Balciunaite, E., Jones, S., Toker, A., Kazlauka s, A. (2000) PDGF initiates two distinct phases of protein kinase C activity that make unequal contributions to the G0 to S transition. Curr. Biol . 10, 261-267. Basu, A., Weixel, K., Saijo, N. (1996) Ch aracterization of the pr otein kinase C signal transduction pathway in cisplantin-sensitive and resistant small cell carcinoma cells. Cell Growth Differ. 7, 1507-1512. Baylin, S.B., Herman, J.G. (2000) DNA hyperm ethylation in tumorigenesis: epigenetics joins genetics. Trends. Genet . 16, 168-174. Beard, C., Li, E., Jaenisch, R. (1995) Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev. 9, 2325-2334. Berrier, A.L., Mastrangelo, A.M., Downward, J ., Ginsberg, M., LaFlamme, S.E. (2000) Activated R-ras, Rac1, PI 3-kinase and PK C-epsilon can each restore cell spreading inhibited by isolated integrin beta 1 cytoplasmic domains. J. Cell Biol . 151, 15491560. Besson, A., Wilson, T.L., Yong, V.W. (2002) The anchoring protein RACK1 links protein kinase C-epsilon to integrin beta chains. Requirements for adhesion and motility. J. Biol. Chem . 277, 22073-22084.
36 Bestor, T., Laudano, A., Mattaliano, R., Ingram , V. (1988) Cloning and characterization of a cDNA encoding DNA methyltr ansferase of mouse cells. J. Mol. Biol. 203, 971-983. Brabender, J., Usadel, H., Danenberg, K.D., Metzger, R., Schneider, P.M., Lord, R.V., Wickramasinghe, K. (2001) Adenomatous polyposis coli gene promoter hypermethylation in non-small cell lung cancer is associated with survival. Oncogene 20, 3528-3532. Cacace, A.M., Ueffing, M., Phillip, A., Han, E. K., Kolch, W., Weinstein, I.B. (1996) PKC epsilon functions as an oncogene by e nhancing activation of the Raf kinase. Oncogene 13, 2517-2526. Cacace, A.M., Guadagno, S.N., Krauss, R.S., Fabbro, D., Weinstein, I.B. (1993) The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 8, 2095-105. Cacace, A.M., Ueffing, M., Han, E.K., Ma rme, D., Weinstein, I.B. (1998) Overexpression of PKC-epsilon in R6 fi broblasts causes incr eased production of active TGF beta. J. Cell. Physiol . 175, 314-322. Cao, X. (2000) Conserved plant genes with similarity to mammalian de novo DNA methyltransferases. PNAS (USA) 97, 4979-4984. Castillo, A., Pennington, D.J., Otto, F., Park er, P.J., Owen, M.J., Bosca, L. (2001) Protein kinase C epsilon is required for macrophage activation and defense against bacterial infection. J. Exp. Med . 194, 1231-1242. Chen, N., Ma, W., Huang, C., Dong, Z. (1999) Translocation of prot ein kinase C epsilon an protein kinase C delta to membrane is required for ultraviolet B-induced activation of mitogen-activated protein kinases and apoptosis. J. Biol. Chem . 274, 15389-15394. Chuang, L.S. (1997) Human DNA-(cytosine5) methyltransferase-PCNA complex is a target for p21 Waf 1 Science 277, 1996-200. Chun, J.S., Ha, M.J., Jacobson, B.S. (1996) Di fferential translocation of protein kinase C epsilon during HeLa cell adhesi on to a gelatin substratum. J. Biol. Chem . 271, 13008-13012. Chute, J.P., Chen, T., Feigal, E., Simon, R., Johnson, B.E. (1999) Twenty years of phase III trials for patients with extensivestage small cell lung cancer: perceptible progress. J. Clin. Oncl. 17, 1794-801. Cross, S.H., Bird, A.P. (1995) CpG islands and genes. Curr. Opin. Genet. Dev. 5, 309314.
37 Dammann, R., Li, C., Yoon, J.H., Chin, P.L., Bate s, S., Pfeiffer, G.P. (2000) Epigenetic inactivation of a RAS asso ciation domain family pr otein from the lung tumor suppressor locus 3p21.3. Nature Genet. 25, 315-319. DeCoy, D.L., Snapper, J.R., Breyer, M.D. (1995) Anti-sense DNA down-regulates protein kinase C-epsilon and enha nces vasopressin-stimulated Na+ absorption in rabbit cortical collecting duct . J. Clin. Invest . 95,2749-2756. Ding, L., Wang, H., Lang, W., Xiao, L. ((2002) Protein kinase C epsilon promotes survival of lung cancer cells by suppressi ng apoptosis through dysregulation of the mitochondrial caspase pathway. J. Biol. Chem . 277:35305-35313. Doll, R., Peto, R. (1981) The causes of cancer : quantitative estimates of avoidable risks of cancer in the United States today. J. Natl. Cancer. Inst . 66, 1191-2308. Eden, S., Hashimshony, T., Keshet, I. (1998) DNA methylation models histone acetylation. Nature 394, 842. Eto, A., Akita, Y., Saido, T.C., Suzuki, K., Kawashima, S. (1995) The role of the calpain-calpastatin system in thyrotr opin-releasing hormone-induced selective down-regulation of a protein kinase C is ozymes, nPKC epsilon, in rat pituitary GH4C1 cells. J. Biol. Chem . 270, 25115-25120. Fagerstrom, S., Pahlman, S., Ge stblom, C., Nanberg, E. (1996) Protein kinase C-epsilon is implicated in neurite outgrowth in differentiating human neuroblastoma cells. Cell Growth Differ. 7,775-785. Genot, E.M., Parker, P.J., Cantrell, D.A. (1995) Analysis of the role of protein kinase Calpha, epsilon, and zeta in T cell activation. J. Biol. Chem . 270, 9833-9839. Greenlee, R.T., Murray, T., Bolden, S., Ni ngo, P.A. (2000) Cancer Statistics. CA Cancer J. Clin . 50, 7-33. Guldberg, J.W. (2002) DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy. J. Oral. Pathol. Med. 31, 443-449. Hiwasa, T., Nakata, M., Ohno, S., Maki, M ., Suzuki, K., Takiguchi, M. (2002) Regulation of transformed state by calpas tatin via PKC-epsilon in NIH3T3 mouse fibroblast. Biochem. Biophys. Res. Commun . 290, 510-517. Hundle, B., McMahon, T., Dadgar, J., Messing, R. O. (1995) Overexpression of epsilonprotein kinase C enhances nerve gr owth factor-induced phosphorylation of mitogen-activated protein kina ses and neurite outgrowth. J. Bio. Chem . 270, 30134-30140. Inoue, M., Kishimoto, A., Takai, Y., Nish izuka, Y. (1977) Studies on a cyclic nucleotide-independent prot ein kinase and its proenzyme in mammalian tissue. J. Biol. Chem . 252:7610-7616.
38 Ivaska, J., Whelan, R.D., Watson, R., Parker, P.J. (2002) PKC epsilon controls the traffic of beta 1 integrins in motile cells. EMBO J. 21, 3608-3619. Jansen, A.P., Verwiebe, E.G., Dreckschmidt, N.E., Wheeler, D.L., Oberley, T.D., Verma, A.K. (2001) Protein kinase C-epsilo n transgenic mice: a unique model for metastatic squamous cell carcinoma. Cancer Res. 61, 808-812. Jemal, A., Chu, K.C., Tarone, R.E. (2001) R ecent trends in lung can cer mortality in the United States . J. Natl. Inst. 93, 277-283. Jobbagy, Z., Olah, Z., Petrovics, G., Eiden, M. V., Leverett, B.D., Dean, N.M., Anderson, W.B. (1999) Up-regulation of the Pit-2 phosphate transporter/retrovirus receptor by protein kinase C-epsilon. J. Biol. Chem . 274, 7067-7071. Johannes, F.J., Prestle, J., Eis, S., Oberha germann, P., Pfizenmaier, K. (1994) PKC is a novel, atypical member of the protein kinase C family. J. Biol. Chem . 269:61406148. Jones, P.A., Laird, P.W. (1999) Ca ncer epigenetics comes of age. Nature. Genet . 21, 163-166. Jones, P.L., Veenstra, G.J., Wade, P.A., Vermack, D., Kass, S.U., Landsberger, N., Strouboulis, J., Wolffe, A.P. (1998) Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genet. 19, 187-191. Kampfer, S., Windegger, M., Hochholdinger, F., Schwaiger, W., Pestell, R.G., Baier, G., Grunicke, H.H., Uberall, F. (2001) Pr otein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras. J. Biol. Chem . 276, 42834-42842. Knauf, J.A., Ward, L.S., Nikiforova, Y.E ., Nikiforova, M., Puxeddu, E., Medvedovic, M., Liron, T., Mochly-Rosen, D., Fagin, J.A. (2002) Isozyme-specific abnormalities of PKC in thyroid cancer: Evidence for post-transcriptional changes in PKC epsilon. J. Clin. Endocrinol. Metab . 87, 2150-2159. Koriyama, H., Kouchi, Z., Saido, T.C., Momoi, T., Ishiura, S. (1999) Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis. Cell. Signal. 11, 831-838 Larsen, E.C., DiGennaro, J.A., Saito, N., Meht a, S., Loegering, D.J., Mazurkiewicz, J.E., Lennartz, M.R. (2000) Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RA W. 264.7 cells. J. Immunol . 165, 2809-2817. Larsen, F., Gundersen, G., Lopez, R., Prydz, H. (1992) CpG islands are gene markers in the human genome. Genomics 13, 1095-1107.
39 Lebitasy, M.P., Hedelin, G., Purohit, A., Mo reau, L., Klinzig, F., Quoix, E. (2001) Progress in the management and outcome of small cell lung cancers in a French region from 1981-1994. Br. J. Cancer. 85, 808-815. Lehel, C., Olah, Z., Jakab, G., Szallasi, Z ., Petrovics, G., Blumberg, P.M., Anderson, W.B. (1995) Protein kinase C-epsil on subcellular localization domains and proteolytic degradation sites. A mode l for protein kinase C conformational changes. J. Biol. Chem. 270, 19651-19658. Lewis, J.D. (1992) Purification, sequen ce, and cellular loca lization of a novel chromosomal protein that bi nds to methylated DNA. Cell 69, 905-914. Li, E., Beard, C., Jaenisch, R. (1993) DNA methylation in genome imprinting. Nature 366, 362-365. Li, E., Bestor, T.H., Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene resu lts in embryonic lethality. Cell 69, 915-926. Mabry, M., Nelkin, B.D., Falco, J.P., Barr, L., Baylin, S.B. (1991) Transitions between lung cancer phenotypes-implications for tumor progression. Cancer Cells. 3, 5358. Mehta, R.D., Radomiska-Pandya, A., Kapoor, G.S., Dave, B., Atkins, B.A. (2002) Critical role of diacylglycer ol and phospholipid-regulated protein kinase C epsilon in induction of low density lipoprotein receptor transcription in response to depletion of cholesterol. Mol. Cell. Biol. 22, 3783-3793. Mellor, H., Parker, P.J. (1998) The extended protein kinase C superfamily. J. Biol. Chem . 332:281-292. Minowa, M.T., Minowa, T., Monswa, F., Si bley, D.R., Mouradian, M.M. (1992) Characterization of 5 flanking region of the human DIA dopamine receptor gene. PNAS (USA) 89, 3045-3049. Mischak, H., Goodmight, J.A., Kolch, W., Ma rtiny, B.G., Schaechtle, C., Kazamietz, M.G., Blumber, P.M., Pierce, J.H., Mushin ski, J.F. (1993) Overexpression of protein kinase C-delta a nd epsilon in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity. J. Biol. Chem . 268, 6090-6096. Nan, X., Campoy, F.J., Bird, A.P. (1997) Me CP2 is a transcripti onal repressor with abundant binding sites in genomic imprinting. Cell 88, 471-481. Nan, X., Ng, H-H., Johnson, C.A., Laherty, C.D., Turner, B.M., Eisenman, R.N., Bird, A.P. (1998) Transcriptional repression of the methyl-CpG binding protein MeCP2 involves a histone deacetylase complex. Nature 393, 386-389.
40 Naveh-Many, T., Cedar, H. (1981) Ac tive gene sequences are undermethylated. PNAS (USA) 78, 4246-4250. Newton, A.C. (1995) Prot ein kinase C: structure, function and regulation. J. Biol. Chem . 270, 28495-28498. Newton, A.C. (2001) Struct ural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions. Chem Rev . 101, 2353-2364. Nishizuka, Y. (1995) Prot ein kinase C and lipid signa ling for sustained cellular responses. Faseb J. 9, 484-496. Ohmichi, M., Zhu, G., Saltiel, A.R. (1993) Nerve growth factor activates calciuminsensitive protein kinase C-epsilon in PC-12 rat pheochromocytoma cells. Biochem J . 295, 767-772. Ohno, S., Akita, Y., Konno, Y., Imajoh, S., Suz uki, K. (1988) A novel phorbol ester receptor/protein kinase C family. Cell 53, 731-741. Okano, M., Beli, D.W., Haber, D.A., Li, W. (1999) DNA methyltransferase DNM3a and DNM3b are essential for de novo methyl ation and mammalian development. Cell 99, 247-257. Okano, M., Xie, S., Li, E. (1998) Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature Genet. 19, 219-220. Oho, Y., Fujii, T., Ogita, K., Kikkawa, U. (1988) The structure, expression, and properties of additional members of the protein kinase C family. J. Biol. Chem . 263, 6927-6932. Palmantier, R., George, M.D., Akiyama, S.K ., Wolber, F.M., Olden, K., Roberts, J.D. (2001) Cis-polyunsaturated fatty acids stim ulate beta 1 integrin-mediated adhesion of human breast carcinoma cells to type IV collagen by activating protein kinases C-epsilon and Â–mu. Cancer Res . 61, 2445-2452 Parrow, V., Fagerstrom, S., Meyerson, G., Nanberg, E., Pahlman, S. (1995) Protein kinase C-alpha and epsilon are enriched in growth cones of differentiating SHSYSY human neuroblastoma cells. J. Neurosci Res 41, 782-791. Plass, C., Soloway, P.D. (2002) DNA methylation, imprinting, and cancer. Europ. J. Hum. Genet . 10, 6-16. Pradhen, S. (1997) Baculovirus-mediated expression and characte rization of the full length murine DNA methyltransferase. Nucleic Acids Res 25, 4666-4673. Pradhen, S., Bacolla, A., Wells, R.D., Robert s, R.J. (1999) Recombinant human DNA (cytosine-5) methyltransferase 1. Expr ession, purification, and comparison of de novo and maintenance. J. Biol. Chem. 274, 33002-33010.
41 Prekeris, R., Mayhew, M.W., Cooper, J.B., Terrian, D.M. (1996) Identification and localization of an actin-binding motif that is unique to the epsilon isoform of protein kinase C and participates in the regulation of synaptic function. J. Cell. Biol. 132, 77-90. Racke, F.K., Wang, D., Zaidi, Z., Kelley, J., Visvader, J., Soh, J.W., Goldfarb, A. N. (2001) A potential role for protein kinase C-epsilon in regulating megakaryocytic lineage commitment. J. Biol. Chem . 276, 522-528. Razin, E., Szallasi, Z., Kazanietz, M.G., Blum berg, P.M., Rivera, J. (1994) Protein kinase C-beta and C-epsilon link the mast cell high-affinity receptor for IgE to the expression of c-fos and c-jun. PNAS (USA) 91, 7722-7726. Robertson, K.D. (1999) The human DNA me thyltransferases (DNMTs) 1, 3a, and 3b: Coordinate mRNA expression in tumors. Nucleic Acid Res. 27, 2291-2298. Robertson, K.D., Ait-Si-Ali, S., Yokochi, T., Wade, P.A., Jones, P.L., Wolffe, A.P. (2000) DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet. 25, 338-342. Robertson, K.D., Hayward, S.D., Ling, P.D ., Samid, D., Ambinder, R.F. (1995) Transcriptional activation of the EpsteinBarr virus latency-C promoter after 5azacytidine treatment: evidence that demethyla tion at a single CpG site is crucial. Mol. Cell . Biol. 15, 6150-6159. Robertson, K.D., Wolffe, A.P. (2000) DNA methylation in health and disease. Nature Rev. Genet . 1, 11-19. Shopland, D.R. (1995) Tobacco use and its c ontribution to early cancer mortality with a special emphasis on cigarette smoking. Environ Health Perspect. 103 Suppl 8:131142 Smit, E., Postmus, P. (1995) Chemothera py of small cell lung cancer. In: D. Carney (ed), Lung Cancer, pp 156-172 Boston, Arnold. Song, J.C., Hrnjez, B.J., Farokhzad, O.C., Matth ews, J.B. (1999) PKC-epsilon regulates basolateral endocytosis in human T84 inte stinal epithelia: ro le of F-actin and MARCKS. Am. J. Physiol . 277, C1239-1249. Swartz, M.N., Trautner, T.A., Kornberg, A. (1962) Enzymatic synthesis of deoxyribonucleic acid. J. Biol. Chem 237, 1961-1967. Swick, A.G., Blake, M.C., Azizkhan, J.C. ( 1989) Functional analysis of GC element binding and transcription in the hamster dihyrofolate reductase gene promoter. Nucleic Acids Res 17, 9291-9304.
42 Tachado, S.D., Mayhew, M.W., Wescott, G. G., Foreman, T.L., Goodwin, C.D., McJilton, M.A., Terrian, D.M. (2002) Regulation of tumor invasion an d metastasis in protein kinase C-epsilon-tran sformed NIH3T3 fibroblasts. J. Cell. Biochem . 85, 785-797. Tate, P.H., Bird, A.P. (1993) Effects of DNA methylation on DNAbinding proteins and gene expression. Curr. Opin. Genet . Dev 3, 226-231. Toker, A. (1998) Signaling through protein kinase C. Front. Biosci . 3, D1134-1147. Traulo, O., Monia, B.P., Dean, N.M., Berk, B.C. (1997) PKC-ep silon is required for mechano-sensitive activation of ERK1/2 in endothelial cells. J. Biol. Chem 272, 31251-31257. Travis, W.D., Linder, J., Mackay, B. (1996) Lung Cancer : principles and practice. Pass HI, Mitchell JB, Johnson, DH (eds) Philade lphia: Lippincott-Ra ven Publishers, pp. 361-395. Travis, W.D., Travis, L.B., Devesa, S.S. (1995) Lung cancer. Cancer 75, 191-202. Tsou, J.A., Hagen, J.A., Carpenter, C.L., Lair d-Offringa, I.A. ( 2002) DNA methylation analysis: a powerful new tool for lung cancer diagnosis. Oncogene 21, 5450-5461. Ueffing, M., Lovric, J., Phillip, A., Mischak, H., Kolch, W. (1997) Protein kinase Cepsilon associates with the Raf-1 kinase and induces the production of growth factors that stimulate Raf-1 activity. Oncogene 15, 2921-2927 Valverde, A.M., Sinnett-Smith, J., Van Lint, J., Rozengurt, E. ( 1994) Molecular cloning and characterization of protein kinase D: a target for diacylg lycerol and phorbol esters with a distinctive catalytic domain. PNAS USA 91, 8572-8576. Volm, M., Pommerenke, E.W. (1995) Associ ated expression of pr otein kinase C with resistance to doxorubicin in human lung cancer. Anticancer. Res 15, 463-466. Wade, P.A., Gegonne, A., Jones, P.L., Ballestar , E., Aubry, F., Wolffe , A.P. (1999) Mi2 complex couples DNA methylation to chromatin remodeling and histone acetylation and histone deacetylation. Nat. Genet 23, 62-66. Wakefield, R.D., Smith, B.D., Nan, X., Free, A., Soteriou, A., Uhrin, D., Bird, A.P., Barlow, P.N. (1999) J. Mol. Biol 291, 1055-1065. WHO. (1982) World Health Organization: Geneva. Xie, S., Wang, Z., Okano, M., Nogami, M., Li, Y., Okumura, K., Li, E. (1999) Cloning, expression and chromosome locations of the human DNMT3 gene family. Gene 236, 87-95.
43 Yeivin, A., Razin, A. (1993) Gene me thylation patterns and expression. In DNA methylation: Molecular Biology and Biol ogical Significance. Edited by Jost JP, Saluz, H.P, Basel, Switzerland : Birkhauser Verlag pgs 523-568. Zeidman, R., Troller, U., Raghunath, A., Pahlma n, S., Larson, C. (2002) Protein kinase C-epsilon actin-binding site is important for neurite outgrowth during neuronal differentiation. Mol. Biol. Cell . 13, 12-24. Zhu, W.G., Dai, Z., Ding, H., Srinivasan, K., Hall, J., Duan, W., Villalona-Calero, M.A., Plass, C., Otterson, G.A. (2001) Incr eased expression of unmethylated CDN2D by 5-Aza-2 -deoxycytidine in human lung cancer cells. Oncogene 20, 7787-7796. Zochbauer-Mulle, S., Gazdar, A., Minna, J.B. (2002) Molecular pa thogenesis of lung cancer. 64, 681-708.
44 BIOGRAPHICAL SKETCH Linnet Akinyi was born in Nakuru, Kenya. Sh e received her Bachelor of Science degree in chemistry from Xavier University of New Orleans, Louisiana in 1990, and a Master of Science in biology from Morgan State University (Baltimore, Maryland) in 2001. She worked as a Biological Scientist for the Department of Energy at the Pacific Northwest Laboratory from 1995 to 1996. She also worked as a Chemist for the Shell Oil Company from 1996 to 1997. Upon completi on of her graduate studies, she will pursue a career in academic research.