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Characterization of zebularine: A novel inhibitor of DNA methylation with clinical potential

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Characterization of zebularine: A novel inhibitor of DNA methylation with clinical potential

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Content CHARACTERIZATION OF ZEBULARINE: A NOVEL INHIBITOR OF DNA METHYLAIION WITH CLINICAL POTENTIAL ©2003 By Jonathan Chi-Hong Cheng A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (BIOCHEMISTRY AND MOLECULAR BIOLOGY) December 2003 Jonathan C. Cheng Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3133249 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, th ese will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 3133249 Copyright 2004 by ProQ uest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United S tates Code. ProQ uest Information and Learning Com pany 300 North Z eeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90089-1695 This dissertation, written by 'ScywdbfiA C hi ~Ha/|a. Chm % under the direction o f h j $ _ dissertation committee, and approved by all its members, has been presented to and accepted by the D irector o f Graduate and Professional Programs, in partial fulfillment o f the requirements fo r the degree o f DOCTOR OF PHILOSOPHY Director D ecem ber 1 7 , 2003 Date D issertatifp Committee Chair Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I would like to acknowledge the following people who made the work in this thesis possible: Dr. Peter A. Jones, my thesis advisor, for his patience and guidance and mentorship. Dr s. Gerry Coetzee and Peter W. Laird for their helpful and constructive criticisms during lab meetings. Drs. Victor E. Marquez, Sheldon Greer, Eric Selker, Torben Orntoft and Thomas Thykjaer for their helpful advice and research collaborations. Felicidad Gonzales, for her unrelenting faith in me, friendship and technical assistance. Daniel Weisenberger, for his true friendship, encouragements and advice on and off the lab. Gangning Liang, for his great scientific discussions and advice. Yvonne Tsai, for her patience and helpfulness in the lab. Sandy Mosteller and the M.D./Ph.D. program, for their commitment and support. Past and present graduate students and post-docs of the Jones lab who I have had the pleasure of working with: Christine B. Yoo, Mihaela Velicescu, Gerda Egger, Mehrnaz Fatemi, Jody Chuang, Sonia Escobar, Connie Cortez, Daiya Takai, Shiro Matsuro, Joy Lin, Ana Aparacio, Martin Friedrich, Shahin Chandrosoma, and Shinwu Jeong. ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF FIGURES vi LIST OF TABLES ix ABSTRACT x CHAPTER 1: EPIGENETICS IN CANCER INTRODUCTION 1 DNA METHYLTION 2 CpG islands 2 Maintenance of DNA methylation 5 Role in development 7 Functions of DNA methylation in normal cells 9 DNA methylation and transcriptional repression 16 METHODS FOR DETECTION OF DNA METHYLATION 17 Methylation-sensitive restriction enzymes for detection of DNA methylation 17 Bisulfite methods of detection of DNA methylation 18 Techniques to globally detect hypermethylated genes 20 CHROMATIN INHERITANCE 21 Histone modification and "histone code" 21 Chromatin remodeling and DNA methylation 24 Link between DNA methylation and chromatin structure 28 CANCER EPIGENETICS 34 CpG island hypermethylation in tumor suppressor genes 36 Global genomic hypomethylation 40 Mutagenesis of 5-methylcytosine 42 REVERSAL OF EPIGENETIC MODIFICATIONS AS A CANCER THERAPY 44 CONCLUSIONS 47 OVERVIEW OF THESIS RESEARCH 48 CHAPTER 2: INHIBITION OF DNA METHYLATION AND REACTIVATION OF SILENCED GENES BY ZEBULARINE INTRODUCTION 52 MATERIALS AND METHODS 56 Neurospora culture 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mammalian cell lines and drug treatments 57 DNA and RNA isolation 58 Southern blot analysis 58 Reverse transcription-polymerase chain reaction (RT-PCR) analysis 59 Quantitation of DNA methylation by Ms-SnuPE analysis 59 In vivo experiments 62 Determination of cytotoxicity 64 Statistical analysis 64 RESULTS 68 Reactivation of a silenced hph gene in Neurospora 68 Inhibition of DNA methylation in Neurospora 71 Induction of the myogenic phenotype and inhibition of DNA methylation in 10T1 / 2 mouse embryo cells 74 Induction of p!6 gene expression and inhibition of DNA methylation in T24 hum an bladder carcinoma cells 78 Effects of zebularine on hum an bladder carcinoma cells in vivo 83 DISCUSSION 94 CHAPTER 3: CONTINUOUS ZEBULARINE TREATMENT EFFECTIVELY SUSTAINS DEMETHYLATION IN HUMAN CANCER CELLS INTRODUCTION 103 MATERIALS AND METHODS 107 Cell lines 107 Drug treatments 107 Nucleic acid isolation 108 RT-PCR analysis 108 Quantitative RT-PCR analysis 111 Western blot analysis of DNMT protein levels 111 Hemimethylation assay 113 Quantitation of DNA methylation levels by methylation- sensitive single-nucleotide primer extension assay 113 Bisulfite genomic sequencing 114 Determination of population doublings and cell growth 115 RESULTS 118 Kinetics of p!6 mRNA induction and demethylation of the 5' region by zebularine 118 Continuous treatment with zebularine sustains the expression and demethylation of the p!6 gene 118 Continuous zebularine causes variable demethylation of the entire p!6 gene locus 129 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Zebularine selectively depletes DNMT1 132 Sequential treatment of T24 cells with 5-Aza-CdR followed by zebularine 138 DISCUSSION 144 CHAPTER 4: PREFERENTIAL DEMETHYLATION OF CPG ISLANDS IN CANCER CELLS BY ZEBULARINE INTRODUCTION 148 MATERIALS AND METHODS 151 Cell lines 151 Drug treatments 152 Nucleic acid isolation 152 RT-PCR analysis 152 Oligonucleotide array analysis 153 cRNA preparation 153 Array hybridization and scanning 153 Western blot analysis of DNMT protein levels 156 Quantitation of DNA methylation levels by methylation- sensitive single-nucleotide primer extension assay 157 Determination of cell doubling time and population doublings 157 RESULTS 159 Zebularine selectively inhibits the growth of cancer cells but not normal fibroblasts 159 Heterogeneous effects of zebularine on p i 6 gene expression and demethylation in normal and cancer cells 163 Differential cellular responses to depletion of DNMT levels by zebularine 166 Zebularine substantially alters gene expression in T24 bladder cancer cells 169 DISCUSSION 177 CHAPTER 5: SUMMARY AND CONCLUSIONS 182 REFERENCES 187 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Cytosine methylation and spontaneous deamination Maintenance of cytosine methylation patterns DNA methylation in normal and cancer cells Complex interplay between two epigienetic marks of heterochromatin: histone H3 lysine 9 methylation and cytosine methylation Changes in the patterns of DNA methylation on different DNA elements from normal cells to cancer cells Structures of cytidine and its analogs, 5-azacytidine and zebularine Reactivation of the silenced hygrom ydn resistance (hph) gene in Neurospora Zebularine inhibits DNA methylation in Neurospora crassa Myotube formation in murine embryonic fibroblast C3H10T1/2 C18 (10T1/2) cells after treatment with cytidine and deoxycytidine analogs In vitro effects of cytidine and deoxycytidine analogs in T24 hum an bladder cardnoma cells Inhibition of methylation of p!6 prom oter/ 5' region in T24 hum an bladder carcinoma cells after treatment with cytidine and deoxycytidine analogs Microscopic effects of zebularineon on hum an bladder carcinoma cells grown in BALE/c nu/nu mice Anti tumor effects of zebularine on hum an bladder carcinoma cells grown BALB/c nu/nu mice In vivo effects on the weight of nude mice by zebularine Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.10 In vivo effects on the reactivation of silenced p!6 gene expression by zebularine 91 Figure 2.11 In vivo effects on the inhibiton of methylation of p!6 p ro m o te r/5 'region by zebularine 93 Figure 2.12 Proposed metabolism of zebularine and 2'-deoxyzebuiarine (2dZeb) 95 Figure 2.13 Covalent binding of DNA methyltransferases (DNMTs) to zebularine-substituted DNA 97 Figure 2.14 Metabolism of 5-azacytidine (5-Aza-CR) and 5-aza-2'- deoxycytidine (5-Aza-CdR) 99 Figure 2.15 Covalent binding of DNA methyltransferases (DNMTs) to aza-substituted DNA 100 Figure 3.1 Kinetics of p!6 mRNA induction by zebularine 119 Figure 3.2 Kinetics of p!6 5' region demethylation by zebularine 120 Figure 3.3 Effects of continuous zebularine treatment on p i 6 mRNA 122 Figure 3.4 Comparison of p!6 expression between T24 cells treated with zebularine and untreated normal LD419 cells 123 Figure 3.5 Effects of continuous zebularine treatment on p!6 5' region demethylation 125 Figure 3.6 Effects of continuous zebularine treatment on cellular growth 126 Figure 3.7 Bisulfite genomic sequencing of the p!6 5' region in T24 cells after continuous treatment with zebularine 128 Figure 3.8 Effects of continuous zebularine treatment on methylation levels of p!6 gene locus 131 Figure 3.9 Distribution of methylation states at the plS intronl after continuous zebularine treatment 133 V ll Reproduced with permission of the copyright owner. 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Figure 3.10 Distribution of methylation states at the plS exon2 after continuous zebularine treatment 134 Figure 3.11 Effects of continuous zebularine treatment on DNMT protein levels in T24 cells 136 Figure 3.12 Effects of continuous zebularine treatment on DNMT mRNA levels in T24 cells 137 Figure 3.13 Effects of continuous zebularine treatment on methylation status of various loci in T24 cells 139 Figure 3.14 Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on p i 6 mRNA expression 140 Figure 3.15 Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on p i6 prom oter/ 5' region methylation 142 Figure 3.16 Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on cellular growth 143 Figure 4.1 Effects of zebularine on growth regulatory genes in normal fibroblasts 161 Figure 4.2 Effects of zebularine on growth regulatory genes in cancer cells 162 Figure 4.3 Effects of zebularine on pl6 gene reactivation and pl6 5' region methylation in normal and cancer cells 165 Figure 4.4 Effects of zebularine on methylation of M4-4 167 Figure 4.5 Effects of zebularine on methylation of D4Z4 168 Figure 4.6 Effects of zebularine on DNMT protein levels in normal cells 170 Figure 4.7 Effects of zebularine on DNMT protein levels in cancer Cells 171 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1.1 Methodologies in detection of methylation patterns and methylation profiles 19 Table 1.2 Genes abnormally methylated in hum an cancers 38 Table 2.1 Conditions and primer sequences for bisulfite PCR and Ms-SNuPE 61 Table 2.2 Inhibition of DNA methylation and induction of myotubes in 10T1/2 cells 76 Table 3.1 Conditions and primer sequences for RT-PCR 110 Table 3.2 Conditions and primer sequences for bisulfite PCR 116 Table 3.3 Conditions and primer sequences for Ms-SNuPE 117 Table 4.1 Conditions and primer sequences for RT-PCR 154 Table 4.2 Effects of zebularine on growth suppression in normal and cancer cells 160 Table 4.3 Genes upregulated > 3 fold in T24 cells 8 days after continuous zebularine treatment 173 Table 4.4 Genes downregulated > 3 fold in T24 cells 8 days after continuous zebularine treatment 174 Table 4.5 Genes upregulated > 3 fold in LD419 cells 8 days after continuous zebularine treatment 175 Table 4.6 Genes downregulated > 3 fold in LD419 cells 8 days after continuous zebularine treatment 176 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors, however, the use of these inhibitors is often limited by their cytotoxicities and non-specific effects on both normal and cancer cells, as well as by their inabilities to permanently reverse methylation. In this study, I characterized a new demethylating agent, zebularine, which is a chemically stable cytidine analog. My results demonstrate the inhibition of DNA methylation and reactivation of genes by zebularine in C3H 10T1/2 C18 mouse embryo cells, T24 bladder carcinoma cells in vitro and in tumors grown in nude mice. The drug was exhibited minimal cytotoxicity in T24 cells in vitro and in tumor-bearing mice as assessed by minimal weight changes. Tumor volumes were also significantly reduced in nude mice treated with high dose zebularine, administered either by intraperitoneai injection or by oral gavage feeding. To exploit the drug's stability, I tested the effects of the continuous administration of zebularine to cultured T24 cells. Continuous application of zebularine to T24 cells maintained pl6 gene expression and sustained demethylation of the entire pl6 gene locus for over 40 days, preventing remethylation from occurring. The drug also caused a complete depletion of x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. extractable DNA methyltransferase 1 (DNMT1), and partial depletion of DNMT3a and DNMT3b3. Moreover, sequential treatment with S-aza-2'- deoxycytidine (5-Aza-CdR) followed by zebularine hindered the remethylation of the pl6 5' region and gene re-silencing, suggesting the possible combination use of both drugs as a potential anticancer regimen. Lastly, I investigated the effects of continuous zebularine treatment on normal and cancer cells. Zebularine preferentially targeted cancer cells versus normal fibroblasts, in terms of cell growth, methylation, DNMT levels, and gene expression. The drug is apparently selective towards cancer cells. Altogether, my results have demonstrated various beneficial properties of zebularine, giving this drug significant potential as a useful pharmacological inhibitor of methylation in biological systems and make it a promising candidate for epigenetic therapy. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 1 EPIGENETICS IN CANCER INTRODUCTION The diverse cell types in multicellular organisms have identical genotypes but are functionally and morphologically different. This is due to differences in gene expression patterns, which are established during development and are subsequently retained through homeostasis. Stable alterations of this kind are known as epigenetic modifications, which are defined as heritable, but potentially reversible changes in gene expression that occur without alterations in the DNA sequence. This chapter will focus on two key epigenetic phenomena that seemingly impact each other: DNA methylation and chromatin inheritance. Accumulating evidence indicates that alterations in DNA methylation and chromatin structure are linked to various hum an diseases, such as cancer and mental retardation syndromes. Elucidation of the epigenetic regulation of chromatin structure, DNA methylation and gene expression in development and in cancer can provide us with insights into the underlying causes of such diseases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNA METHYLATION CpG islands DNA methylation involves the addition of a methyl (-CH3 ) group to the S'-carbon on a cytosine in DNA and is a major contributor to the stability of gene expression states (Figure 1.1). The majority of 5-methylcytosine (5mC) in mammalian DNA is present in the context of the CpG dinucleotide (Bird 1978/ Cedar et al. 1979, Riggs and Jones 1983); however, non-CpG sequences such as CpNpG (Clark et al. 1995) or non-symmetrical CpA and CpT sequences (Woodcock et al. 1987) may also exhibit methylation, but generally at a much lower frequency. Non-CpG methylation is more prevalent in mouse embryonic stem than somatic cells (Ramsahoye et al. 2000), plants and fungi (Lindroth et al. 2001, Selker et al. 1993). The distribution of 5mC and of the CpG dinucleotide itself are neither uniform nor random (Cooper and Krawczak 1989). CpG is the only dinucleotide to be severely under-represented in the hum an genome, and this is thought to be due to the increased spontaneous deamination rate of 5mC into thymine (Figure 1.1). Approximately 70% of the CpGs that are present in the genome are methylated, whereas the majority of unmethylated CpGs occur in small clusters known as CpG islands (Cooper and Krawczak 1989), which are often found within or near promoters and first exons of genes (Bird 1986, 2 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f the copyright owner. Further reproduction prohibited without permission. NH, Methylation CH, i N Deaminatio C CH, Cytosine (in DNA) 5-methylcytosine (in DNA) Thymine (in DNA) Figure 1.1: Cytosine methylation and spontaneous deamination. A methyl group is added to the 5-carbon position of cytosine residues in DNA to become 5-methylcytosine (5mC) in the DNA. 5mC can undergo spontaneous hydrolytic deamination to cause cytosine to thymine transition mutations in DNA (Coulondre et al. 1978). Modified from Cheng and Jones, "Chapter 5: Epigenetics Events in Cancer," Cellular and Molecular Biology of Cancer, 4th Edition, Oxford University Press, In Press. Gardiner-Garden and Frommer 1987, Larsen et al. 1992, Takai and Jones 2002). CpG islands, which comprise 1-2% of the genome, are sequences of approximately 0.5-4 kb in length, with a GC content [(number of C bases + number of G bases)/sequence length] of over 55% (in contrast to a genome- wide average of about 40%), and an observed/expected ratio for the occurrence of CpG > 0.65 {[number of CpG sites/(num ber of C bases x number of G bases)]} (Frommer et al. 1992, Takai and Jones 2002). There are an estimated 45,000 CpG islands in the genome, and approximately 50-60% of all genes contain a promoter-associated CpG island (Antequera and Bird 1993a, Antequera and Bird 1993b). While most CpG islands are unmethylated and associated with transcriptionally active genes, such as "housekeeping" genes, certain CpG islands are methylated, including those associated with imprinted genes and genes on the inactive X chromosome in females (Bird 1992, Paulsen et al. 2001). Since the promoter CpG islands of most genes are generally unmethylated in the germ line, they are less susceptible to deamination and thus have retained the expected frequency of CpG dinucleotides. Conversely, the methylation of cytosines in the majority of the genome makes them more susceptible to deamination, which thereby reduces the overall frequency of CpGs in the bulk genome (Bird 1992, Bird 1980). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M aintenance of DNA methylation DNA methylation in mammals is carried out by at least three DNA methyltransferase (DNMT) enzymes known to be cataiytically active out of a family of five known members, DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L (Bestor 2000, Hsieh 1999, Robertson 2002). These enzymes exhibit two distinct functions but vary in their abilities to perform one or the other. Maintenance methyltransferase activity is responsible for copying methylation patterns onto newly synthesized strands of DNA based on the methylation status of the template parent strand (Figure 1.2). Thus a pattern of methylated and unmethylated CpGs along a DNA strand tends to be copied, and this provides a way for passing epigenetic information between cell generations. The second function, de novo methylation, is responsible for the methylation of CpG sites that were previously unmethylated (Figure 1.2). DNMT1 is believed to be primarily a maintenance methyltransferase (Bestor 2000, Li et al. 1993, Li et al. 1992), whereas de novo methylation of DNA sequences is mediated by DNMT3A and 3B (Okano et al. 1999). These methyltransferases have also been shown to exhibit a great degree of cooperativity in the methylation of certain classes of DNA repeats (Fatemi et al. 2002, Kim et al. 2002, Liang et al. 2002, Rhee et al. 2002). Despite extensive in vitro and in vivo analysis of the various DNMTs, the mechanisms by which specific DNA sequences are targeted for methylation by the various DNMTs remain poorly understood at this time. 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f the copyright owner. Further reproduction prohibited without permission. Replication Denovo Maintenance < 3\ Figure 1.2: Maintenance of cytosine methylation patterns. During DNA replication, the methylation patterns are copied onto the newly synthesized strands of DNA based on the template parental strand by DNA methyltransferase (DNMT). De novo methylation involves the addition of methyl groups on previously unmethylated CpG sites. Each circle represents a CpG dinucleotide. Open circles are unmethylated CpGs, while black circles are methylated CpGs. Grey circles represent de novo methylated CpGs which can occur on either one or both strands, however the mechanism of which is still unclear. Interestingly, DNMT3A was recently demonstrated to be a strand asymmetric hemi-methylase by preferentially methylating one strand of DNA without concurrent methylation of the CpG site on the complementary strand (Lin et al. 2002). Figure 1.1: Cytosine methylation and spontaneous deamination. A methyl group is added to the 5-carbon position of cytosine residues in DNA to become 5-methylcytosine (5mC) in the DNA. 5mC can undergo spontaneous hydrolytic deamination to cause cytosine to thymine transition mutations in DNA (Coulondre et al. 1978). Modified from Cheng and Jones, "Chapter 5: Epigenetics Events in Cancer," Cellular and Molecular Biology of Cancer, 4th Edition, Oxford University Press, In Press. In the adult, the amount and pattern of methylation are tissue- and cell type-specific (Futscher et al. 2002, Shiota and Yanagimachi 2002), and ageing- related methylation changes of CpG islands in the promoter of genes have been demonstrated, such as in the estrogen receptor gene and MYOD1 (Issa 2000). Variations in the methylation patterns of certain genomic regions appeared tissue-specific and were reproducible after transmission through the germ line, suggesting that distinct blueprints for the tissue-specific regulation of methylation may exist in the homologous chromosomes (Silva and White 1988). The establishment and maintenance of methylation patterns are hence regulated both temporally and spatially. Disruption of proper DNA methylation has been shown in several hum an disorders, including ATRX (X- linked alpha-thalassemia and mental retardation), Fragile X, and ICF (Immune deficiency, Centromeric instability, and Facial anomalies) syndromes (Robertson 2002). Role in development DNA methylation is essential for mammalian embryogenesis and methylation patterns are established during defined phases of development. The methylation profile of the early embryo shows an initial wave of genome wide demethylation, which removes most of the pre-existing patterns of methylation inherited from parental DNA, from fertilization up until the eight 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell stage of blastocyst formation (Razin and Shemer 1995, Reik and Walter 2001). This is followed after implantation by a wave of de novo methylation (Kafri et al. 1992, Monk et al. 1987). The importance of DNMTs in development has been clearly established by the generation of null mice as well as by the study of the hum an ICF syndrome. DnmtT7 ' mice die early in embryonic development, and exhibit severe genomic hypomethylation (Lei et al. 1996, Li et al. 1992). Furthermore, conditional deletion of D nm tl from mouse fibroblasts results in p53 dependent apoptosis and massive dysregulation of gene expression (Jackson-Grusby et al. 2001). Mouse embryos having homozygous DnmtSA deletions die approximately 4 weeks after birth (Okano et al. 1999). Homozygous deletion of Dnmt3B, however, results in early embryonic lethality w ith a pronounced loss of genomic DNA methylation in pericentromeric heterochromatin repeats. This defect is similar to that seen in lymphocytes of ICF syndrome patients who have immune defects that frequently lead to death at an early age. DNMT3B has been found to be mutated in patients with ICF syndrome (Hansen et al. 1999, Okano et al. 1999, Xu et al. 1999). Loss of DNMT3B function results in hypomethylation and chromatin decondensation of the repetitive satellite DNA in pericentromeric heterochromatin regions on chromosomes 1, 9 and 16, as well as changes in gene expression (Ehrlich et al. 2001). From these observations, it is obvious that DNA methylation plays a critical role in 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development, and that the disruption of methylation can lead to detrimental consequences. Functions of DNA methylation in normal cells DNA methylation serves as an essential mechanism for permanent, heritable silencing of gene transcription in mammalian development, most notably exhibited in genomic imprinting, transcriptional silencing of parasitic sequence elements, and X-chromosome inactivation (Figure 1.3) (Jones and Baylin 2002, Li et al. 1993, Walsh and Bestor 1999, Walsh et al. 1998). An important regulatory role of DNA methylation has been established in genomic imprinting. Differential DNA methylation is a critical signal for mammalian gene imprinting, leading to monoallelic expression of these genes (Plass and Soloway 2002). The functional differences between the paternal and maternal genomes are attributed to the differential expression of the respective alleles of several dozen imprinted genes during development. In many clusters of imprinted genes, one allele is very highly methylated and the other unmethylated or methylated at only a small percentage of CpGs in a 1-5 kb CpG-rich differentially methylated region (DMR). The methylation patterns of DMR exhibited gamete-specific differences, which are usually partially retained during embryogenesis and appeared to generally be the primary imprinting mark (Ehrlich 2003). The paternal alleles of the H19 (transforming-suppressing 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.3: D N A methylation in normal and cancer cells. In normal cells, DNA methylation is known to play important roles in tissue-specific methylation, genomic imprinting, X-chromosome inactivation, and silencing of parasitic sequences. On the other hand, cancer cells are usually found to be associated with hypermethylation of CpG islands of tumor suppressor genes, global genomic hypomethylation and increased mutagenicity due to spontaneous deamination of 5mC residues. Modified from Cheng and Jones, "Chapter 5: Epigenetics Events in Cancer," Cellular and Molecular Biology of Cancer, 4th Edition, Oxford University Press, In Press. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. £ z m u Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RNA) and Rasgrfl (guanine nucleotide exchange factors) genes are methylated in their 5' upstream regions in the male germ cells during embryogenesis, whereas the other known imprinted genes, such as Igflr (anti-apoptotic growth factor) and Snprn (encodes Smn protein involved in RNA splicing), acquire their methylation imprints from the oocyte; the deletion of such differentially methylated regions results in the loss of imprinting (Feinberg 1999, Shemer et al. 1997, Tremblay et al. 1995, Wutz et al. 1997, Yoon et al. 2002). The importance of methylation in genomic imprinting has been well demonstrated in homozygous mice with a targeted disruption of D nm tl, which can result in decreased global methylation levels and major perturbations in the expression of several imprinted genes (Li et al. 1993, Li et al. 1992). In m utant embryos, the normally silent paternal allele of the H19 gene was activated, whereas the normally active paternal allele of the Igf-2 gene and the active maternal allele of the Igflr gene were repressed. These results suggest that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprinted genes. A prominent example of transcriptional regulation by DNA methylation in genomic imprinting involves the role of the CTCF protein at the H19/Igf2 locus in chromosome llp lS in mice (Bell and Felsenfeld 2000, Feinberg 1999, Hark et al. 2000, Szabo et al. 2000, Takai et al. 2001). CTCF, associated with transcriptional domain boundaries (Bell et al. 1999), can safeguard a promoter 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from being affected by remote enhancers. When CTCF binds between the promoter and a downstream enhancer, the maternally-derived copy of the Igf2 gene becomes transcriptionally silent. In contrast, these CpG-rich binding sites are methylated at the paternal locus, which prevent CTCF from binding and thus allow the activation lgf2 expression by the downstream enhancer. Although the H19/Igf2 imprinting may involve additional processes (Ferguson- Smith and Surani 2001), the role of CTCF represents one of the clearest examples of transcriptional regulation by DNA methylation. Methylation has also been implicated as a possible genome defense mechanism against mobile genetic elements. CpG dinucleotides found outside CpG islands are mostly methylated and many of these CpGs reside within repetitive DNA sequences or retrotransposons, such as endogenous retroviruses, LI and Alu sequences (Yoder et al. 1997). Since these repetitive sequences comprise almost 40% of the human genome, it has been proposed that DNA methylation may have evolved as a genomic defense system to suppress these sequences and prevent their spread through the genome (Yoder et al. 1997). DNA retrotransposons may pose a significant threat to the integrity and stability of the genome by mediating recombination between non-allelic repeats, which can cause chromosome rearrangements or translocations, and actively integrating into and disrupting genes (Kazazian and Moran 1998, Montagna et al. 1999, Robertson and Wolffe 2000). Many retrotransposons have 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. potentially active promoters that, if integrated within a transcriptional unit, could result in internal initiation. Depending on the orientation of the integration, the consequences can vary and be quite harmful. When the integration is in the "sense" orientation (relative to the normal direction of transcription of targeted genes), this could result in a truncated transcript. Conversely, when the integration in the "antisense" orientation, this could potentially inhibit gene expression by either transcriptional interference or an antisense mechanism (Robertson and Wolffe 2000, Yoder et al. 1997). The mobility of these retrotransposons depend on the expression their encoded genes, however, the transcription of these genes have been shown to be silenced by the methylation of retrotransposon promoters (Kochanek et al. 1995). Apparently, methylation of promoters of intra-genomic parasites can function to inactivate these sequences, and moreover, the mutagenicity of 5mC can introduce C to T transition mutations resulting in the disabling of many retrotransposons. In female mammals, dosage compensation is mainly achieved by X- chromosome inactivation, a process that silences one of the two X chromosomes during embryogenesis. An X chromosome is converted from active euchromatin into transcriptionally silenced and highly condensed heterochromatin during inactivation through a sequence of events that include the coating of the X chromosome by X1ST (X inactive specific transcript) RNA, 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNA methylation and histone modification (Avner and Heard 2001). Recently, it was shown that histone H3 lysine 9 (H3-K9) methylation is a very early event in the process of X inactivation (Mermoud et al. 2002). The choice of the inactive X chromosome and initiation of the inactivation process also depend on the expression of XIST RNA [a noncoding transcript that originates at the X inactivation center (XIC)], accumulation of this transcript, and then spreading to coat the entire inactive X chromosome (Mermoud et al. 2002). This ultimately leads to chromosome-wide transcriptional silencing, condensation and the late replication of the inactive X. The expression of the XIST gene appeared to correlate with the methylation status of its promoter, but XIST is unmethylated and expressed from the inactive X and methylated and silent on the active X (Goto and Monk 1998). In fact, in D NM TTh embryonic stem cells, the normally silenced XIST gene on the active X chromosome in males became reactivated (Panning and Jaenisch 1996). It is still unclear whether DNA methylation initiates the process in vivo or if methylation is a secondary effect following the formation of transcriptionally inactive chromatin. Nevertheless, X-chromosome inactivation is an important process, which is mediated and controlled by XIST RNA, DNA methylation, as well as histone modification which will be discussed in greater detail. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNA m ethylation and transcriptional repression There are several ways by which DNA methylation may repress transcription (Karpf and Jones 2002). One mechanism involves DNA methylation sterically hindering the binding of activating transcription factors to gene promoters (Clark et al. 1997, Gaston and Fried 1995, Umezawa et al. 1997). A second mechanism involves the activity of methyl-CpG binding domain (MBD) proteins, which specifically bind methylated DNA and prevent subsequent binding of transcription factors (Hendrich and Bird 1998). At least three members of this protein family (MBD1, MBD2, and MeCP2) can directly repress transcription (Nan et al. 1997, Ng et al. 1999). These MBD proteins have been shown to recruit transcriptional co-repressors, including histone deacetylases (HDAC1 and 2) and chromatin-remodelling activities (Sin3A and Mi-2), to methylated DNA (Jones et al. 1998, Ng et al. 1999). A third mechanism of methylation silencing involves a non-enzymatic transcriptional repression by DNMT proteins (Karpf and Jones 2002). DNMT1, 3A and 3B have all been shown to contain transcriptional repressor domains. Furthermore, similar to MBDs, each of these DNMTs can recruit HDACs an d /o r other co-repressor proteins to DNA (Bachman et al. 2001, Fuks et al. 2000, Robertson and Wolffe 2000, Rountree et al. 2000). Therefore, in the current view of gene silencing by DNA methylation, DNMTs and MBDs may act as direct repressors that also facilitate the assembly of repressed, condensed chromatin structures onto DNA. 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. METHODS FOR DETECTION OF DNA METHYLATION M ethylation-sensitive restriction enzymes for detection of DNA methylation. Among the various methods for studying DNA methylation patterns of specific regions of DNA without base modifications include methods based on the use of methylation-sensitive and -insensitive restriction endonucleases which are widely used (Cedar et al. 1979, Fraga and Esteller 2002). One of the restriction enzymes of the isoschizomer pair cleaves the DNA only when its target is unmethylated, whereas the other is insensitive to methylated cytosines. The most common isoschizomers used are the H pall/M spI pair. Both enzymes cleave the DNA at the CCGG target, but Hpall is unable to cut when the second cytosine is methylated (CmCGG) in a double-stranded DNA template. After the DNA has been digested with methylation-sensitive endonucleases, the methylation status of a gene can subsequently be identified using Southern blot hybridization or PCR procedures. These methods for the quantification of DNA methylation patterns are simple, rapid, and can be used for any known sequence genomic DNA region. Despite the fact that these methods are extremely specific, their limitation to specific restriction sites reduces their value. 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bisulfite m ethods for detection of DNA methylation Southern blot analysis of DNA digested with methylation-sensitive restriction endonucleases has previously been an indispensable tool in the study of DNA methylation, however, they are unable to provide the critical information required for a complete understanding of the role of methylcytosine in a more specific sequence context. These isoschizomer-based methods have now been replaced by PCR methods that are based on initial modification of DNA with bisulfite. Bisulfite modification of the DNA is used to address these concerns (Clark et al. 1994) by selectively deaminating cytosine residues into uracil in genomic DNA, whereas the methylated cytosine residues are resistant to this modification (Wang et al. 1980). This bisulfite-modified DNA can then be used as a template in a standard PCR using primers specific for the gene of interest (Frommer et al. 1992). Bisulifte genomic sequencing of the resulting PCR product provides an accurate display of methylated cytosines (Frommer et al. 1992), but may be technically difficult and labor intensive. Various PCR methods have been developed (Table 1.1), including methylation- specific PCR (Herman et al. 1996), methylation-sensitive single nucleotide primer extension (Ms-SNuPE) (Gonzalgo and Jones 1997), real-time PCR-based MethyLight (Eads et al. 2000), and methods based on the use of restriction endonucleases (Sadri and Hornsby 1996, Xiong and Laird 1997), which are simple to use but all suffer from the drawback that only a limited number of 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Table 1.1* Methodologies in detection of methylation patterns and methylation profiles Technology M ethylation discrim ination priniciple Quantitative/ Q ualitative A pplication D isadvantages References M e th y la tio n P atterns MSP Bisulfite conversion Qualitative Rapid sensitive detection of m ethylation at specific sequences Poor resolution at the nucletide level (Herman et al. 1996a) Ms-SNUPE Bisulfite conversion/ Radioactive incorporation Quantitative Detect specific CpG methylation at specific sequences PCR bias/Difficulty with liigh CpG-rich regions (Gonzalgo and Jones 1997) MethyLight Bisulfite conversion/ Fluorescence-b ased, real-time PCR Quantitative Rapid high throughput analysis of m ethylation at specific sequences Inability to distinguish in detail different m ethylation patterns in present at the same location (Eads et al. 2000) Bisulfite-SSCP Bisulfite conversion/ SSCP Semi-quantitative Sensitive detection of high-resolution polymorphisms in hum an coding loci Detection of single-base changes requires a minimal level of alteration (Poduslo et al. 1991) (M aekawa et al. 1999) COBRA Bisulfite conversion/ Restriction enzyme digestion Semi-quantitative Analysis of methylation status at specific l egions in any DNA sample Confuted to restriction targets (Xiong and Laird 1997) In-Tube Fluorescence Melting Curve Bisulfite conversion/ Melting analysis Quantitative Detection of overall methylation status of a CpG island No information is provided on the methylation status of individual cytosines (Worm and Guldberg 2001) Bisulfite Genomic Sequencing Bisulfite conversion/ Cloning Qualitative Positive identification and localization of 5mC in genomic DNA Passible clonal selection bias (Frommer et al. 2992) M e th y la tio n P rofiles RLGS Restriction digestion/ 2D Fractional Electrophoresis Qualitative Genome-wide scan for changes in DNA methylation in CpG islands CpG island detection are sometimes not in the promoter regions of genes (H atadaetal. 1991) MS AP-PCR Restriction digestion/ Arbitrarily prim ed PCR Qualitative Rapid identification of CpG islands that are differentially methylated in different tissues M ost CpG islands detected are not in the prom oter regions of genes (Gonzalgo et al. 1997) DMH Restriction digestion/ CpG island array hybridization Qualitative Identiification of hypermethylated sequences in tum or cells Defining exact transcriptional start site can be laborious (Huang etal. 1999) Gene Re-Expression Arrays Drug agent activation (5-Aza-CdR, TSA)/ cDMA M icroarray analysis Qualitative Detection of hyperm ethylated sites is linked to the transcriptional status of genes Identifying hyperm ethylated CpG island, which is associated w ith the gene promoter, is genomic databases (Suzuki etal. 2002) (Liang et al. 2002) MSO Bisulfite conversion/ Oligonucletide M icroarray analysis Quantitative Rapid screening of multiple CpG sites in m any gene promotel's Possible cross-hybridization between imperfect-match probes and targets (Gitan et al. 2002) ICEAMP Methyl-CpG binding colum n/ Subtractive hybridization Qualitative Identification methylation changes in genomic regions during tumorigenesis Possible PCR amplification bias; may not detect all methylation changes in or near a repetitive element (Brock e t a l 2001) MCA-RDA Restriction digestion/ Subtractive hybridization Qualitative Detect large numbers of CpG islands wliich are mostly associated w ith genes Defining exact transcriptional start site can be laborious (Toyota et al. 1999a) Modified from Cheng and Jones, "Chapter 5: Epigenetic Events in Cancer," Cellular and Molecular Biology of Cancer, 4th Edition, Oxford University Press, In Press VO CpG sites can be analyzed in each assay. Different methods which are commonly used to detect DNA methylation are compared in Table 1.1. It is important to note that there is no one technique or general approach that is universally superior over the others, since the ultimate goals of quantitative accuracy, sensitive detection, high local or global informational content, compatibility with formalin-fixed tissues and compatibility with automation are not all found in a single or specific technique (Laird 2003). Therefore, the method of choice will depend on the desired application. Techniques to globally detect hyperm ethylated genes In the past, the lack of sequence information and the presence of multiple candidate genes in amplified or deleted regions have hampered the rapid identification of novel cancer genes. Fortunately, with the availability of human genome sequences, the large insert genomic clone resources, and the development of various new genomic scanning techniques, there is now growing interest in the discovery of novel cancer genes. Since most cancers are associated with abnormally hypermethylated genes, a large number of techniques have been developed to screen the cancer cell genome for these genes (Table 1.1). Some of these techniques are aimed specifically at identifying genes by discovering aberrantly hypermethylated CpG islands, since the identification of these CpG islands will allow the discovery of those genes 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. disrupted during tum or progression. On the other hand, other techniques are aimed at identifying regions of abnormal methylation per se rather than specific genes (Costello et al. 2000, Gonzalgo and Jones 1997, Huang et al. 1999, Toyota et al. 1999) (Table 1.1). The search for differences between tum or tissue and equivalent histologically normal tissue from the same patient, as well as different stages of disease progression from various patients, has often been of great interest to researchers. This type of screening approach can lead to identification of methylation markers that are useful for the sensitive detection of disease or markers associated with disease progression (Laird 2003). CHROMATIN INHERITANCE Histone modification and "histone code" Genes in eukaryotic cells are complexed with core histones and other chromosomal proteins in the form of chromatin. Gene transcription in mammalian cells does not occur on naked DNA, but instead occurs in the context of chromatin. The basic repeating unit of chromatin, the nucleosome, includes two copies of each of the four core histones H2A, H2B, H3, and H4 wrapped by 146 bp of DNA. Chromatin is not uniform with respect to gene distribution and transcriptional activity. Two discrete types of chromatin are known, heterochromatin and euckromatin. Heterochromatin represents a 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tightly packed, condensed conformation of chromosome and is usually associated with transcriptional repression and inactivity (Grunstein et al. 1995, Karpen 1994). On the other hand, euchromatin contains less condensed regions of chromosomal DNA and is generally associated with transcriptional activity (Grunstein et al. 1995). Histones are basic proteins consisting of a globular domain and a "tail" that protrudes out of the nucleosome. Recent studies indicate that epigenetic processes, such as histone modifications on the N- terminal tails and chromatin remodeling, can cooperate to control chromatin structure and ultimately cellular processes such as gene expression and DNA methylation itself (Berger 2002, Fahrner et al. 2002). These histone tails are targets for diverse post-translational modifications, which include acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation (Geiman and Robertson 2002, Lachner et ah 2003). The roles of acetylation, phosphorylation and methylation of various amino residues on histones H3 and H4 have been described (Lachner et al. 2003). The acetylation and deacetylation of conserved lysine residues present in histone tails has long been linked to transcriptional activity and has been one of the most well-studied histone modification. Histone acetylation is usually associated with transcriptionally active chromatin and deacetylated histones with inactive chromatin. Histone acetyitransferases (HATs) acetylate lysine residues to create an accessible and open chromatin configuration that 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. facilitates transcriptional activity, whereas histone deacetylases (HDACs) remove acetyl groups to facilitate chromatin compaction that leads to transcription repression. Histone deacetylase inhibitors, such as trichostatin A (TSA), can activate the transcription of certain genes (Table 1.1)(Cameron et al. 1999). In addition to acetylation, another histone modification which has also been shown to be critical to the function of the chromatin from which they occur includes methylation. Methylation of lysine residues on the histone tails of H3 and H4 appears to provide an additional layer of control over the chromatin structure, and ultimately over gene expression. A group of enzymes called histone methyltransferases (HMTase) is responsible for catalyzing histone lysine methylation (Lachner and Jenuwein 2002). Members of this group contain a conserved SET domain that is flanked by cysteine-rich regions (Rea et al. 2000). Some of the more prominent histone tail modifications include the acetylation of lysine 9 and methylation of lysine 4 [specifically, trimethylation of lysine 4; (Santos-Rosa et al. 2002)] of histone H3, both of which were shown to be associated with an open chromatin configuration. In contrast, methylation of lysine 9 of H3 (H3-K9) is a marker of condensed, inactive chromatin as with the inactive X-chromosome (Boggs et al. 2002, Heard et al. 2001, Maison et al. 2002, Peters et al. 2002) and is also found to be associated with aberrant gene silencing in cancer cells (Fahrner et al. 2002, Kondo and Issa 2003, Nguyen et al. 2002). The link between H3-K9 methylation 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and repressive chromatin is clearly evident, however this heterochromatin is shown to be associated with lack of transcriptional initiation but not inhibition of elongation by RNA polymerase II (Nguyen et al. 2002). It is becoming increasingly apparent that characteristic modification patterns, or combinations thereof, constitute a code that defines actual or potential transcriptional states (Jenuwein and Allis 2001, Wu and Grunstein 2000). The histone code hypothesis predicts that a pre-existing modification affects subsequent modifications on histone tails, and these modifications act as marks for the recruitment of different proteins or protein complexes to regulate various chromatin functions, such as gene expression, chromosome segregation, and DNA replication (Jenuwein and Allis 2001, Strahl and Allis 2000). In order to realize its full information carrying potential, the code must use various combinations of modifications. This requires not only proteins that can read such combined modifications, but also mechanisms by which they can be initiated and maintained. Interestingly, this intricate interplay between various covalent modifications occurring in different sites on the histone tails appears to ultimately impact gene expression. Chromatin remodeling and DNA m ethylation DNA methylation and histone modification are vital to the control of gene expression, however, chromatin remodeling proteins also play a crucial 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. role in the regulation of this process. The SNF2 family of chromatin remodeling proteins, which utilize ATP to alter the structure of chromatin through the disruption of the histone/DN A contacts, acts in various cellular processes such as gene expression, replication, DNA repair, and recombination (Geiman and Robertson 2002, Havas et al. 2001, Vignali et al. 2000). Depending on the SNF2 factor and the proteins with which it interacts, SNF2 family members function in transcriptional activation as well as repression. Some of these family members have been recently connected to the process of maintaining proper DNA methylation patterns in organisms as diverse as Arabidopsis thaliana and humans (Geiman and Robertson 2002). The first connection between a member of the SNF2 helicase/ATPase family and DNA methylation was the Arabidopsis thaliana protein DDM1 (decrease in DNA methylation) (Jeddeloh et al. 1999). Mutation of this gene results in a 70% reduction in global DNA methylation levels, which becomes more pronounced with successive generations. In DDM1 mutants, this loss of DNA methylation altered the expression and mobility of transposable elements, most likely by disrupting the formation of silent heterochromatin (Miura et al. 2001). The DDM1 gene is closely related to the mammalian lymphoid specific helicase (Lsh) (Hells, PASG) gene, which is first identified as an SNF2 helicase family member highly expressed in fetal thymus and activated lymphocytes (Jarvis et al. 1996). Mice with a homozygous deletion of Lsh show perinatal 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lethality and global defects in the level of DNA methylation, both in repetitive elements and single copy genes (Dennis et al. 2001). Chromatin remodelers, such as DDM1 and Lsh, appeared to be important in the control of genomic DNA methylation levels, as evidenced by the substantial loss of methylation in both DDM1 and Lsh mutants. Another member of the SNF2 family of chromatin remodeling proteins is ATRX, the gene for which is m utated in ATRX syndrome (Gibbons et al. 2000). Mutation of the ATR X gene on the X chromosome leads to an unusual form of thalassemia, presumably due to a 30-60% decrease in alpha globin gene expression levels (Gibbons et al. 2000). Even though no changes in the methylation pattern of the alpha globin gene have been reported in ATRX patients, both hyper- and hypomethylation changes in highly repetitive elements such as satellite DNA have been demonstrated (Geiman and Robertson 2002, Gibbons et al. 2000). Apparently, both of these chromatin remodeling family members are essential for proper DNA methylation, however, the mechanisms by which they function may differ, probably due to a different complement of interacting proteins. This connection between chromatin remodeling and DNA methylation supports the notion that there are multiple layers of epigenetic modifications to heritably modulate gene expression. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chromatin structure is closely linked to gene expression, and deregulation of chromosome-remodeling activity may thus interfere with many critical cellular processes, resulting in development of diseases (Luo and Dean 1999). A classic example demonstrating the cancer-chromatin connection is the fact that a well-known tumor suppressor, retinoblastoma gene (Rb), is closely linked to histone deacetylation. Most Rb mutants found in cancers affect the integrity of the pocket, which is the region that binds to HDAC1 and HDAC2 (Dahiya et al. 2000). Hence, Rb mutants are no longer able to recruit histone deacetylases to maintain a repressed chromatin structure. Another example is acute promyelocytic leukemia which is caused by a chimeric m utant of the retinoic acid receptor associated with HDAC1 (Grignani et al. 1998, Lin et al. 1998). Treatment of patients with acute promyelocytic leukemia with a histone deacetylase inhibitor and retinoic acid re-induced remission (Warrell et al. 1998). Moreover, truncating mutations region found in the hSMF5/INIl gene, which encodes a member of the human chromatin-remodelling SWI/SNF multiprotein complexes, have also been linked to the development of malignant rhabdoid tumors (Versteege et al. 1998). Finally, the finding that dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities (Tong et al. 1998, Zhang et al. 1998) further demonstrates the broad spectrum of diseases associated with deregulation of chromosome structure. 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Link betw een DNA m ethylation and chromatin structure Cytosine methylation changes the interactions between proteins and DNA, leading to alterations in chromatin structure and either a decrease or an increase in the rate of transcription. The processes of histone deacetylation and DNA methylation have turned out to be tightly linked through protein complexes containing DNMTs and HDACs (Rountree et al. 2000). This link provides a plausible mechanism between DNA methylation and histone deacetylation in transcriptional repression, since the recruitment of DNMTs and their associated HDACs to methylated DNA would cause local deacetylation of core histone tails, thereby resulting in tight chromatin compaction and limited access of transcription factors to their binding sites (Robertson and Wolffe 2000). Recently, four of the methyl-CpG binding domain-containing proteins, MeCP2, MBD1, MBD2, and MBDS, have been associated with aspects of the chromatin remodeling machinery in addition to HDACs. In Xenopus eggs, for instance, MBDS is a component of the Mi-2 chromatin remodeling complex which also includes RpdS (Xenopus HDAC1/2) and two Rb-associated histone- binding proteins, RbAp46/48 (Wade et al. 1999). The mechanistic link between DNA methylation and histone deacetylation has also been supported by combination treatment of tumor cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-Aza-CdR), and the histone deacetylation inhibitor, trichostatin A (ISA). Low doses of 5-Aza-CdR resulted in low level of 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gene reexpression and minimal demethylation of hypermethylated CpG-island- associated genes, but a combination of 5-Aza-CdR and TSA resulted in robust activation of these same genes whereas TSA alone had no effect (Cameron et al. 1999). This supported the idea that DNA methylation and histone deacetylation worked cooperatively to silence gene transcription. The associations between DNA methylation and chromatin modification processes are not only mediated by HDACs. Interestingly, histone methylation provides another crucial link. In Neurospora crassa, mutations of the D IM S (defective in methylation 5) gene, which encodes a SET domain-containing H3-K9 methyltransferase, resulted in the complete loss of genomic DNA methylation (Tamaru and Selker 2001). When wild-type Neurospora H3-K9 is replaced by H3 with an altered amino acid at position 9 that cannot be methylated, the genomic DNA methylation is also reduced (Tamaru and Selker 2001). Specifically, it was shown that trimethylated H3-K9, not dimethylated H3-K9, marks the chromatin regions for cytosine methylation and that DIM-5 is the enzyme responsible for creating this mark (Tamaru et al. 2003). Similar observations were made in Arabidoposis thaliana, in which mutations of an H3-K9 methyltransferase [encoded by kryptonite (kyp)] abolished methylation of CpNpG sites, but not CpG sites (Jackson et al. 2002). Furthermore, in Arabidopsis, the loss of DNA methylation in DDM1 mutants was may be the consequence of reduced H3-K9 methylation in heterochromatin (Gendrel et al. 2002). Interestingly, in 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Arabidopsis, the interaction between DNMT chromomethylase 3 (CMT3) with the methyl-lysine binding protein, heterochromatin protein 1 (HP1) provides a specific mechanism for targeting DNA methylation to genomic regions containing histone methylation (Jackson et al. 2002). From these studies, it appeared that the establishment and maintenance of DNA methylation is dependent upon histone H3-K9 methylation in both of these organisms and may be a common eukaryotic epigenetic mechanism (Figure 1.4). Histone H3 lysine 9 methylation and DNA methylation are generally both associated with transcriptionally silent heterochromatin. Methylated lysine 9 of histone H3 is a binding site for HP1 (Bannister et al. 2002, Lachner et al. 2001), and the histone H3 lysine 9 methyltransferase SUV39H1 colocalizes and interacts with HP1 in regions of heterochromatin (Lachner et al. 2001). As a major component of heterochromatin, HP1 contributes to the establishment and maintenance of the transcriptionally repressed state of heterochromatin. In murine embryonic stem cells, it is interesting to find that DNMT3 proteins co- localize with HP1 to pericentromeric heterochromatin regions, indicating that DNA methylation may be targeted to heterochroma tic sites through HP1 (Bachman et al. 2001). Therefore, in addition to methylation abilities, DNA methyltransferases may function as transcriptional repressors and serve as scaffolds to direct other chromatin-modifying activities in establishing heterochromatin (Burgers et al. 2002). In addition, MeCP2 was recently 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.4: Complex interplay between two epigenetic marks of heterochromatin: histone H3 lysine 9 methylation and cytosine methylation. The establishment of permanent, heritable silencing of gene transcription and heterochromatin involves the intimate connection between histone H3 lysine 9 methylation and cytosine methylation, irrespective of which comes first. Recent evidence from N. crassa (Tamaru and Selker 2001) and A. thaliana (Jackson et al. 2002) suggests that histone H3 lysine 9 methylation, specifically trimethylated lysine 9 (Tamaru et al. 2003), drives cytosine methylation, presumably through heterochromatin protein 1 (HP1) and DNA methyltransferases (DNMTs) (Bachman et al. 2001, Bannister et al. 2002, Jackson et al. 2002). The mechanism by which this occurs in mammalian cells is still unclear. Cytosine methylation may also facilitate histone H3 lysine 9 methylation through methyl-CpG binding domain proteins (MBDs), histone deacetylases (HDACs), and histone H3 lysine 9 methylase (H3-K9 methylase), thereby reinforcing the repressive function of these two distinct epigenetic markers (Bird and Wolffe 1999, Fujita et al. 2003, Fuks et al. 2003, Lachner et al. 2001, Soppe et al. 2002). Modified from Cheng and Jones, "Chapter 5: Epigenetics Events in Cancer," Cellular and Molecular Biology of Cancer, 4th Edition, Oxford University Press, In Press. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. [ > * * , C C i •5 D < S p a D 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrated to be associated with H3-K9 methylation in vitro as well as in vivo, thereby providing another mechanistic bridge between DNA methylation and histone methylation in establishing the repressed state (Fuks et al. 2003). Furthermore, MBD1, which possesses an MBD involved in mediating DNA methylation-dependent transcriptional repression, was shown to direct SUVH1- HP1 complex to methylated DNA regions, suggesting a potential pathway from DNA methylation to histone methylation for epigenetic gene regulation (Fujita et al. 2003). To further corroborate the idea that cytosine methylation directs H3-K9 methylation, it was shown that DNA methylation precedes and controls H3-K9 methylation and heterochromation in Arabidopsis using DDM1 and MET1 (encodes a maintenance methyltransferase) mutants (Soppe et al. 2002). Even though this appeared to contradict previous results in Neurospora crassa (Tamaru and Selker 2001) and Arabidopsis (Jackson et al. 2002), which show that DNA methylation is dependent on H3-K9 methylation, this discrepancy may be due to differences in the function between the methylases involved (Soppe et al. 2002). Together, these data provide new insights into the link identified between DNA methylation and histone methylation and open up new avenues by which DNA methylation might be connected to the chromatin structure to bring about gene silencing. Nevertheless, the mechanism by which histone methylation regulates CpG methylation in mammalian cells is still unknown. It 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is also yet to be determined whether the maintenance of DNA methylation patterns depends on histone methylation or vice versa in mammalian cells. Since mammalian cells contain several CpG methyltransferases and H3-K9 methyltransferases, the interaction is likely to be more complicated than that in Neurospora and Arabidopsis. The molecules that target histone methyltransferases and DNA methyltransferases to specific genomic loci also remain to be elucidated. CANCER EPIGENETICS Aberrant patterns of cytosine methylation have been found to be associated with an increasing number of cancers over the past few decades. Two distinct patterns have been well-described: (1) global genomic hypomethylation (loss of methylation at normally methylated sequences) (Esteller et al. 2001, Feinberg et al. 1988, Gama-Sosa et al. 1983, Lapeyre and Becker 1979) and (2) localized hypermethylation (gain of methylation) usually in normally unmethylated CpG islands (Figure 1.5) (Baylin et al. 2001, Jones and Baylin 2002, Jones and Laird 1999). These disparities can be found together in a single tumor, yet the overall effect is commonly a drop in total methylation levels. It is currently unknown whether genomic hypomethylation and CpG island hypermethylation are linked by a common underlying mechanism or 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Normal Cell Chromosome ““ ' T E x o n l ■■ i r h h ' J ' - W fv V J D e N o v o Transposon Methylatio Demethylation ^ I Gene Silencing Activated Transposon? 5mC—> T Mutations I Mutated Gene Satellite Demethylation Chromosome Instability L I N E Cancer Cell Chromosome Figure 1.5: Changes in the patterns of D N A methylation on different D N A elements from normal cells to cancer cells. The de novo methylation of promoter or 5' region of genes, demethylation of transposon, mutagenesis of 5mC and demethylation of satellite sequences can all possibly contribute to the genesis of altered chromosomes in cancer. Modified from Figurelin Hsieh and Jones, N at Cell Biol, 2003. result from distinct abnormalities in the cancer cell. Both of these changes in methylation patterns can, however, precede malignancy, suggesting that they are not simply a consequence of the malignant state (Costello and Plass 2001). In addition, the mutagenicity of 5mC and secondary effects of DNA methylation can also influence tumorigenesis via different mechanisms. Altogether, the study of epigenetic alterations in cancer is commonly referred to as cancer epigenetics (Figure 1.3). CpG island hyperm ethylation in tum or suppressor genes CpG island hypermethylation in the promoter regions of cancer-related or tumor suppressor genes has been commonly associated with their silencing. Since methylation of the associated CpG islands corresponds with inactivation of these genes in the tumors, hypermethylation has been included as an alternative mechanism to genetic mutation an d /o r deletion in eliciting allelic gene silencing of tumor suppressor genes in cancer (Jones and Laird 1999). Abnormal methylation of CpG islands can efficiently repress transcription of the associated gene in a manner similar to mutations and deletions, thereby acting as one of the "hits" in the Knudsen two-hit hypothesis for tumor (Baylin and Herman 2000, Jones and Baylin 2002, Jones and Laird 1999). Biallelic inactivation of a tumor suppressor or cancer-related gene may result from either genetic and epigenetic mechanisms alone or combinations thereof. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Interestingly, demethylating agents are capable of restoring gene activity and tumor suppressor function in cultured tumor cells (Baylin and Herman 2000, Jones and Baylin 2002). Numerous genes involved in fundamental pathways, such as cell cycle regulation, DNA repair, drug resistance and detoxification, differentiation, apoptosis, angiogenesis, metastasis, and invasion, have been shown to be inappropriately silenced by methylation (Table 1.2). It is clear that hypermethylation is a significant alteration in the cancer genome, however the mechanisms responsible for eliciting this change are not well understood. Two models have been suggested for the abnormal CpG island methylation of various tumor-suppressor genes in cancer (Costello and Plass 2001, Jones and Baylin 2002, Tycko 2000). One proposed mechanism involves the loss of protective factors that normally bind to CpG islands and prevent them from methylation. The protective factors, such as structural proteins (Zardo and Caiafa 1998) or transcription factors (Brandeis et al. 1994), could compete with DNMTs for sites within the CpG island to prevent methylation. An example supporting this mechanism includes the SP1 transcription factor, whose recognition sites for binding are usually found within most CpG islands, and mutation of its site in transgenic mice resulted in methylation of the associated transgene CpG island (Brandeis et al. 1994, Mummaneni et al. 1998). Nevertheless, in SP2'A mouse embryos, CpG islands remain unmethylated (Marin et al. 1997). Undoubtedly other transcription factors may function in a 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. \ hum an cancels L a D O Function Gene Abbreviation(s) Genes Tumor Time(s) Location References Apoptosis DAPK D eath associated protein kinase Uterine, cervix, lym phoma, colon, lung 9q34.1 (Katzenellenhogen et al. 1999) CASP8 Caspase S Primary PN ET/ medulloblastoma 2q33-34 (Teitzetal.2000) TMS1 Target of methylation induced silencing Breast 16pll.2-12.1 (McConnell and Vertino 2000) Angiogenesis THBS1 Thrombospondin-1 Glioblastoana multiforme 15ql5 (Li et al.1999) Cell Cycle RB Retinoblastoma Glioblastomas 13ql4 (Sakai et al. 1991) (Greger et aL 1989) pUARF p l4 Alternative reading frame Colorectal, stomach, kidney 9p21 (Esteller et al. 2000c) (Zheng et al. 2000) CDKN2A/pl61NK4a Cyclin-dependenl kinase 2A Solid tumors 9p21 (Merlo et al. 1995) (Costello et al. 1996) C DKN2 B/p 15INK4b Cyclin-dependent kinase 2B Hematological malignancies 9p21 (Herman et al. 1996b) (N guyen et al. 2000) p27 p27/K IPl Melanoma 12pl3 (Worm et al. 2000) TP73, p73 p73 (TP73) Lymphomas lp36 (C om et al.1999) SF N /14-3-3-cr Sbatifin Breast, gastric, colorectal, hepatocellular ip (Ferguson et al. 2000) (Suzuki et al. 2000) Differentiation MYOD Myogenic differentiation antigen-1 Colorectal llp l5 .4 (jones PA et al. 19%) (Ahuja et al. 1998) PAX6 Paired box gene 5 Colon, bladder H p l3 (Salem et al. 2000) RA R fi2 Retinoic acid receptor Nasopharyngeal 3p24 (Bovenzi et al. 1999) (Virmani et al. 2000) WT1 Wilms tum or 1 Wilms' Tumor llp l3 (Malik etal. 2000) DNA Repair hM U il H um an MutL homologue 1 Colon, gastric 3p23-p21.3 (Esteller et al. 1998b) (Fleisher et al. 1999) o 6-m g m t O-6-methylguanine-DNA mcthyltransferase Colon, gastric, lymphoma 10q26 (Pieper et al.1990) (Harris et al. 1994) (Costello et al. 1994) (Watts et al. 1997) Detoxification/ GSTP1 Glutatluone S-transferase % Breast, prostate, kidney llq l3 (Lee et al. 1994) (Esteller et al. 1998a) d rag resistance MDR1 M ulti-drug resistance 1 H um an T cell leukemia 7q21.1 (K antharidisetal. 1997) Invasion/ Metastasis com E-cadherin Breast, gastric, leukemia 16q22.1 (Graff et al. 1995) (Kanai et al. 1997) CDH13 H-cadherin Colorectal, breast, lung 16q24 (Toyooka etal. 2001) TEMP-3 Tissue inhibitor of metalloproteinase 3 Colon, renal 22ql2.3 (Bachman et al. 1999) Maspin/PIS M aspin (protease inhibitor 5) Breast 18q21.3 (Domann et al. 2000) Signal transduction APC Adenomatous polyposis of the colon Colon, gastric 5q21-22 (Tsuchiya et al. 2000) PTEN Phosphatase and tensen homologue deleted on chromosome 10 Prostate 10q23-3 (Salvesen et al. 2001) (Cairns et aL 1997) AR Androgen receptor Prostate Xqll-12 (Jarrard et al. 1998) ESR1 Estrogen receptor 1 Breast 6q25.1 (Issa et al. 1994) (Ottaviano et al. 1994) PR Progesterone receptor Breast, prostate llq22 (Lapidus et a l 1996) (Yan et al. 2001) RASSF1A Ras association dom ain family member 1 N asopharyngeal, ovarian, renal 3p21.3 (Dammann et al. 2000) STK11/LKB1 Serine/ threonine protein kinase 11 Colon, breast, lung 19pl3.3 (Esteller et al. 2000) (Trojan et al. 2000) Transcription/ VHL Von Hippel-Lindau syndrome Renal, hemangioblastoma 3p26~p25 (Herman et al. 1994) (Kuzmin et al. 1999) transcription factors HIC-1 Hypermethylated in cancer Breast 17pl33 (Wales et al. 1995) (Fujii et al. 1998) BRCA1 Breast cancer, typel Breast, ovarian 17q21 (Esteller et al. 2000) (Rice et al. 1998) SRBC BRCAl-binding protein Breast, lung lp l5 (Xu etal. 2001) SYK Spleen tyrosine kinase Breast, lymphoblastic leukemia 9q22 (Y uanetal. 2001) SOCS-1 Suppressor of cytokine signaling 1 Hepatoblastomas; multiple myeloma 16pl3.13 (Yoshikawa et al. 2001) Other CD44 CD44 antigen Prostate llp ter-p l3 (Lou et al. 1999) C.OX2 Cyclo-oxygnease 2 Colon, gastric lq25.2-25.3 (Toyota et al. 2000) CACNA1G Calcium channel, voltage dependent, T type, alpha-IG subunit Colorectal, gastric 17q22 (Toyota et al. 1999b) CALC A Calcitonin Chronic myelogenous leukemia llpl5.2-15.1 (N elk in etal 1991) (B aylinetal 1986) FHFT Fragile histidine tr iad gene Esophageal, cervical, breast 3p l4 2 (T arakaetal. 1998) TERT Telomerase reverse transcriptase Colorectal 5pl5.33 (Dessain et al. 2000) (Devereux et al. 1999) TPEF/HPP1 Transmembrane protein containing epidermal grow th factor and foilistatin domains Bladder, colon 2q33 (Liang et al. 2000) CSPG2 Chondroitin sulphate proteoglycan 2 Colon 5ql2-14 (Adany and Iozzo 1990) R1Z1 Retinoblastoma protein -interacting zinc finger Breast, Liver, Nasopharyngeal Ip36 (Du etal. 2001) " Modified fro m C h en ^ and Jones, “C hapter 5: Epigenetic Events in C anon," C ellular and M olecular Biology'of C ancer,4th Edition, O xford U niversity Press, In Press similar matter, but the fact that even CpG islands from non-expressed genes remain unmethylated in normal cells suggests that factors other than those associated with active transcription may also be involved in protecting some CpG islands from methylation (Costello and Plass 2001). In hum an tumor cells, the loss of protective factors may provide a mechanism by which methylation can spread into the CpG island from flanking densely methylated sequences that often contain Alu elements (Graff et al. 1997, Magewu and Jones 1994). Even though most CpG islands of tumor suppressor genes are unmethylated in normal tissues, these islands are embedded between heavily methylated flanking regions containing multiple Alu repeats (Graff et al. 1997). Together, these data suggest that abnormal CpG island methylation in cancer can possibly result from the loss of protective factors and the encroachment of methylation from flanking methylated regions into the boundaries that apparently exist at both ends of the unmethylated CpG island. A second model suggests that aberrant CpG island methylation is an active process and causes inappropriate gene silencing (Clark and Melki 2002). In support of this model, there is considerable evidence showing the overexpression of all DNMTs at the mJRNA levels in several cancers (Belinsky et al. 1996, De Marzo et al. 1999, Issa et al. 1993, Kautiainen and Jones 1986), and increased DNMT1 expression in normal human fibroblasts cause aberrant de novo methylation of CpG islands (Vertino et al. 1996) and promote cellular 39 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transformation in NIH3T3 cells (Bakin and Curran 1999, W u et al. 1993). Conversely, low levels of DNMT1 appear to have protective effects. For instance, hypomorphic alleles of D nm tl lead to the complete suppression of intestinal polyp formation in M in mice, which are genetically predisposed to colonic polyp formation (Eads et al. 1999, Laird et al. 1995). Furthermore, inhibition of the methyltransferase using an antisense to DNMT1 reduces the tumorigenicity of murine adrenocortical tumor cells (MacLeod and Szyf 1995, Ramchandani et al. 1997). However, other reports indicate that there is no correlation between CpG island hypermethylation and DNMT1 levels (Eads et al. 1999, Nass et al. 1999). Alterations in DNA function will result in gains and losses in DNA methylation as well as cause variations in DNA methylation patterns (Dennis et al. 2001). Global genomic hypom ethylation Malignant cells can have 20-60% less genomic 5mC than their normal counterparts (Gama-Sosa et al. 1983, Lapeyre and Becker 1979). Global DNA hypomethylation has also been reported in almost every hum an malignancy (Bedford and van Helden 1987, Feinberg and Vogelstein 1983, Tsujiuchi et al. 1999). Interesting, the majority of hypomethylation events occur in repetitive elements localized in satellite sequences or centromeric regions, which are normally methylated (Ji et al. 1997). In tumors, the extent of genome wide 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hypomethylation parallels closely the degree of malignancy, although this may be tumor type-dependent. In various tumors, such as breast, cervical, ovarian, and brain tumors, hypomethylation increases progressively with increasing malignancy grade (Costello and Plass 2001, Gama-Sosa et al. 1983, Kim et al. 1994, Narayan et al. 1998, Qu et al. 1999). In addition, another study on breast lesions demonstrated a significant correlation between the extent of hypomethylation and disease stage, tumor size, and degree of malignancy (Soares et al. 1999). Hence, hypomethylation may have prognostic values as a biological marker. The significance of hypomethylation in tumorigenesis is becoming increasingly recognized. DNA hypomethylation can contribute to tumorigenesis in several ways. Loss of methylation in cancers could induce expression of the normally repressed transposons scattered throughout the genome leading to deleterious transposition events (Yoder et al. 1997). There is evidence that cancer-related hypomethylation results in increased expression of some of these elements, but whether this increased expression is harmful to the genome either directly or through transposition of the elements is not clear (Florl et al. 1999). Second, genomic hypomethylation in cancer has been linked to chromosome instability (Chen et al. 1998, Gaudet et al. 2003), and may favor mitotic recombination leading to loss of heterozygosity, as well as promoting karyotypically detectable rearrangements (Chen et al. 1998, Hernandez et al. 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1997, Rizwana and Hahn 1999). Though genomic demethylation may be protective against some cancers such as intestinal tumors in the ApcM in mouse model (Laird et al. 1995), it may act as a double-edged sword by also promoting chromosome instability and loss of heterozygosity (LOH), as well as increasing the risk of cancer in other tissues, as seen in hypomethylated m utant mice (Chen et al. 1998, Gaudet et al. 2003). Third, evidence for activation of oncogenes by specific gene demethylation in cancer is poor, however, hypomethylation has been reported in the coding regions of some oncogenes, including cMYC and H-RAS (Vachtenheim et al. 1994). Hypomethylation has also been reported to be responsible for the activation of the MAGE and related genes (De Smet et al. 1996). These genes are germline-specific and their promoters are normally methylated and silent in all adult somatic tissue but can become aberrantly activated in a number of tumors. Finally, the loss of methyl groups can affect imprinted genes, such as the H19/Igf2r locus, where the disturbance of methylation may cause overexpression of Igf2r and loss of H19 in certain childhood tumors (Feinberg 1999). All four mechanisms mentioned above are possible means in which hypomethylation can contribute to tumorigenesis. 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M utagenesis of 5-methylcytosine Cytosine methylation can also influence tumorigenicity via mechanisms other than through though hypo- and hypermethylation. 5mC can undergo spontaneous hydrolytic deamination to cause C to T transition mutations (Coulondre et al. 1978) and an estimated 31% of germline mutations which lead to genetic disorders can be attributed to 5mC to T transitions at methylated CpG dinucletotides (Cooper and Youssoufian 1988, Lengauer et al. 1997, Shen et al. 1994). Moreover, a high incidence of C to T transition mutations occurring at methylated cytosines was also observed in the human p53 tumor suppressor gene in somatic cells (Rideout et al. 1990). The spontaneous deamination of 5mC to T generates a G /T mismatch, which can be recognized and repaired by G /T mismatch thymine DNA glycosylase (Brown and Jiricny 1988). This type of transition mutation may also arise from the spontaneous deamination of cytosine to uracil, generating a G /U mismatch, which can be recognized by uracil DNA glycosylase. An accumulation of either G /U or G /T mismatches, which are not appropriately repaired by their respective enzymes, uracil- (or thymine) DNA glycosylases, will increase the frequencies of C to T transition mutations in the genome. Studies have shown that bacterial methyltransferases can enhance C to T transition mutations by blocking the repair of G /U mismatches by uracil DNA glycosylase, and can mediate 5mC to T transition mutations at CpG 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. dinucleotides under specific conditions which include increased DNMT expression or decreased cellular S-adenosylmethionine (SAM) levels (Bandaru et al. 1995, Lindahl and Nyberg 1974, Shen et al. 1992, Wyszynski et al. 1994, Yang et al. 1995). There is, however, no evidence as yet that this particular SAM-deficient mechanism plays a significant role in inducing mutation in hum an cells. The major cause of the high mutation rate at CpG dinucleotides is still likely to be spontaneous deamination of 5mC (Schmutte et al. 1996). There are other causes for mutation induction secondary to DNA methylation. For instance, the UV absorption wavelength of cytosine is shifted by methylation into the range of incident sunlight, thereby increasing CC to TT mutations that commonly occur in skin cancers (Pfeifer 2000). In addition, methylated CpG dinucleotides show enhanced binding of benzo[a]pyrene diol epoxide and other carcinogens found in tobacco smoke, which causes DNA adduct formation and G to T transversion mutations (Denissenko et al. 1997, Denissenko et al. 1994, Yoon et al. 2001). Methylation can therefore directly contribute to the induction of mutations that cause cancer. REVERSAL OF EPIGENETIC MODIFICATIONS AS A CANCER THERAPY Inactivation of tumor suppressor genes by genetic and epigenetic mechanisms are functionally equivalent in many ways, however, there are some fundamental differences between these two pathways that may 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. potentially be important for anticancer therapy. Genetic hits confer a fixed, irreversible state of gene inactivation, whereas epigenetic events do not change the information content of the affected genes and are potentially reversible. This feature makes epigenetic modifications good targets for therapeutic interventions in cancer patients. Epigenetic silencing may be alleviated at two different levels: inhibition of DNA methylation and inhibition of chromatin modification. Many inhibitors of DNA methylation or histone deacetylase are available that can modulate gene transcription in vitro and in vivo. A potent specific inhibitor of DNA methylation, 5-Aza-CdR, has been widely used as a demethylating agent in vitro (Constantinides et al. 1977). 5- Aza-CdR is specific inhibitor of DNA methyltransferase, which forms an irreversible covalent complex with the enzyme after incorporation into DNA (Christman 2002, Juttermann et al. 1994, Santi et al. 1984). 5-Aza-CdR exhibits promising activity as an anticancer agent, and is used clinically in the treatment of acute leukemias and myelodysplasia (Lubbert 2000). However, this demethylating agent and its ribose analog, 5-azacytidine (5-Aza-CR), are unstable in neutral aqueous solutions and quite toxic, thereby complicating their clinical use. Zebularine, a cytidine analog, was shown recently to be a stable, minimally toxic demethylating agent capable of reactivating an epigenetically silenced gene by oral administration in tumors grown in nude mice (Cheng et al. 2003). The characterization of this drug and its role as a 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demetylating agent will be the main focus of this thesis. Other drugs include the antihypertensive and antiarrhythmic agents, hydralazine and procainamide, respectively, both of which are non-nucleoside analogs that have been shown to inhibit DNA methylation and reactivate silenced genes in cultured cells and in mice (Lin et al. 2001, Scheinbart et al. 1991, Segura-Pacheco et al. 2003). Inhibition of DNA methylation as a target for cancer therapy is further supported by the interesting observation that antisense to DNMT1 shows in vitro antitumor activity and some potential to reverse the malignant phenotype (MacLeod and Szyf 1995). This antisense oligonucleotide also inhibits tumor growth in an animal model (Ramchandani et al. 1997). Likewise, cell culture experiments have shown that histone deacetylase inhibitors [i.e. butyrates, suberoylanilide hydroxamic acid (SAHA), valproic acid (VPA) (Marks et al. 2001)] can reactivate a range of epigenetically silenced genes, and several of these agents are now in clinical trial (Marks et al. 2001). Unfortunately, a major problem of drugs targeting the DNA methylation or histone methylation machineries is the lack of specificity (Worm and Guldberg 2002). The potential activation of genes that are normally silenced, but contribute to tumor initiation or progression in the activated state, may significantly limit the beneficial effects of these drugs. Future studies need to address these concerns and to focus on developing better strategies in reactivating specific genes. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CONCLUSIONS DNA methylation is critical for the permanent, heritable silencing of gene transcription in mammals. Exciting new observations point to the importance of multiple layers of epigenetic modifications in the control of chromatin structure and gene expression. The complexities of the silencing process are just beginning to be elucidated in relationship to other epigenetic mechanisms. The complex interplay between these various epigenetic mechanisms will need to be elucidated. With all the recent discoveries related to the interactions between DNMT with various proteins or protein complexes, the roles of the methylation machinery in transcriptional regulation, chromatin structure, DNA repair, and genome stability will become a significant focus in the methylation field. By characterizing these interactions, the nature of the methylation defect in cancer cells is likely to be clarified, with the potential of finding novel therapies to reverse aberrant methylation patterns and restore growth control in cancers. Investigation on the complex interaction between various epigenetic modulators can potentially shed new light on the fundamentals of various epigenetic processes, and the problems associated with their deregulation as they pertain to inherited hum an disorders and cancer. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OVERVIEW OF THESIS RESEARCH Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. The frequent silencing of tumor suppressor genes by altered cytosine methylation and chromatin structural changes makes this process an attractive target for epigenetic therapy. Unfortunately, the most well-known and widely used inhibitors of DNA methylation, such as 5-azacytidine (5-Aza-CR) and 5-aza-2/-deoxycytidine (5-Aza-CdR), are chemically unstable, rather toxic both in vitro and in vivo, and cannot be given orally. Furthermore, most demethylating agents affect both normal and cancer cells indiscriminately, and activated genes usually become resilenced after drug withdrawal. This thesis describes a detailed characterization of a novel cytidine analog, zebularine, as a demethylating agent that offers various beneficial properties over traditional demethylating agents. Chapter 2 describes investigations on the role of zebularine, a chemically stable cytidine analog that is stable in both acidic and neutral solutions, as a new demethylating agent in vitro and in vivo. The ability of zebularine to inhibit DNA methylation in Neurospora crassa and in mammalian cells, C3H 10T1/2 C18 (10T1/2) mouse embryo cells and T24 hum an bladder carcinoma cells, as well as human bladder carcinoma cells grown in BALB/c nu/nu mice, was 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. demonstrated. The work shows the ability of zebularine to inhibit DNA methylation, reactivate a previously silenced hph gene in Neurospora crassa, induce the myogenic phenotype in 10T1/2 cells (a phenomenon unique to DNA methylation inhibitors), reactivate the silenced pl6 gene, and demethylate the pl6 prom oter/5 ' region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. The results also demonstrate that treatments of nude mice with zebularine, via either oral or inixaperitoneal (i.p.) adminstrations, lead to the suppression of tumor growth and reduction of tumor sizes in some groups. In addition, the drug exhibits minimal cytotoxicity in T24 cells in vitro and in tumor-bearing mice as assessed by minimal weight changes. My results provide the first functional evidence that zebularine is a stable DNA demethylating agent and the only drug in its class able to reactivate an epigenetically silenced gene by oral administration. In Chapter 3 ,1 sought to develop a more effective method in potentiating the effects of zebularine by exploiting zebularine's stability and applying the drug continuously to cells. This chapter examines the effectiveness of continuous zebularine treatment in sustaining DNA demethylation and preventing remethylation. The global effects on demethylation and the specific effects on DNA methyltransferase (DNMT) levels were also examined using this method. The work establishes strong evidence on the efficacy of this method in sustaining DNA demethylation, globally demethylating methylated 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regions and selectively depleting DNMT1. Furthermore, the chapter presents data showing that sequential treatment with 5-aza-2'-deoxycytidine (5-Aza- CdR) followed by establish zebularine can hinder the remethylation rate of p l6 5' region and gene re-silencing, suggesting the possible combination use of both drugs as a potential anticancer regimen. Chapter 4 describes the analysis on the differential effects of zebularine in normal hum an fibroblasts and hum an cancer cells. The effects of continuous zebularine treatment on cell growth, p i 6 gene activation, methylation of various methylated loci and DNMT levels were investigated in a panel of 3 normal fibroblasts (LD98 and LD419 bladder fibroblasts and T-l skin fibroblasts) and 5 cancer cells (CALU-1 lung cancer, PCS prostate cancer, CFPAC-1 pancreatic cancer, T24 bladder cancer and HCT15 colon cancer). The results show that zebularine selectively suppresses the cell growth in all cancer cell lines but not normal fibroblasts. In addition, zebularine demethylates various methylated genes and depletes DNMT1 in 3 out of 5 cancer cell lines but not normal fibroblasts. Moreover, the effects of zebularine on global gene expression profile in the T24 bladder cancer cells and LD419 normal fibroblasts were analyzed using microarray gene chip analysis. The work shows that zebularine strongly alters gene expression in T24 cells by activating tumor suppressor and cancer / testis-specific antigen genes but induces few changes in LD419 normal 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fibroblasts. The chapter demonstrates that zebularine can be selective towards cancer cells and may hold clinical promise as an anticancer therapy. The studies in this thesis have important biological and clinical implications because they divulge invaluable information about the potential utilization of a novel DNA methylation inhibitor which is shown to be stable, minimally toxic, effective in vitro and in vivo, and selective towards cancer cells. They also provide insight into the mechanism and biochemical aspects of DNA methylation, cellular response to demethylating agents and potential combinatorial treatments with other drugs. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CH A PTER 2 IN H IB IT IO N OF DNA METHYLATION AND REACTIVATION OF SILENCED GENES BY ZEBULARINE INTRODUCTION Aberrant de novo DNA methylation is commonly associated with cancer (Jones and Baylin 2002) and several studies have shown that de novo methylation of CpG islands in regulatory sequences of tumor suppressor genes can result in their silencing (Herman et al. 1995, Issa et al. 1994, Ottaviano et al. 1994). Thus DNA methylation may lead to abnormal growth of cancer cells. Examples of regulatory genes that are commonly hypermethylated in cancer cells include the RBI gene in retinoblastomas (Greger et al. 1989, Sakai et al. 1991), the VHL gene in sporadic renal cell carcinomas (Herman et al. 1994), the H19 gene in Wilms' tumors (Zhang et al. 1993), the pl5 gene in leukemias (Herman et al. 1996b), and the pl6 gene in hum an tumor cell lines (Gonzalez- Zulueta et al. 1995, Herman et al. 1996b). Given the high frequency of hypermethylation of genes critical to the control of cell proliferation (Herman et al. 1995, Zingg and Jones 1997), pharmacological use of DNA methylation inhibitors should be considered to reactivate anti-proliferative, apoptotic, and differentiation-inducing genes in cancer cells. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The most well-characterized and widely used drugs to inhibit DNA cytosine methylation and reactivate silenced genes are nucleoside analogs, such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR) (Harris 1982, Jones and Taylor 1980), and non-nucleoside drugs, such as procainamide (Lin et al. 2001, Scheinbart et al. 1991). Both 5-Aza-CR and 5-Aza-CdR have nitrogens in place of carbons at position 5 of the pyrimidine ring (Figure 2.1). These nucleoside analogs were originally developed as ■ cancer chemotherapeutic agents (Vesely and Cihak 1975) and are powerful inducers of genes silenced by DNA methylation (Jones 1985). Although 5-Aza-CR and 5- Aza-CdR are both being tested in international clinical trials, especially for the treatment of acute myeloid leukemia and myelodysplastic syndrome (Lubbert 2000, Silverman et al. 1993, Wijermans et al. 2000), their instability in neutral solutions has complicated their clinical use. With a short half-life of about 90 min at 50°C in phosphate-buffered saline (PBS) at pH 7.4 (Constantinides et al. 1977), 5-Aza-CR is well-known for its instability in neutral aqueous solutions, and the hydrolysis products have been well characterized (Beisler 1978). This chemical instability encouraged the development of other analogs such as 5,6- dihydro-5-azacytidine (1976) and pseudoisocytidine (Chou et al. 1979), which possessed more stable ring systems but have not been clinically useful. 5- Fluoro-2'-deoxycytidine also inhibited DNA methylation and reactivated silenced genes when incorporated into DNA (Jones and Taylor 1980), but 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. generated 5-fluorodeoxyuridine and its metabolites, which may result in toxicity (Boothman et al. 1989). Thus, there is a need for an effective, stable, and minimally toxic inhibitor of DNA methylation. In the search for new DNA methylation inhibitors, other collaborators and I became interested in a cytidine deaminase inhibitor, zebularine. Zebularine is a cytidine analog containing a 2-(lH)-pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor because it lacks an amino group at position 4 of the pyrimidine ring (Figure 2.1) (Kim et al. 1986). In addition, unlike aza nucleosides, zebularine is stable in aqueous solution up to a pH of 12 (Barchi et al. 1992, Kelley et al. 1986). In vitro experiments have shown that synthetic oligonucleotides containing zebularine form tight complexes with bacterial methyltransferases (Hurd et al. 1999), leading to a potent inhibition of DNA methylation. To assess the ability of zebularine to inhibit DNA methylation, the ability of zebularine to reactivate an antibiotic gene (hph) silenced by DNA methylation in the filamentous fungus Neurospora crassa was assessed (Irelan and Selker 1997, Selker 1998). I then tested whether zebularine is an effective inhibitor of mammalian DNA methylation by using the mouse embryo (10T1/2) and hum an bladder carcinoma (T24) cell lines. Finally, I examined the effects of zebularine of tumor growth in vivo, and its ability to reactivate a silenced p!6 gene in hum an bladder carcinoma cells grown in BALB/c nu/nu mice. 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f the copyright owner. Further reproduction prohibited without permission. NH. Ribose Ribose Ribose Cytidine Fig. 2.1: Structures of cytidine and its analogs, 5-azacytidine and zebularine. 5-azacytidine contains a nitrogen in position 5 and zebularine contains a 2-( 1 H)-pyrimidinone ring. Both ring systems have been shown to form covalent bonds with DNA methyltransferase enzymes after incorporation into DNA. U l MATERIALS AND METHODS Neurospora cultures N. crassa strains N644 (mat A am132 [{am/hph/am)ec^ - PP2jRIP77 N242 (al-2 matA) and N613 (al-2 matA; dim-2) were grown in liquid or solidified media using standard procedures, as previously described (Selker 1998). Approximately 2000 corddia (asexual spores) of the indicated strains, grown on minimal medium supplemented with alanine, were used for each plate. Drugs (trichostatin A [ISA], 5-Aza-CR, and zebularine) in varying concentrations were administered from a 4-mm diameter no. 1 paper disk (Whatman International Ltd., Maidstone, U.K.) placed in the middle of each plate shortly after plating the conidia. To test for resistance to hygromycin, 5 mg hygromycin B (Calbiochem, San Diego, CA) was added in 5 mL solidified medium after the plate was incubated 24 hours at 32°C. The plates were then incubated for 2 days at 32°C and photographed. To test the effect of TSA, 5-Aza-CR, or zebularine on DNA methylation, DNA was isolated from cultures grown to saturation (2-3 days) in the continuous presence of the drugs and processed as described (Selker 1998). TSA (Wako Chemicals USA Inc., Richmond, VA) was dissolved in dimethyl sulfoxide (DMSO), whereas 5-Aza-CR (Sigma Chemical Co., St. Louis, MO) and zebularine, synthesized as previously described (Barchi et al. 1995), 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were both dissolved in water. This experiment was performed by Cindy Matsen and Dr. Eric Selker (University of Oregon, OR). Mammalian, cell lines and drug treatments All cultures were grown in a humidified incubator at 37°C in 5% COr Stock cultures of the mouse embryonic cell line C3H 10T1/2 Cl 8 (10T1/2) (American Type Culture Collection, Manassas, VA) between passages 7 and 15 were grown in 75 cm2 plastic flasks (BD Biosciences Discovery Labware, Billerica, MA) in Eagle's basal media supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U /m L penicillin and 100 pg/m L streptomycin (Gibco/Life Technologies, Inc., Palo Alto, CA). The hum an bladder carcinoma cell line T24 was obtained from the American Type Culture Collection (Manassas, VA) and EJ6 bladder carcinoma cell line was provided by Dr. Eric J. Stanbridge (University of California at Irvine, CA). Both cell lines were cultured in DMEM supplemented with 10% heat-inactivated FCS, 100 U /m L penicillin and 100 pg/m L streptomycin. To observe the myogenic phenotype, we plated 10T1/2 cells (2500 cells/60-mm dish) and treated the cells 24 hours later with the indicated concentration of either 5-Aza-CdR, 5-Aza-CR, or zebularine. Controls are not treated with any drugs. The medium was changed 24 hours after the initial 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. drug treatment and every 3 days thereafter until the myogenic phenotype is observed, which is approximately 9 to 10 days after initial drug treatment. For methylation analysis, 10T1 /2 cells and T24 cells were plated (3 x 10s cells/100-mm dish) and were treated 24 hours later with the indicated concentration of either 5-Aza-CdR, 5-Aza-CR, or zebularine. Controls are not treated with any drugs. For 1011/2 cells, the medium was changed 24 hours after the initial drug treatment, whereas for 124 cells, the medium was changed either 24 hours or 48 hours after the initial drug treatment as indicated. DNA and RNA were harvested from 1011/2 cells 72 hours after initial drug treatment and from 124 cells 96 hours after initial drug treatment. Two separate and independent experiments were done, both in duplicates. DNA and RNA isolation DNA was isolated from Neurospora as previously described (Foss et al. 1993), and DNA and RNA from cultured cells and tumor cells were isolated using the NucleoBond RNA/DNA Midi Kit (Clontech Laboratories, Inc., Palo Alto, CA) according to the manufacturer's recommended protocol. Southern blot analysis Liquid cultures of N. crassa strain N644 inoculated at a concentration of 7 x 104 conidia/mL were grown in the absence or presence of ISA (0.33 and 3.3 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. pM), 5-Aza-CR (12 and 24 pM), or zebularine (20-310 pM) for 4 days at 32°C with shaking. Because of the instability of 5-Aza-CR, the indicated doses of this drug were added at time zero and after 1 and 2 days of growth. DNA was isolated and Southern hybridizations were performed on 1 fig DNA samples that were digested with Dpnll or SauSAl, fractionated by gel electrophoresis, transferred to a nylon membrane and probed for am™’ or ¥63 sequences. A control hybridization indicated that the digests were complete (not shown). This experiment was performed by Cindy B. Matsen and Dr. Eric Selker (University of Oregon, OR). Reverse transcription-polymerase chain reaction (RT-PCR) analysis The protocol for RT-PCR analysis to detect p l6 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) mRNA expression, including the primer sequences, probe sequences, and PCR conditions has been previously described (Gonzalez-Zulueta et al. 1995). Quantification of DNA m ethylation b y Ms-SNuPE analysis The average methylation at defined CpG sites was quantified using the Ms-SNuPE assay as described (Gonzalgo and Jones 1997). Briefly, genomic DNA (4 pg) was digested overnight with 4 units of EcoRI or with 4 units of Rsal to cut outside the pi 6 prom oter/ 5' region or the endogenous retroviral 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequence Class Il-d, respectively. After digestion, the DNA was denatured for 20 minutes at 95°C and then treated with 3 M NaOH for 20 minutes at 45°C This solution was then treated 3.6 M sodium bisulfite and 0.1 M hydroquinone for 16 hours at 55°C in the dark. Treatment of DNA with bisulfite converts unmethylated cytosine residues to uracil, which is then converted to thymine after the primary bisulfite-specific polymerase chain reaction (PCR), leaving methylated cytosines unchanged. Bisulfite-converted DNA was purified with the Wizard Plus Minipreps DNA Purification System (Promega Corp., Madison, WI), desulfonated by the addition of 3 M NaOH for 15 minutes at 40°C, and ethanol precipitated. The sequences of the primers used for bisulfite-treated DNA PCR amplification are summarized in Table 2.1. The PCR products (templates) were separated by electrophoresis through a 2% agarose gel and purified from the gel using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). Each template was eluted in 30 /xL H2 0. Duplicates of 4 |xL of template were then added to 6 fiL of a reaction mixture consisting of IX PCR buffer, 1 fiM SNuPE primers, 1 fiCi [3 2 P]dCTP or dTTP, and 1 unit 1:1 Taq/TaqStart antibody (Clontech, Palo Alto, CA). The sequences for SNuPE primers and conditions are summarized in Table 2.1. The reaction mixtures were combined with 4 \\L of stop solution [95% formamide, 20 mM EDTA (pH 8.0), 0.05% bromophenol blue, and 0.05% xylene cyanol] before being denatured at 95°C for 5 min and loaded onto a 15% 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2.1 Conditions and primer sequences for bisulfite PCR and Ms-SNuPE Bisulfite PCR Region Conditions Nucleotide Sequence Class Il-d (P3) 95° 2 mirt 95° 1 m in 50° 50 sec »4q x 72° 1 m in 72° 10 m in 5'-GTTTATAGGTTTAGAGG'i'iT 1-3' (sense) 5'-AACACATAAACCTATTTTAAACTTA-3' (antisense) p l6 Promoter! 5' Region 94° 3 m in 94° 45 sec 67° 45 sec MOX 72° 45 sec J 72° 10 m in 5'-GTAGGTGGGGAGGAGTTTAGTT-3' (sense) 5'-TCTAATAACCAACCAACCCCTCCT-3' (antisense) Ms-SNuPE Class Il-d (P3) 95°C 1 m in 46°C 30 sec 72°C 20 sec 5'-GGTATAGTTTGAGTAT-3' 5'-TTTT ATTT ATTGTT ATT ATGG-3' 5'-TATTTTTTAATAGTATTATTTTTTAT-3' plS PromoterI 5 * Region 95°C 1 m in 50°C 2 m in 72°C 1 m in 5'-TTTGAGGGATAGGGT-3' 5’ -TTTTAGGGGTGTTATATT-3' 5'-TTTTTTTGTTTGG A A AG AT AT -3' 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. denaturing polyacrylamide gel (IX TBE buffer, 14.25% acrylamide, 0.75% bis- acrylamide, 7 M urea). Methylation levels were quantified on a Phosphoimager analysis system (Molecular Dynamics, Sunnyvale, CA). The reported methylation status of the pi 6 prom oter/ 5" region and Class Il-d (P3) represents the averaged methylation levels of three CpG sites from duplicates samples from two independent experiments. Methylation levels, defined as the percent of DNA that is methylated, were measured by Ms-SNuPE as described previously (Gonzalgo and Jones 1997). In vivo experiments EJ6 cells (5 x lQ5 /injection) suspended in PBS were inoculated subcutaneously into the right and left flanks (along the midaxillary lines) of 4- 6-week old male BALB/c nu/nu mice (Harlan, San Diego, CA). Mice (n = 30) were randomly divided into six groups (intraperitoneal control group, oral control group, intraperitoneal zebularine at 500 m g/kg, oral zebularine at 500 m g/kg, intraperitoneal zebularine at 1000 m g/kg, and oral zebularine at 1000 m g/kg). Each group consisted of five mice (at least 6 tumors per group; one or two mice per group were randomly killed at earlier time points to determine the length of the experiment). After approximately 2-3 weeks and once macroscopic tumors (50-200 mm3 ) had formed, zebularine or control treatments were initiated. Zebularine, at doses of 500 m g/kg or 1000 m g/kg, was 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. administered daily by intraperitoneal injection in a solution of 0.45% saline or oral gavage feeding over a period of 18 days. Two control groups were used, one mock-treated with 0.45% saline administered by intraperitoneal injection, and the other group mock-treated with 0.45% saline administered by oral gavage feeding over the same period of 18 days. Tumors were measured with calipers at the indicated days and tumor volumes (TV) were calculated using the following formula: tumor volume = LDz/2 (where L is the longest diameter and D is the shortest diameter). The fold differences in tumor growth between the various mice groups, were calculated as the relative tumor volume (RTV) as follows: RTV = TVn/TV0 , where TVn is the tumor volume in mm3 at a given day n and TVo is the tumor volume in mm3 at day 0 (initial treatment). All other mice were killed 24 hours after the last treatment. Tumors were removed and divided into two separate portions. One portion of each tumor was immediately fixed with neutral-buffered formalin, embedded in OCT compound, frozened and then sectioned. The frozen sections were stained with hematoxylin and eosin (H&E). All histological examinations were carried out by light microscopy using a Leica DM LB microscope (Leica Microsystems, Inc., Bannockburn, IL). From the other portion of each tumor, DNA and total RNA were isolated to analyze the methylation status of pl6 prom oter/ 5' region by Ms-SNuPE and gene expression by RT-PCR analyses, respectively. All experimental protocols were approved by the Institutional 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Animal Care and Use Committee, in compliance with the Guide for the Care and Use of Laboratory Animals, University of Southern California. Determ ination of cytotoxicity Cytotoxicity was assessed using a colony formation assay. Both 10T1/2 (250 cells /60-mm dish) and T24 cells (100 cells /60-mm dish) were plated in triplicates and treated after 24 hours later with the indicated concentration of either 5-Aza-CdR, 5-Aza-CR, or zebularine. Control plates are not treated with any drugs. After cell colonies were visible (approximately after 12-14 days), cells were fixed in 100% methanol and stained with 10% Giemsa stain. The total number of colonies was counted and the percentage of average plating efficiency was determined by dividing the mean colony number on the treated plates or the control plates by the number of cells seeded in the dishes X 100. In this cytotoxicity assay, a decrease in the size of the colonies is an indication of growth inhibition, whereas a decrease in the number of the colonies is an indication of cytotoxicity. Two separate and independent experiments were done. Statistical analysis For 10T1/2 cells and T24 cells, analysis of variances (ANOVA) were performed to test differences in DNA methylation and average plating 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. efficiency among the treatment groups, using the experiment-to-experiment variability as the error term. The least significant difference (LSD) m ethod was used for the pair-wise comparisons, once the overall F-test was significant at the 0.05-level (Snedecor and Cochran 1973). The p-values from the overall F-test of the treatment effect were 0.001 for DNA methylation and <0.001 for average plating efficiency in 10T1/2 cells (Table 2.2) and <0.0001 for both DNA methylation and average plating efficiency in T24 cells. Pearson correlation coefficient was calculated to test the association between pi 6 promoter methylation status and pi 6 / GAPDH ratio for T24 cells. For the in vivo experiment, tumor volumes of mice were determined for two tumors from each mouse. Prior to analysis, the logarithms of the ratios of tumor volumes at day 18, day 14, day 8, and day 4 divided by the tumor volume at day 0 were taken. For each mouse, the rate of the change in log ratio with day (slope) was estimated using a linear regression model with tumor and day as the covariates. A lack of fit test and visual inspection of the data indicated that the linear pattern fit remarkably well for animals receiving the control and 1000 m g/kg zebularine, and reasonably well for the mice receiving 500 m g/kg zebularine. Then, ANOVA was done using the calculated slope as the dependant variable to investigate the effects of route of administration and dose of zebularine and their interaction on the daily change in (log) ratio of tumor volume (relative to day 0). Pair-wise comparison of the slopes was 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. performed using the LSD method, once the overall F-test was significant at the 0.05 level (Snedecor and Cochran 1973) The effects of dose, route of administration and their interaction on weight of mice were investigated by ANOVA on the ratio between the weight at day 18 and the baseline weight at day 0. The LSD method was used for multiple comparisons, once the overall F-test was significant at the 0.05 level (Snedecor and Cochran 1973). The ratio of pl6/G A P D H mRNA expression in EJ6 tumors in vivo was measured for each mouse in the six groups as described above. The methylation status of pl6 prom oter/ 5' region was measured only for control oral, control intraperitoneal, 1000 m g/kg oral and 1000 m g/kg intraperitoneal, for one tumor per mouse in two of the three mice in each group and two tumors in a third mouse. ANOVA was performed to test the effects of route of administration and dose of zebularine and their interaction on the pl6/G A P D H ratio and methylation status. In the analyses, a mouse was nested within the treatment group and for the methylation status a tumor was further nested within the mouse. The mouse-to-mouse variability was used as the error term. The LSD method was used for the pair-wise comparisons, once the overall F- test was significant at the 0.05-level (Snedecor and Cochran 1973). All reported p-values were two-sided. All confidence intervals were calculated using the pooled estimates of the experiment-to-experiment or 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mouse-to-mouse variability as calculated by the ANOVA. The SAS software package (SAS institute, Inc., Cary, NC) was used for all statistical analyses. 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Reactivation of a silenced hph gene in Neurospora To elucidate the mechanism of action for zebularine, the analysis was performed on an artificial locus in N. crassa, which includes a copy of the bacterial hygromycin resistance gene hph that had been silenced by DNA methylation as a result of the operation of repeat-induced point mutation on flanking duplicate copies of the Neurospora am gene (Irelan and Selker 1997). The strain (N644) bearing this construct was previously used to demonstrate that the histone deacetylase inhibitor TSA causes selective loss of DNA methylation in Neurospora (Selker 1998). In the present study, zebularine was applied to a paper disc in the middle of a plate containing approximately 2000 N644 conidia, and challenged the cells to grow in the presence of hygromycin. A dilution series showed that as little as 2.5 nmoles of zebularine applied to the center of the plate were sufficient to permit growth of nearby cells, while 20 nmoles of zebularine permitted growth that was comparable to that observed in the presence of 5-Aza-CR (Figure 2.2). Some inhibition of growth near the site of application was observed at higher doses of zebularine (Figure 2.2). This observation suggested that zebularine, like the known demethylating agent 5- Aza-CR, caused reactivation of the silenced hph gene. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.2: Reactivation of the silenced hygromycin resistance (hph) gene in Neurospora. N. crassa strain N644, which has a single copy of the E. coli hph gene that was silenced by cytosine methylation (Irelan and Selker 1997), was plated on agar plates, treated with various quantities of zebularine applied to the central paper disc on each plate, and challenged with hygromycin 24 hours later. The active hph gene confers hygromycin resistance. The demethylating drug 5-azacytidine (5-Aza-CR) was used as a positive control. With the exception of the "no hygromycin" plate, all plates had a top agar layer containing hygromycin. Representative data are shown. This experiment was performed by Cindy B. Matsen and Dr. Eric Selker (University of Oregon, OR). 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Inhibition of DNA methylation in Neurospora To verify that reactivation of hph by zebularine resulted from an inhibition of DNA methylation, methylation-sensitive restriction endonucleases and Southern hybridization was used to assess methylation in representative methylated chromosomal regions as well as in the overall genome. Liquid cultures of strain N644 were grown in the presence or absence of zebularine, TSA and 5-Aza-CR. Methylation was then examined in the ¥63 region, which was known to be affected by 5-Aza-CR but not TSA, and in the am sequences flanking hph, which were known to be affected by both drugs (Selker 1998). Digests with DpnU and Sau3Al, which both recognize GATC but differ in that only Sau3Al is inhibited by cytosine methylation, revealed that zebularine dramatically reduced methylation in both amw p and ¥63 regions (Figure 2.3). Substantial loss of methylation was observed with the lowest concentration tested (20 pM) and cultures grown with 78-310 pM zebularine showed virtually no DNA methylation. This suggested that zebularine was acting as a general methylation inhibitor, similar to 5-Aza-CR, and was supported by inspection of the total genomic DNA digested with DpnU or Sau3Al and stained with etfridium bromide. The spectra of fragments produced by DpnU or Sau3Al digests appeared equivalent with DNA samples from cultures grown in the presence of either 5-Aza-CR or zebularine, in contrast with DNA from cultures grown in the presence of TSA or in the absence of drugs (data not shown). 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.3: Zebularine inhibits D NA methylation in Neurospora crassa. Genomic DNA of N. crassa strain N644 grown in the continuous presence or absence of 5- azacytidine (5-Aza-CR), trichostatin A (TSA,) or zebularine was digested with Dpn II (D) or SauSAl (S) and probed for am® (left panel) or ¥63 (right panel) sequences, which are normally methylated, by Southern blot analysis. The ramp symbols represent increasing concentrations of drug: 12 and 24 gM 5-Aza-CR, 0.33 and 3.3 ptM TSA and 20, 39, 78,160 and 310 pM zebularine. Because of the instability of 5-Aza-CR, cultures treated with this drug were given booster doses at the same concentrations after one and two days of growth. The positions of selected size standards (kb) are indicated. This experiment was performed by Cindy B. Matsen and Dr. Eric Selker (University of Oregon, OR). 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. j c Jsd O 1 0 CO CM Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thus, zebularine is a global inhibitor of DNA methylation, similar to 5-Aza-CR, rather than a selective inhibitor, such as TSA, an inhibitor of histone deacetylase (Selker 1998, Selker and Stevens 1985). Induction of the myogenic phenotype and inhibition of DNA methylation in 10T1/2 mouse embryo cells I next determined whether zebularine could inhibit DNA methylation in mammalian cells. Cytidine analogs with modifications in the 5 position of the ring are powerful inhibitors of DNA methylation and can induce the formation of striated muscles cells in the non-myogenic 10T1/2 mouse embryo cell line (Constantinides et al. 1977, Jones and Taylor 1980). I tested the ability of zebularine to induce 10T1/2 cells to undergo the myogenic switch (Figure 2.4). Control 10T1/2 cells formed flat even monolayers and appeared epithelioid (Figure 2.4, A), whereas 10T1/2 cells treated with 5-Aza-CR (Figure 2.4, B) or with 5-Aza-CdR (Figure 2.4, C) formed multinucleated myotubes approximately 9-10 days after initial drug treatment. I found that zebularine also induced muscle formation in 10T1/2 cells (Figure 2.4, D). The extent of muscle formation in cultures treated with zebularine was less than that induced by either 5-Aza-CdR or 5-Aza-CR (Table 2.2). The muscle phenotype has only been reported to be induced by inhibitors of cytosine methylation (Constantinides et al. 1977, Constantinides et 74 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f the copyright owner. Further reproduction prohibited without permission. Figure 2.4: Myotube formation in murine embryonic fibroblast C3H 10T1/2 Cl 8 (10T1/2) cells after treatment with cytidine and deoxycytidine analogs. A-D) Phase-contrast micrographs (x 400) of 10T1/2 cells that were not treated with any drugs (A), treated with 0.3 pM 5-aza-2'- deoxycytidine (B), treated with 3 pM 5-azacytidine (C), or treated with 30 pM zebularine (D). Cells were treated for only 24 hours. Photographs were taken 9-10 days after the treatments began. Notice the formation of multinucleated myotubes in panels B-D. Reproduced w ith permission o f the copyright owner. Further reproduction prohibited without permission. Table 2.2 Inhibition of DNA methylation and induction of myotubes in 10T1/2 cells Treatment Concentration (pM) Muscle Formation Methylation (%) Average Plating Efficiency (%) Control 0 0 86.3 (79.5-93.0) 22.3 (19.7-24.8) 5-Aza-CdR 0.3 +++ 54.3 (47.5-61.0) 5.0 (2.4-7.6) 5-Aza-CR 1 ++ 64.8(58.0-71.5) 21.3 (18.7-24.8) 3 +++ 59.0 (52.3-65.7) 15.5 (12.9-18.1) Zebularine 10 + 74.8 (68.0-81.5) 20.3 (17.7-22.8) 30 ++ 58.5 (51.8-65.2) 18.3 (15.7-20.8) 10T1/2 cells were treated with the indicated cytidine and deoxycytidine analogs for 24 hours and scored for the presence of muscle cells 9-10 days later as + to +++, with + representing minimal muscle formation and +++ representing maximal muscle formation. To determine the inhibition of DNA methylation, Class Il-d locus was assayed for the level of methylation using methylation-sensitive single nucleotide primer extension (Ms-SNuPE) analysis. For each treatment, two separate and independent experiments were done starting from cell treatment. For each experiment, the samples were ran in duplicates. Results are represented as the mean (95% confidence intervals) and are obtained from duplicates of two experiments. Cell killing was determined by the lowering of plating efficiency in similarly treated cultures containing 250 cells and stained with Giemsa stain after 12-14 days. Results are represented the mean (95% confidence intervals) and are obtained for triplicate dishes in two separate experiments. O n al. 1978), suggesting that zebularine inhibited DNA methylation in 10T1/2 cells. I tested this possibility directly by using the Ms-SNuPE method (Gonzalgo and Jones 1997). Zebularine, like 5-Aza-CR and 5-Aza-CdR, did indeed inhibit the methylation of an endogenous retroviral sequence (Class Il-d) (Liang et al. 2001) in 10X1/2 cells in a dose-dependent manner (Table 2.2). Treatment with 0.3 pM 5-Aza-CdR or 3 pM 5-Aza-CR reduced methylation of the Class Il-d sequence from the control level of 86.3% to 54.3% (difference = 32%, 95% Cl of the difference = 22.5% to 41.5%, Pc.001) and 59.0% (difference = 27.3%, 95% Cl of the difference = 17.7%-36.8%, Pc.001), respectively. Zebularine (30 pM) also reduced methylation of the Class Il-d sequence to 58.5% (difference = 27.8%, 95% Cl of the difference = 18.2% to 37.3%, Pc.001). Two factors that limit the clinical potential of 5-Aza-CdR are its instability and its toxicity. I therefore compared the cytotoxicity of zebularine, 5-Aza-CdR, and 5-Aza-CR by measuring their effects on 10T1/2 cell plating efficiency (Table 2.2). A dose of 0.3 pM 5-Aza-CdR reduced plating efficiency from a control level of 22.3% (95% Cl = 19.7% to 24.8%) to 5.0% (95% Cl = 2.4% to 7.6%, Pc.001) and was considerably more cytotoxic than either 3 pM 5-Aza- CR (plating efficiency = 15.5%, 95% Cl = 12.9% to 18.1%, Pc.001) or 30 pM zebularine (plating efficiency = 18.3%, 95% Cl = 15.7 to 20.8%, Pc.001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Induction of p l6 gene expression and inhibition of DNA m ethylation in T24 hum an bladder carcinoma cells I next tested whether zebularine can induce expression of a hum an tumor suppressor gene that had been silenced by methylation. The T24 hum an bladder carcinoma-derived cell line contains a transcriptionally silent hypermethylated p i6 gene prom oter/ 5' region (Bender et al. 1998, Gonzalez- Zulueta et al. 1995) that can be demethylated and reactivated by 5-Aza-CdR, a deoxyribonucleoside analog (Bender et al. 1998, Gonzalgo et al. 1998). I examined whether 5-Aza-CR and zebularine, which are ribonucleoside analogs, could also induce pl6 expression in T24 cells (Figure 2.5). Untreated control cells showed no pl6 expression, whereas cells treated with 3 pM 5-Aza-CdR showed robust expression of pl6. Both 5-Aza-CR and zebularine successfully induced p i 6 expression in a dose-dependent manner, although not as effectively as 5-Aza-CdR. It is interesting to note that zebularine showed time- dependent induction of pl6 expression, in that treatment with 300 pM zebularine for 24 hours was insufficient to induce expression but treatment with 300 pM zebularine for 48 hours was sufficient to induce pl6 gene expression. 5-Aza-CR, 5-Aza-CdR, and zebularine also induced similar levels of pl6 protein, as confirmed by Western blot analysis (data not shown). Thus, my findings suggest that zebularine inhibited DNA methylation in T24 cells. 78 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. I directly investigated the effectiveness of zebularine and 5-Aza-CR in demethylating the promoter CpG island/ 5' region of the pl6 gene (Figure 2.6). 5-Aza-CdR (3 pM) decreased methylation of the pl6 prom oter/ 5' region from a control level of 95.8% to 51.5% (difference = 44.3%, 95% Cl of difference= 40.7% to 47.8%, Pc.001). Likewise, increasing concentrations of 5-Aza-CR also decreased methylation to 89.5 % (3 pM 5-Aza-CR, difference=6.3%, 95% Cl of difference=2.7% to 9.8%) and further to 67.8% (100 pM 5-Aza-CR, difference = 28%, 95% Cl of difference = 24.5% to 31.5%, Pc.001). Treatment with zebularine decreased methylation in a dose- and time-dependent manner, with the most effective treatment (1 mM zebularine for 48 h) reducing methylation to 70.3% (difference = 25.5%, 95% Cl of difference = 21.9% to 29.0%, Pc.001). The methylation status of the p l6 prom oter/ 5' region has a statistically significant negative association with the pl6/G A P D H ratio, with a Pearson correlation coefficient of -0.81 (P = .001) (Figures 2.5,2.6). The cytotoxicity of 5-Aza-CdR, 5-Aza-CR, and zebularine in T24 cells was assessed by measuring the average plating efficiency. A dose of 3 pM 5- Aza-CdR decreased plating efficiency by 55.7% in average (95% Cl = 51.5% to 59.8%). 5-Aza-CR decreased plating efficiency in a dose-dependent manner, with 100 pM producing the highest cytotoxicity (39.7% decrease, 95% Cl = 35.5% to 43.8%). Interestingly, the increasing cytotoxicity of zebularine associated with increasing dose is greater than what is observed with the 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. U H < Z D « 5-Aza-CR Zebularine r _ ~ n i _ _ _ _ _ | Concentration (jrM) — — 3 3 10 30 100 300 500 1000 Treatment (h) — — 24 24 24 24 24 ^24 48^ 24 48 24 48 GAPDH Figure 2.5: In vitro effects of cytidine and deoxycytine analogs in T24 human bladder carcinoma cells. RT-PCR analysis was performed on the total cellular RNA (5 fig) isolated from T24 cells that were treated with the indicated drugs as shown in the figure. Expression of p!6 was analyzed by RT-PCR with RNA isolated 96 h after drug treatment. GAPDH expression served as a control for the input cDNA. Untreated T24 cells and no cDNA samples were analyzed similarly as negative controls. O O o Figure 2.6: Inhibition of methylation o fp l6 promoter/ 5' region in T24 human bladder carcinoma cells after treatment with cytidine and deoxycytidine analogs. T24 cells treated with the indicated drugs are evaluated for their inhibition of DNA methylation and cytotoxicity. Top, partial map of 5' region of the p!6 gene. Tick marks below the line, CpG sites; bent arrow, transcription start site; arrows, CpG sites analyzed by Ms-SNuPE analysis. The methylation status of the p i 6 prom oter/ 5' region, defined as the percent of DNA that is methylated, was analyzed 96 hours after the indicated drug treatments by using methylation- sensitive single nucleotide primer extension (Ms-SNuPE) analysis as described previously (Gonzalgo and Jones 1997). Bars represent the means of duplicates from two separate experiments performed. Cytotoxicity was determined by measuring the average plating efficiency in cultures initially plated with 100 cells. Bars represent the means of triplicate dishes from three separate experiments. All error bars are the 95% confidence intervals. Solid bars = p i6 prom oter/ 5' region methylation status (%); open bars = average plating efficiency (%). 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. p16/CDKN2A 5s Region * i i i i i i 'iii i»' i' i r B p i6 Promoter/ 5’ Region Methylation Status (%) □ Average Plating Efficiency (%) 24 3 o u £ a U 8 w u N m 24 3 24 10 24 24 | 24 48 | | 24 48 | | 24 48 | Treatment (h) 30 100 ■ I 300 500 1000 i Concentration (pM) 5-Aza-CdR Zebularine DO to increasing cytotoxicity associated with increasing time of drug treatment. The most cytotoxic treatment regimen observed in T24 cells was 1 mM zebularine for 48 hours, which resulted in a decrease in plating efficiency of 17.0% (95% CI=12,8% to 21.2%). Zebularine was clearly less toxic than 5-Aza-CR in T24 cells at concentrations which induced similar levels of p i6 prom oter/ 5' region demethylation (Figure 2.6). Both agents decreased methylation by an approximately 27%~29% at concentrations of 100 pM 5-Aza-CR and 1 mM zebularine for 48 hours; however, the plating efficiency for cells treated with 5- Aza-CR was lower than that of zebularine (34.3%, 95% Cl = 31.4% to 37.3% versus 57.0%, 95% Cl = 54.1% to 59.9%, respectively, Pc.001). Thus, zebularine was minimally cytotoxic in T24 cells, even at high concentrations. Effects of zebularine on hum an bladder carcinoma cells in vivo Because zebularine is an inhibitor of DNA methylation in Neurospora and cultured animal cells, I next examined the ability of the drug to reactivate silenced mammalian genes in vivo. I used a tumorigenic derivative of T24 cells, EJ6 cells, which also have a methylated p i6 gene prom oter/ 5' region. EJ6 bladder cells were inoculated subcutaneously into the right and left flanks of male BALB/c nu/nu mice at 4-6 weeks of age. When macroscopic tumors were evident (approximately 2-3 weeks later), the mice were treated with zebularine at concentrations of 500 m g/kg or 1000 m g/kg, administered either by 83 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. intraperitoneal injection or oral gavage feeding. I decided to examine oral gavage feeding as a possible route of administration, because zebularine has a half-life of about 44 hours at 37°C in PBS at pH 1.0 and about 508 hours at pH 7.4 (data not shown). Frozen sections of resected tumors from control mice and mice treated with zebularine were stained with hematoxylin and eosin to observe for any morphologic changes. Tumors from control mice had a high ratio of tum or cells to stroma, whereas mice treated with either 1000 m g/kg orally or 1000 m g/kg intraperitoneally had a much lower ratio of tumor cells to stroma (Figure 2.7). Moreover, tumor growth appeared inhibited in all treated groups compared with tumor growth in the control group. Compared with the growth of tumors from the control mice, tumor growth was statistically significantly reduced in the groups treated either with 1000 m g/kg orally (Pc.001) or intraperitoneally (Pc.001) (Figure 2.8). Weight loss was minimal in all control and treated groups. The average maximum weight loss observed in the group treated with 1000 m g/kg zebularine by intraperitoneal administration was approximately 11% (95% 0 = 4 % to 19%) (Figure 2.9), suggesting that zebularine was minimally toxic in these mice. In addition, no death was observed for any of the mice in all of the groups (data not shown). I also assessed whether p l6 was reactivated in the tumors. Compared with tumors from the control groups which did not express pl6, tumors from the four groups of treated mice showed that the dormant p l6 gene could be 84 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 1000 mg/kg oral 1000 mg/kg i.p. Control Figure 2.7: M icroscopic effects o f zebularine on human bladder carcinoma cells grown in BALB/c nu/nu mice. Tumor sections from control or treated mice groups were cut and analyzed by H&E staining. Images were representative of the whole field of tumor sections taken from each group (magnification, x 400). Notice a lower ratio of tumor cells to stroma in treated versus untreated tumor sections. DO Figure 2.8; Antitumor effects of zebularine on human bladder carcinoma cells grown in BALB/c nu/nu mice. Tumor volume was measured for all mice groups at the indicated time points, averaged, and plotted as the relative tumor volume versus days of treatment for each group. The relative tumor volume is defined as the tumor volume at any given time (TVn in mm3 ) divided by the tumor volume at the start of the treatment (TVo in mm3 ). Data represents the mean tumor volume (in RTV) ± 95% confidence intervals {error bars). Statistically significant decreases in tumor growth between the treated mice group and the control group are indicated by asterisks (* Pc.001). 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 40 -^ —Control — o— 500 m g/kg oral - -a ■ 1000 m g/kg oral — 500 m g/kg ip. ■ -m - 1000 m g/kg i.p. 3.0 2.0 1.0 " i L 0.0 Days of Treatment o o Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. — •— Control — a— 500 m g / kg oral - - a - 1000 mg/kg oral — 500 m g /k g ip , ■ -a - 1000 mg/kg ip . Days After Treatment Figure 2.9: In vivo effects on the weight of nude mice by zebularine. Each group of mice (n = 3) was weighed at the indicated time points and average body weight was plotted against days of treatment. Data represents the mean body weight ± 95% confidence intervals (error bars). GO O O effectively reactivated by the drug in vivo via both intraperitoneal and oral routes of administration (Figure 2.10). The highest level of p i 6 expression was observed in the group treated with 1000 m g/kg zebularine via intraperitoneal injection, then 1000 m g/kg via oral gavage feeding, then 500 m g/kg via intraperitoneal, and finally 500 m g/kg via oral gavage feeding (Figure 2.10). The pl6/G A P D H ratio, which reflects the actual p i6 expression levels after normalization with GAPDH, was statistically significantly affected by both route of administration (P = .027) and dose of zebularine (Pc.001). However, the interaction between route and dose was not statistically significant (P = .18); that is, the effect of dose was similar for both routes of administration. Finally, I assessed the methylation status of the pl6 prom oter/ 5' region and confirmed that the p l6 prom oter/ 5' region was statistically significantly hypomethylated (Pc.001) in the DMAs obtained from tumors of both representative treated groups relative to the methylation in the DNAs from the control group (Figure 2.11). Thus, zebularine delivered by both routes of administration was effective in demethylating the pl6 prom oter/ 5' region in a dose-dependent manner. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.10: In vivo effects on the reactivation of silenced p!6 gene expression by zebularine. After treating the BALB/c nu/nu mice for 18 days with zebularine, total RNA (5 fig) was isolated from EJ6 tumor cells obtained for all the mice (n =3 per group; 6 tumors) in each group. Expression of p!6 was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis using primers specific for hum an p i6 (top panel). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured to control for the quantity and integrity of the input RNA (top panel). The control group represents tumors obtained from mice mock-treated with either with 0.45% saline by intraperitoneal injection or oral gavage feeding. The data for both groups were combined to represent the control, because the results are exactly the same. Results from one of six similar independent tumors are shown from each group. The level of p i6 expression for each tumor in a group was normalized to the level of GAPDH using Phospholmager Quantitation and plotted as the p i6 to GAPDH ratio (bottom panel). The results represent the mean ratios for six tumors in each group ± 95% confidence intervals (error bars). 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. . Jf s m n o g o § rH i » 1 i 1 I O J IU 0 3 VNCP °M 1 A A vo ^ 'S . © o i 1 ---------- 1 ---------- i ---------- 1 — in en to evi w m <N rH I ■ I “1 --- m © o HQdVD/9ld- jo ofura 91 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 0 0 m g/kg 1 0 0 0 m g/kg 5 0 0 m g flcg 1 0 0 0 m^kg o ral oral i.p. i.p. Figure 2.11: In vivo effects on the inhibition of methylation of p i 6 promoter/ 5' region by zebularine. The methylation status of the p i6 prom oter/ 5' region in DNA isolated from EJ6 tumor cells from the representative groups was quantified by using methylation-sensitive single nucleotide primer extension (Ms-SNuPE) analysis (Gonzalgo and Jones 1997). Solid bar = control group; hatched bar = mice group treated with 1000 m g/kg zebularine by oral gavage feeding; open bar = mice group treated with 1000 m g/kg zebularine by intraperitoneal administration. The bars represent the average of three CpG sites from four independent tumors in each group performed in duplicates. Error bars are the 95% confidence intervals. Statistically significant decreases in methylation status of treated group versus the control group are indicated by asterisks (Pc.001). 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. \ © o © o m oo o o is © uoiSa^j ,£ /mornoid 9ld j i g a, o o o c 0 u 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION My study has established zebularine as an effective general inhibitor of DNA methylation. In N. crassa, zebularine functions as a global inhibitor of DNA methylation similar to 5-Aza-CR, and unlike TSA, which functions as a selective inhibitor of DNA methylation. Zebularine induced the expression of the myogenic phenotype in mouse embryonic fibroblast cells and inhibited the methylation of specific loci in both the mouse Class H-d and human pl6 prom oter/ 5' region. In Neurospora, cultured mammalian cells, and tumor cells in BALB/c nu/nu mice, zebularine was able to reactivate silenced genes, consistent with our findings that zebularine inhibits DNA methylation. The mechanism of action of zebularine as a DNA methylation inhibitor presumably requires incorporation into DNA after phosphorylation of zebularine to the diphosphate level and conversion to a deoxynucleotide (Figure 2.12). After the conversion to the deoxy-zebularine triphosphate and subsequent incorporation into DNA in place of a cytosine base, the 2-(lH)- pyrimidinone would most likely base pair with guanine, resulting in two Watson-Crick hydrogen bonds instead of three. It is unlikely that zebularine would be incorporated in place of thymine, because this would lead to a single Watson-Crick hydrogen bond, which is very weak and unstable. Replacing cytosine by 2-(lH)-pyrimidinone would result in the formation of a tight complex that can lead to potent inhibition of DNA methylation, as reported by 94 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. DNA RNA Ribonucleotide Reductase ZMP Deoxycytidine Kinase Uridine - Cytidine Kinase Phosphatase 2dZeb Zebularine Figure 2.12: Proposed metabolism o f zebularine and 2 ’ -deoxyzebularine (2dZeb). Zebularine is activated by uridine-cytidine kinase to the monophosphate level and finally after a series of enzymatic processes incorporated into both RNA and DNA. 2dZeb, however, is not recognized by deoxycytidine kinase, thereby preventing its incorporation into the DNA. H urd et al. (Hurd et al. 1999) (Figure 2.13). In fact, this structural formation was recently demonstrated by Zhou et al. (Zhou et al. 2002) in a crystallized complex showing that the 2-(lH)-pyrimidinone ring can be flipped out of the helix to form a covalent bond with the active cysteine group in the DNA methyltransferase. Furthermore, my preliminary experiments have shown a marked decrease in the level of DNA methyltransferase 1 protein (Bestor 1988) in cells treated with zebularine (data not shown). The novelty of my study is that I demonstrate that zebularine can act as a pro-drug leading to inhibition of DNA methylation and gene activation in fungal and mammalian cells. Although the potency of zebularine was similar to that of 5-Aza-CR in N. crassa, it was less effective than either 5-Aza-CR or 5-Aza-CdR in the animal cell in vitro systems. Higher doses of zebularine were required for the induction of pl6 gene expression and demethylation of its promoter sequence. Because the strength of the covalent complex between a DNA methyltransferase and modified DNA containing 5-Aza-CdR (Momparler 1985) or 2-(lH)- pyrimidinone (Hurd et al. 1999) would be expected to be comparable with each other, differences in transport or metabolic activation of zebularine and 5-Aza- CR in mammalian cells could account for the differences in potencies between the two drugs. 5-Aza-CdR (or 5-Aza-CR) m ust be phosphorylated to its nucleotide form by deoxycytidine kinase (or uridine-cytidine kinase) and subsequently incorporated into replicating DNA to inhibit DNA 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Covalent binding of DNMTs to zebularine-substituted DNA DNMT DNA DNA Figure 2.13: Covalent binding o f DNA methyltransferases (DNMTs) to zebularine-substituted DNA. Once incorporated into the DNA, zebularine-substituted DNA can form a covalent bond with DNMT to inactivate the enzyme level and activity (Hurd et al. 1999, Zhou et al. 2002b). vo methyltransferase (Figures 2.14, 2.15) (Constantinides et al. 1978, Gabbara and Bhagwat 1995, Lee et al. 1974, Momparler and Derse 1979, Santi et al. 1984). Presumably, zebularine is metabolized in the same way but perhaps the uridine-cytidine kinase has a lower binding affinity for zebularine than for 5- Aza-CR, resulting in less activation and incorporation of zebularine into DNA, thus necessitating higher dosages of the drug. Because 5-Aza-CdR is active at one-tenth the concentration of 5-Aza-CR (Jones and Taylor 1980), we investigated the possibility of the deoxyribose form of zebularine, 2/-deoxyzebularine, would be a more effective demethylating agent than its corresponding ribonucleoside form. It was found that 2'- deoxyzebularine neither induced the myogenic phenotype nor inhibited DNA methylation of the Class Il-d locus in 10T1/2 cells nor the hph locus in Neurospora (data not shown). Its inactivity in mammalian cells may be a reflection of different specificities or binding affinities of the enzymes for their corresponding substrates. Perhaps deoxycytidine kinase, the most likely enzyme to phosphorylate 2'-deoxyzebularine, does not recognize or bind well to this compound because it lacks the 4-amino group on the pyrimidine ring (Figure 2.12). Zebularine is probably phosphorylated by uridine-cytidine kinase, which may be insensitive to the missing 4-amino group. Because only 2/-deoxyzebularine is expected to be incorporated into DNA, the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. RNA t 5-aza-CTP it 5-aza-CDP it 5-aza-CMP Phospbatase I ^ 5-aza-CR i 5-azauridine Uridine - Cytidine Kinase Cytidine Deaminase DNA t 5-aza-dCTP Ribonucleotide w ^ ► 5-aza-dCDP it 5-aza-dCMP Phosphatase I | 5-aza-CdR Reductase Deoxycytidine Kinase i Cytidine Deaminase 5 -aza-2 ’ -deoxyuridine V O v o Figure 2.14: Metabolism o f 5-azacytidine (5-Aza-CR) and 5-aza-2 ’ -deoxycytidine (5-Aza-CdR). 5-Aza-CR is activated by uridine-cytidine kinase to the monophosphate level, while 5-Aza-CdR is activated by deoxycytidine kinase. 5-Aza-CR is subsequently incorporated into both RNA and DNA, whereas 5-Aza-CdR is only incorporated into the DNA. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Covalent binding of DNMTs to aza-substituted DNA NH. 1 DNMT + SAM DNA DNMT DNA Figure 2.15: Covalent binding o f DNA methyltransferases (DNMTs) to aza-substituted DNA. Once incorporated into the DNA, aza-substituted DNA can form an irreversible, covalent bond with DNMT to inactivate the enzyme level and activity (Momparler 1985). © o ribonucleotide reductase could be an additional rate-limiting step (Figure 2.12). The metabolic activation of zebularine to 2'-deoxyzebularine-5'-triph.osphate is currently under investigation. In addition, cytidine and uridine are known to be strong competitive inhibitors of 5-Aza-CR phosphorylation (Liacouras and Anderson 1979); it is therefore possible that both cytidine and uridine can competitively inhibit the phosphorylation of zebularine to a greater degree than that of 5-Aza-CR, thus accounting for the tenfold difference in potency in animal cells. Other possibilities include the potential that cytidine deaminase may sequester zebularine to a certain extent, thus lowering the effective concentration of the drug, or the potential that cytidine deaminase inhibition may lead to increased levels of deoxycytidine and cytidine levels, hence affecting the anabolism of zebularine. It is also important to consider that zebularine is probably incorporated into RNA as well as into DNA. Indeed, the growth inhibitory effects of zebularine on N. crassa were found to be independent of the dim-2 DNA methyltransferase (Dr. Eric Selker, data not shown), which is the only DNA methyltransferase active in vegetative tissues of N. crassa (Kouzminova and Selker 2001). Perhaps the toxicity of zebularine reflects incorporation into RNA. The finding that zebularine was able to induce p!6 expression in a time- dependent manner suggests that this drug may be usefully administered over extended periods. The instability and toxicity associated with 5-Aza-CR and 5- 101 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Aza-CdR were shown in a previous study that demonstrated the time- dependent treatment of T24 cells with 5-Aza-CdR was limited to 24 hours (Bender et al. 1999). The stability of zebularine in both acidic and neutral solutions was presumably responsible for its more extended inhibitory effect on methylation of the p i 6 prom oter/ 5' region. This finding offers new opportunities for the application of zebularine in future in vitro and in vivo studies. My studies with BALB/c nu/nu mice demonstrated that silenced genes can be reactivated by zebularine in vivo, as shown previously with 5-Aza-CdR (Bender et al. 1998). However, what is unique and exciting about zebularine is that this is the first time a methylation inhibitor has been shown to exhibit an in vivo antitumor effect via oral administration. This observation further supports the notion that the drug is add-stable. Additionally, the apparent decreased tumorigenicity of zebularine-treated EJ6 cells, and minimal weight loss observed in these zebularine-treated mice, further support the clinical potential of zebularine. These findings raise the possibility that this drug, or a related DNA methylation inhibitor with minimal toxicity, may be clinically useful to reverse aberrant DNA methylation, restore critical gene function in vivo and thereby treat certain cancers. 102 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTERS CONTINUOUS ZEBULARINE TREATMENT EFFECTIVELY SUSTAINS DEMETHYLATION IN H U M A N CANCER CELLS INTRODUCTION The abnormal de novo methylation of promoter CpG islands in numerous tumor suppressor and other cancer-related genes has been shown to be associated with their silencing during carcinogenesis (Baylin et al. 2001, Jones and Baylin 2002, Jones and Laird 1999, Momparler and Bovenzi 2000). This frequent alteration in hum an cancer cells may represent an alterative mechanism to mutations and chromosomal deletions for gene inactivation during tumorigenesis. The prevalence of aberrant methylaton in cancer has encouraged the search for therapeutic agents which can inhibit methylation and may thus be utilized to reverse this effect by reactivating genes which have become abnormally silenced. 5-Azacytidine (5-Aza-CR) and its deoxy analog, 5-aza-2'-deoxycytidine (5-Aza-CdR), are two of the most well-known DNA methylation inhibitors (Harris 1982, Jones and Taylor 1980). Both drugs are nucleoside analogs which have been widely used for studying the role of DNA methylation in biological processes as well as for clinical treatments of patients with acute myeloid 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. leukemia (AML) and myelodysplastk syndrome (MDS) (Lubbert 2000, Pinto and Zagonel 1993, Wijermans et al. 2000). 5-Aza-CR and 5-Aza-CdR are potent mechanism-based inhibitors of DNA methyltransferases (DNMTs), and function by substituting for cytosine residues during DNA replication and forming covalent bonds with the DNA methyltransferase (DNMT), which ultimately leads to the inhibition of the DNMT's activity (Constantinides et al. 1978, Gabbara and Bhagwat 1995, Momparler and Derse 1979, Santi et al. 1984). Unfortunately, these drugs are both unstable in aqueous solutions and toxic (Beisler 1978, Constantinides et al. 1977, Santi et al. 1984), and these characteristics have complicated their clinical use, hence there is the need for an effective, stable, and minimally toxic inhibitor of DNA methylation. Previously, others and I have characterized zebularine (l-(beta-D- ribofuranosyl)-l,2-dihydropyrimidin-2-one) as a novel inhibitor of DNA methylation, which is stable and minimally toxic both in vitro and in vivo (Cheng et al. 2003). Zebularine is a cytidine analog containing a 2-(lH)- pyrimidinone ring that was originally developed as a cytidine deaminase inhibitor because it lacks an amino group on position 4 of the ring (Kim et al. 1986, Laliberte et al. 1992). Studies with synthetic oligonucleotides containing the 2-(lH)-pyrimidinone ring have demonstrated the formation of a tight complex with bacterial methyltransferases in vitro (Hurd et al. 1999), and this was further corrobated by a recent study demonstrating the crystallization of a 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bacterial DNA methyltransferase with the 2-(lH)-pyrimidinone ring forming a covalent bond at the active site (Zhou et al. 2002b). In a previous study, we have shown that zebularine can be orally administered to cause reactivation and demethylation of a silenced and hypermethylated p l6 gene in human bladder tumor cells grown in nude mice (Cheng et al. 2003). Nonetheless, one of the major challenges with the usage and application of nucleoside analogs as inhibitors of DNA methylation is the problem of remethylation of genes that were demethylated after treatment with these agents, which eventually results in their re-silencing (Bender et al. 1999). This remethylation phenomenon makes the clinical application of these drugs quite limited following cessation of drug treatment. Here I demonstrate that the single treatment of T24 bladder carcinoma cells with zebularine resulted in a rapid induction of the pl6 gene, followed by its re-silencing and remethylation of its 5' region. I therefore examined the possibility of exploiting zebularine's stability to achieve an effective demethylation of aberrantly silenced genes and maintain their expression over extended time periods, and found that zebularine can effectively sustain demethylation of the pi 6 5' region and prevent gene re-silencing when administered in a continuous fashion to cultured cancer cells. Continuous zebularine treatment also caused demethylation of the entire pl6 gene locus, which was most pronounced in CpG-depleted regions. Furthermore, zebularine 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. induced a complete depletion of extractable DNMT1 but not DNMT3a and 3b proteins in T24 cells. Lastly, I found that sequential treatment of T24 cells with an initial dose of 5-Aza-CdR followed by a sustained low dose of zebularine hindered the remethylation of pl6 5' region and re-silencing of p i 6 gene expression. My results suggest new strategies for cancer therapy using prolonged and continuous zebularine treatment, as well as a combination therapeutic regimen of 5-Aza-CdR and zebularine. 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MATERIALS AND METHODS Cell lines The T24 human bladder transitional carcinoma-derived cell line was obtained from the American Type Culture Collection (Rockville/ MD), and cultured in McCoy's 5A medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 units/m l penicillin, and 100 pg/m l streptomycin (Gibco/Life Technologies, Inc., Palo Alto, CA). Normal LD419 hum an bladder fibroblasts were generated in our laboratory and cultured as previously described (Velicescu et al. 2002). D rag treatments For kinetic studies, T24 cells were plated (3 x 105 cells/100-mm dish) and treated 24 h later with 5 x IQ "4 M zebularine. The medium was changed 48 h after drug treatment, and DNA and RNA were harvested at the indicated time points for methylation and RT-PCR analyses, respectively. For the continuous drug treatment, T24 cells were plated (3 x 10s cells/100-mm dish) and treated 24 h later with 10"4 M zebularine. The medium was changed every 3 days, along with fresh zebularine treatment, for up to 40 days. Cells were either treated continuously for 40 days with 10- 4 M zebularine or with increasing concentration of zebularine from IQ- 4 M to 4 x Iff4 M (an 107 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. incremental increase of 5 x 10’ 5 M every 6 days), DNA and RNA were harvested at the indicated time points for methylation and RT-PCR analyses, respectively. Protein lysates were collected at indicated time points for Western blot analysis of the DNMT protein levels. For sequential drug treatment, T24 cells were plated (3 x 105 cells/100- mm dish) and treated 24 h later with either (1) 5 x 10'5 M zebularine continuously for 30 days, (2) 1 C T 6 M 5-Aza-CdR for 24 h, or (3) 10'6 M 5-Aza-CdR for 24 h followed sequentially by 10'4 M zebularine for up to 30 continuous days. DNA and RNA were harvested at the indicated time points for methylation and RT-PCR analyses, respectively. Nucleic acid isolation RNA was collected and extracted from cultured T24 and LD419 cells using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's recommended protocol. DNA was collected as previously described (Gonzalez-Zulueta et al. 1995). RT-PCR analysis Total RNA (5 pg) extracted from cultured cells was reverse-transcribed using MMLV reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamers (Amersham-Pharmacia, Piscataway, NJ) in a total volume of 25 pi. 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The reverse transcription was performed as previously described (Gonzalez- Zulueta et al. 1995). cDNA was amplified with primers specific for either pl6 or GAPDH. The reverse transcription (RT)-PCR conditions were as follows: for pl6, 94°C for 3 min, 28 cycles of 94°C for 1 min, 56°C for 30 s, 72°C for 40 s, and a final extension step at 72°C for 5 min; for GAPDH, 94°C for 1 min, 19 cycles of 94°C for 1 min, 58°C for 30 s, 72°C for 45 s, and a final extension step at 72°C for 2 min. The primer sequences are as follows: p i 6 sense, 5'-AGC CTT CGG CTG ACT GGC TGG-3'; pl6 antisense, 5'-CTG CCC ATC ATC ATG ACC TGG A-3'; GAPDH sense, 5'-CAG CCG AGC CAC ATC GCT CAG ACA-3'; and GAPDH antisense, 5'-TGA GGC TGT TGT CAT ACT TCT C-3'. RT-PCR amplification reactions of each of the expressed genes was performed with 200 ng cDNA, 10% dimethylsulfoxide (DMSO), 100 pM dNTPs, Taq DNA polymerase (Sigma), and 1 pM primers. The RT-PCR conditions, primers, and sequences for DNMT1, 3a and 3b are performed as previously described (Robertson et al. 1999) (Table 3.1). All reactions were analyzed in the linear range of amplification. PCR products were resolved on 2% agarose gels and subsequently transferred to a nylon membrane (Zetaprobe; Bio-Rad, Richmond, CA) under alkaline conditions. All blots were hybridized with a y-3 2 P-labeled internal oligonucleotide probe for pl6 as previously described (Gonzalez-Zulueta et al. 1995, Robertson et al. 1999). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.1 Conditions and prim er sequences for RT-PCR D N M T l D N M T3a DNM T3b Conditions 95° 3 m m 95° l i 40° l i 7 2 ° . 72° 10 m in '1 m in "I ' 1 m in r ' 1 m in J 95 1 m in 95° 1 m in 56° 1 m in 72° 1 m in 72° 10 m in 24X 95° 3 m in 95° 1 m in 46° 1 m in |»28X 72° 1 m in 72° 10 m in 1 } 28X 94° 3 m in 94° 1 m in 56° 30 sec ? ■ 28X 72° 45 sec 72° 5 m in 94° 2 m in 94° 1 nun 58° 30 sec i l 9 X 72° 45 sec 72° 2 m in }■ Nucleotide Sequence 5'-GATCGAATTCATGCCGGCGCGTACCGCCCCAG-3' (sense) 5'-ATGGTGGTTTGCCTGGTGC-3 (antisense) 5'-GGGGACGTCCGCAGCGTCACAC-3' (sense) 5'-CAGGGTTGGACTCGAGAAATCGC-3* (antisense) 5'-CCTGCTGAATTACTCACGCCCC-3' (sense) 5'-GTCTGTGTAGTGCACAGGAAAGCC-3’ (antisense) 5'-AGCCTTCGGCTGACTGGCTGG-3' (sense) 5'-CTGCCCATCATCATGACCTGGA-3' (antisense) 5'-CAGCCGAGCCACATCGCTCAGACA-3' (sense) 5'-TGAGGCTGTTGTCATACTTCTC-3' (antisense) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Quantitative RT-PCR analysis The quantitation of mRNA levels was carried out using a real-time fluorescence detection method as described previously (Eads et al 1999, Held et al. 1996). Briefly, after RNA isolation, cDNA was prepared from each sample as described above. The specific cDNA of interest (pi 6) and reference cDNA (GAPDH) were PCR-amplified separately using an oligonucleotide probe with a 5' fluorescent reporter dye and a 3' quencher dye (Livak et al. 1995). The 5' to 3' nuclease activity of Taq DNA polymerase cleaved the probe and released the reporter, whose fluorescence could be detected by the laser detector of the ABI Prism 7700 Sequence Detection System (Perkin-Elmer Corp., Foster City, CA). All of the samples were normalized to the reference GAPDH gene. The experiment was performed in duplicates. Western blot analysis of DNMT protein levels Cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed by the addition of radioimmunoprecipitation (RIPA) buffer (PBS, 0.1% SDS, 0.5% nonidet P-40 and 0.5% sodium deoxycholate). Cells were scraped off dishes and placed on ice for 30 min. The mixture was centrifuged at 13000 rpm for 30 min at 4°C, and the supernatant was used for western blot analysis. Approximately 60 |ig total protein extract was loaded onto 4-15% gradient Tris-HCl gels (BioRad, Hercules, CA), electrophoresed in Tris-glycine- SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS; pH 8.3) and transferred to PVDF membranes in Tris-glycine buffer (25 mM Tris, 192 mM 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SDS running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS; pH 8.3) and transferred to PVDF membranes in Tris-glycine buffer (25 mM Tris, 192 mM glycine; pH 8.2) overnight at 4°C. The membranes were hybridized with antibodies against human DNMT1 (1:1000 dilution; New England Biolabs, Beverly, MA), human DNMT3b (T-16; 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and PCNA (1:4000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) in Tris-Buffered Saline-Tween (TBS-T) buffer (0.1 M Tris, 1.5 M NaCl, and 1% Tween 20) with 5% non-fat dry milk overnight at 4°C. The hum an DNMT3a was kindly provided by Dr. Guo-Liang Xu (Shanghai, China). The membranes were washed 3 times with TBS-T buffer at room temperature, and incubated with secondary antibodies as follows: anti-mouse-IgG-HRP (1:3000 dilution for PCNA; Santa Cruz); anti-rabbit-IgG-HRP (1:2000 dilution for DNMT1; Santa Cruz); anti-goat-IgG-HRP (1:10000 for DNMT3b; Calbiochem, San Diego, CA). All were incubated with the membrane for 1 hr at room temperature. Afterwards, the membranes were washed 5 times with TBS-T at room temperature. Proteins were detected with the ECL chemiluminescent detection kit (Amersham-Pharmacia, Piscataway, NJ) and by exposure to Kodak X-OMAT AR film (Rochester, NY). Autoradiograms from two independent Western gels were analyzed by scanning densitometry using a Model GS-710 Imaging Densitometer (Bio-Rad). 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hemimethylation assay Hemimethylation analysis was performed as previously described (Liang et al. 2002a, Nguyen et al. 2002, Velicescu et al. 2002). Undigested or Hpall-digested DNA from T24 cells was subjected to bisulfite modification. Hpall digests unmethylated DNA but does not cut a fully or hemimethylated configuration of its CCGG target sequence. Bisulfite-treated DNA was then amplified by PCR using primers that flanked the first Hpall site in p!6 intron 1 and p!6 exon 2. The CpG site targeted by the intron 1 SNuPE primer is also located in a Hpall site, so this same primer was also used for hemimethylation analysis (Velicescu et al. 2002). The equations used to determine hemimethylation levels were as described previously (Liang et al. 2002a). The H:F ratio represented the ratio of the percentage of hemimethylated (H) molecules to the percentage of fully methylated (F) molecules. Quantitation of DNA methylation levels by methylation-sensitive single- nucleotide primer extension assay The mean cytosine methylation levels of CpG sites in the fragment were determined by treatment of genomic DNA (4 jag) with sodium bisulfite according to Frommer et al. (Frommer et al. 1992). Methylation analysis was performed using the methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) assay (Gonzalgo and Jones 1997). The bisulfite-PCR and the 113 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. qualitative methylation-sensitive single nucleotide primer extension (Ms- SNuPE) assay for pl6 prom oter/ 5' region (Cheng et al. 2003) and p l6 regions 1- 8 (Velicescu et al. 2002) were performed as previously described. The bisulfite- PCR primers and conditions for p53 Alu, D4Z4, and M4-4 are shown in Table 3.2. The Ms-SNuPE primers and conditions for p53 Alu, D4Z4, and M4-4 are shown in Table 3.3. Bisulfite genomic sequencing The bisulfite genomic sequencing was used to determine methylation levels in individual molecules of DNA. The region of interest, the pl6 5' region, was PCR amplified using DNA that had undergone sodium bisulfite conversion, as described above (Frommer et al. 1992). The PCR amplified region of the promoter was 530 bp long and 28 CpG dinucleotides, including CpGs both upstream of the transcriptional start site and in the first exon. PCR conditions have been previously described (Pao et al. 2000). Primers for the pl6 5' region were as follows: 5'-GGT GGG GTT TTT ATA ATT AGG AAA GAA TAG TTT TG-3' (sense) and 5'-TCT AAT AAC GAA CCA ACC CCT CC-32 PCR products were then cloned in into the pCR2.1 vector, followed by chemical transformation and plating, all performed using the TOPO-TA Cloning Kit (Invitrogen) and following manufacturer's instructions. After positive clone identification, plasmid purification was performed using the Qiagen Plasmid 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Mini Kit (Qiagen). Sequencing was then performed at Laragen, Inc. (Los Angeles, CA) Determination of population doublings and cell growth Cells were counted with a Z1 Coulter Particle Counter (Beckman Coulter Corporation, Hialeh, FL) at the indicated time points. Untreated cells were analyzed under similar conditions as a control. The average cell number from two plates was determined, and the mean cell numbers plotted to define population doublings. Initial drug treatment was started 24 h after seeding. 115 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.2 Conditions and prim er sequences for bisulfite PCR Region Conditions Nucleotide Sequence D4Z4 95° 3 m in 95° 1 m in % 58° 45 sec L ox 72° 45 sec J 72° 10 m in 5'-GGGTTGAGGGTTGGGTTTAT-3' (sense) 5'-AACTTACACCC 1TCCCTACA-3' (antisense) p53 Alu 95° 3 m in 95° 1 m in "j 52° 45 sec 1-40X 72 ° 45 sec J 72 ° 10 m in S'-TGGGTTTAATTATTGTATAGTTGAA-S' (sense) 5'-CTCAACTCACTACAAACTCCA-3' (antisense) M4-4 95° 2 m in 95° 1 m in ■ % 56° 30 sec l»42X 72° 1 m in J 72° 10 m in 5'-ATGGTTTGAGGGTTTAGATTAGGT-3’ (sense) 5’ -ACATCAAAATAAACTTCCTCTTACCA-3’ (antisense) 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3 J Conditions and prim er sequences for Ms-SNuFE Region Conditions Nucleotide Sequence D4Z4 95° 2 m in 50° 2 m in 72° 1 m in S’ -TTTGGG AGGTT A AGGT AGG-3' S'-GTTTTTATTGAAAAATATAAAAAAAAATTAGT-S' 5'-GAAGGAGAATGGTGTGAATTTGGG-3' p53 Alu 95° 2 m in 50° 2 m in 72° 1 m in 5'-TTTGGGAGGTTAAGGTAGG-3' 5'-GTTTTTATTGAAAAATATAAAAAAAAATTAGT-3' 5'-GAAGGAGAATGGTGTGAATTTGGG-3' M4-4 95° 1 m in 46° 30 sec 72° 20 sec 5-GTAA T AAGG ATT ATTTG A AT AG-3' 5'-TAATAATGTGGATTTGTTTAAATT-3' 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Kinetics of p t6 mRNA induction and demethylation of the 5' region by zebularine The kinetics of pl6 mRNA induction and the demethylation of 5' region of p i 6 by 5-Aza-CdR have been studied in detail (Bender et al. 1999), so that it was of obvious interest to evaluate these parameters in T24 cells treated with zebularine. Treatment of T24 cells with 5 x 10'4 M zebularine for 48 h induced a slight expression of pi 6 by day 2, which increased dramatically up to day 5, and began to diminish thereafter (Figure 3.1). This re-silencing of the pl6 gene was not surprising, since the same phenomenon was previously demonstrated with 5-Aza-CdR, presumably due to the remethylation of the 5' region of p!6 gene (Bender et al. 1999, Velicescu et al. 2002). The methylation of the 5' region of pl6 decreased from 97% at day zero to 75 % at day 3, and then slowly increased thereafter, and this paralleled the decrease in p!6 expression (Figure 3.2). Thus, remethylation is a common problem that is observed with zebularine as well as other inhibitors of DNA methylation and is a potential complication in the clinical applications of these drugs. Continuous treatm ent w ith zebularine sustains the expression and demethylation of the p l6 gene To circumvent the problems of remethylation, I took advantage of 118 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o f th e copyright owner. Further reproduction prohibited without permission. 0 1 2 3 4 5 7 9 12 14 p l6 GAPDH i' Figure 3.1: Kinetics of p i 6 mRNA induction by zebularine. T24 cells were exposed to zebularine (5 x 10-4 M) for 48 h. RNA was isolated at 24-h intervals after drug addition. Expression levels of p i 6 mRNA at each time point were determined by RT-PCR analysis. GAPDH mRNA expression levels were m easured to control for relative cDNA input. v o Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 0 1 2 3 4 5 7 9 12 14 D ays After Treatment (5 x 10-4 M) Figure 3.2: Kinetics o fp l6 5' region demethylation by zebularine. Demethylation of the pl6 5' region by zebularine (5 x 10"4 M) at the indicated time point, was quantitated by Ms-SNuPE analysis. DNA was isolated at 24-h intervals after drug addition. Methylation percentage represents the average of three individual CpG sites in the pl6 5' region as assayed by Ms-SNuPE analysis from two independent experiments. Error bars, the standard deviation of four determinations. to o zebularine's stability and minimal cytoxicity (Barchi et al, 1992, Cheng et al. 2003, Kelley et al. 1986) to investigate the effects of continuous drug treatment on p i 6 expression in T24 cells. Continuous zebularine treatment (1C4 M) for up to 40 days led to an induction of pl6 expression at day 5, which increased over time (Figure 3.3). Treatment with increasing concentrations of zebularine (from 1C4 M to 4 x 1 C T 4 M) for 40 days led to an even greater level of pl6 gene expression (Figure 3.3). I compared the extent of pl6 reactivation from these two regimens to that in a normal fibroblast cell line (LD419), which expresses pl6 and is unmethylated in the 5' region of the gene. Reactivation by zebularine led to gene reactivation, which was 31% (IB4 M) to 43% (increasing doses) of levels seen in LD419 controls (Figure 3.4). This finding is not surprising since the methylation of the 5' region with the increasing doses was reduced to approximately 63% over the same period (Figure 3.5). T24 cells were found to be growth suppressed by drug treatment, which is likely due to the reactivation of the p i6 gene (Figure 3.6). Bisulfite genomic sequencing of DNA obtained from continuously treated cells showed substantial demethylation of the p i 6 5' region, especially around the transcription start site (Figure 3.7). The percentage of methylation from all molecules (69%) was relatively consistent with the result obtained from the Ms-SNuPE analysis (71%) (Figures 3.5,3.7). 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Zebularine Zebularine (1(H M) (1(H M - > 4x 10"4 M) Days 0 5 7 14 21 28 33 40 5 7 14 21 28 33 40 P16 [ GAPDH i Figure 3.3: Effects of continuous zebularine treatment on pl6 mRNA expression. T24 cells were treated with either zebularine (104 M) or an increasing dose of zebularine from 104 M to 4 x 104 M concentration over a period of 40 continuous days. For the increasing dose of zebularine, the drug was added in increments of 5 x 10'5 M every 6 days starting from 104 M. RNA was isolated at the indicated time points. Expression levels of p l6 mRNA at the indicated time point were measured by RT-PCR analysis. GAPDH mRNA expression levels were measured to control for relative cDNA input. HO to Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 400 ■ 5 o g 80 o O 60 r 4 0 m S 2 0 T24 (10-4 M) T24 (^dose) Figure 3.4: Comparison of p i 6 expression between T24 cells treated with zebularine and untreated normal LD419 cells. Relative expression levels of pl6 mRNA (normalized to the reference GAPDH gene) of each indicated drug regimens at day 40 were measured by quantitative real time RT-PCR analysis. The levels are reported as percent (%) expression of normal LD419 cells. Results represent averages and ranges of two separate determinations. N > U ) Figure 3.5: Effects of continuous zebularine treatment on pl6 5’ region demethylation, T24 cells were treated with either ( ■ ) zebularine (10'4 M) or ( □ ) an increasing dose of zebularine from 1 C T 4 M to 4 x K X 4 M concentration over a period of 40 continuous days. For the increasing dose of zebularine, the drug was added in increments of 5 x 10'5 M every 6 days starting from 10'4 M. DNA was isolated at the indicated time points. The methylation level of the p l6 5' region for the indicated time point after either treatment was measured by Ms-SNuPE analysis. Methylation percentage represents the average of three individual CpG sites in the p l6 5" region as assayed by Ms-SNuPE from two independent experiments. Error bars, the standard deviations (SD) from four determinations. 124 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o o o as o C O o IN O V O o in [%) u o T iF iiip a jM Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Days o f Continuous Treatment Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Control 104 M Zebularine ^ Zebularine J" G> I 40 q 3 0 Days After Seeding Figure 3.6: Effects of continuous zebularine treatment on cellular growth. T24 cells were treated with either zebularine (104 M) or an increasing dose of zebularine from 104 M to 4 x 104 M concentration over a period of 40 continuous days. Cellular growths after treatment with the indicated drug regimens were plotted as population doublings against time. Initial drug treatment was started 24 h after seeding. to O s Figure 3.7: Bisulfite genomic sequencing of the pl6 5' region in T24 cells after continuous treatment with zebularine. Cells were either untreated or treated with 10"4 M zebularine continuously for 40 days. DNAs were then isolated, treated with sodium bisulfite, followed by cloning and sequencing of individual molecules. Each horizontal line with a string of circle represents the methylation profile of one molecule. Top diagram is a schematic of the region analyzed and indicates CpG density distributions. Bent arrow is transcriptional start site. White circles = unmethylated CpG; black circles = methylated CpG. Total methylation of all molecules is noted to the right of the molecules and is noted as a percentage. Distances between CpGs are roughly to scale. 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 01 M M 0000 128 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Continuous zebularine treatm ent causes variable demethylation of the entire p%6 gene locus We then assessed the effects of continuous zebularine treatment for 40 days on the methylation levels of several other regions within the pl6 gene locus (Velicescu et al. 2002) (Figure 3.8). Regions 1 and 2 are CpG poor sequences, region 3 is a CpG island containing AJw-repetitive elements located upstream of the pl6 promoter, region 4 is the CpG island of the promoter, regions 5-7 include 3 individual CpGs residing in intron 1 of pl6, and region 8 is the CpG island of the second exon. All regions analyzed showed measurable demethylation, however, the CpG sites in regions 5 and 6, which are located in CpG-depleted areas, showed preferential demethylation (Figure 3.8). To better understand this preferential demethylating effect of zebularine, I performed hemimethylation assays to determine the distribution of fully methylated, hemimethylated and unmethylated sites (Liang et al. 2002a) at both the CpG-depleted region 5 (pl6 intron 1) and the CpG-rich region 8 (pl6 exon 2) (Figures 3.9, 3.10). In region 5, the levels of fully methylated sites substantially decreased after continuous treatment with zebularine, and unmethylated sites were increased dramatically over the 40-day time course (Figure 3.9). Methylation of the CpG site in region 5 is therefore poorly maintained after zebularine treatment, as most fully methylated CpGs were converted to the unmethylated state by day 40. The percentage of hemimethylated sites 129 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3*8: Effects of continuous zebularine treatment on methylation levels of p i6 locus, Methylation levels of regions within the pl6 gene locus in T24 bladder cancer cells either before or after continuous treatment with zebularine as measured by Ms-SNuPE analysis. T24 cells were treated with KT4 M zebularine for 40 continuous days and DNA/RNA were harvested immediately afterwards. Top, eight regions of various CpG densities were identified upstream and downstream of the pl6 promoter region (region 4) (Velicescu et al. 2002). Regions 1 and 2 are CpG poor sequences (three CpGs analyzed in region 1 and four CpGs in region 2), region 3 is a CpG island containing Alu- repetitive elements located upstream of the pl6 prom oter/ 5' region (three CpGs analyzed), and regions 5-7 consist of three individual CpG dinucleotides residing in intron 1 of pl6, whereas region 8 is the CpG island of the second exon (three CpGs analyzed). The transcription start site for the pl6 gene is indicated by the bent arrow on the map. Vertical arrows, the specific CpG sequences analyzed by Ms-SNuPE. Bottom, the methylation values for each region were measured by Ms-SNuPE in T24 cells, before (H ) and after ( □ ) continuous treatment with zebularine for 40 days. 130 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. %) uoueiAmew Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. p16 D N A Region increased slowly over the course of continuous zebularine treatment, but eventually decreased back to starting levels (Figure 3.9). Zebularine appeared to preferentially target this CpG-depleted region, presumably by inhibiting the DNMT(s) responsible for maintaining its modification. The ratio of hemimethylated to fully methylated sites rose dramatically by day 21 and slowly decreased thereafter, implicating the effectiveness of the drug in this region. In contrast, most of the DNA molecules in the CpG island of pl6 exon 2 remained fully methylated throughout 40 days, with a transitory increase in hemimethylated molecules and a slow increase in unmethylated molecules during the course of treatment (Figure 3.10). The ratio of hemimethylated to fully methylated sites remained low throughout the treatment, possibly due to the recruitment of other DNMT(s) to this region that may methylate these hemimethylated molecules. My results suggest that zebularine may target CpG- depleted regions more efficiently than CpG-rich regions. Zebularine selectively depletes DNMT1 Previous work has shown that DNMTs work cooperatively to facilitate and maintain DNA methylation patterns in mammalian cells (Liang et al. 2002a, Rhee et al. 2002). Since zebularine appears to target CpG-depleted regions, I next assessed the levels of DNMT1,3a and 3b in T24 bladder cancer cells before and during drug treatment. Western blot analysis showed a drastic depletion of 132 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Percent (% ) Region 5 (Intron 1) Hpall site 100 ■ Fully M ethylated (F) H Hemimethylated (H) [~| Unmethylated (U) O H :F Days of Continuous Treatment Figure 3.9: Distribution of methylation states at the pl6 intronl after continuous zebularine treatment. Levels of fully methylated (F), hemimethylated (H), and unmethylated (U) DNA at the first Hpall site of p!6 intron 1 (region 5) after continuous treatment with 10"4 M zebularine. Region 8 (Exon 2) Hpall site 5 7 14 21 28 33 40 Days of Continuous Treatment ■ 0.4 0.3* ■ M - 4 ■0.2 © s o □ o 0 Fully M ethylated (F) Figure 3.10: Distribution of methylation states at the pl6 exonl after continuous zebularine treatment. Levels of fully methylated (F), hemimethylated (H), and unmethylated (U) DNA at the first Hpall site of pl6 exon 2 (region 8) after continuous treatment with 10-4 M zebularine. extractable DNMT1 by day 1 of treatment in T24 cells, and virtually no extractable DNMT1 protein was present in cells growing in the presence of the drug, even after 40 days (Figure 3.11). DNMTSa and 3b3 were also affected in T24 cells, and this was most pronounced after 3 days of continuous zebularine treatment, yet both proteins gradually recovered thereafter (Figure 3.11). T24 cells express DNMT3b3 almost exclusively, and the hum an DNMT3b isoform, DNMT3b3, was recently shown to have reduced catalytic activity (Weisenberger et al, submitted; (Soejima et al. 2003)), possibly explaining the partial depletion of the enzyme by zebularine. DNMTSa, unlike DNMT1 or DNMTSb, is expressed throughout the cell cycle (Robertson et al. 2000), and might therefore only interact with zebularine when it is incorporated into DNA during the S-phase. The levels of DNMT mRNA transcripts were found to be unaffected by drug treatment as detected by semi-quantitative RT-PCR (Figure 3.12), supporting the idea that the depletion in DNMT protein levels was due to trapping of enzymes to the zebularine-substituted DNA, rather than an inhibition of transcription or cell proliferation. Moreover, the sustained methylation of CpG-rich regions may be the result of the less efficient depletion of DNMT3b3, since a recent study showed that DNMT1 and DNMTSb cooperate to methylate CpG islands in cancer cells (Rhee et al. 2002). I next assessed the effectiveness of continuous zebularine on the methylation status of other methylated loci in T24 cells. The methylation levels 135 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Western Blot Zebularine (10s 4 M ) Days DNMT1 > DNMT3a > DNMT3b3> PCNA > 1 8 40 ' ' : !■ ! :■ — g j I— ^ "ft. : i l j 3 B 100 a > ® 80 C I I 60 IS 40 I 2 0 & . -O- DNMT1 ■A- DNMT3a O DNMT3b3 1 2 3 8 40 Days of Continuous T r e a t m e n t Figure 3.11: Effects of continuous zebularine treatment on D NM T protein levels in T24 cells. (Left) Levels of DNMT1, 3a and 3b3 proteins after continuous zebularine treatment in T24 cells. PCNA was used as a control for cell proliferation in the Western blot analysis. (Right) Relative protein expression levels of DNMT1, 3a and 3b3 (normalized to PCNA) were measured and quantitated by scanning densitometry. The initial protein expression levels at day 0 were set as 100% for each DNMT to which the levels of protein expression from other days of treatment were compared. The percentage values represent the average of two independent experiments. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Days DNMT1 DNMT3a D N M T3h3> j GAPDH >! RT -PCR Zebularine (10'4 M) 1 8 40 Figure 3.12: Effects of continuous zebularine treatment on D N M T mRNA levels in T24 cells. Semi-quantitative RT-PCR of D NM T mRNA levels in T24 cells under untreated or treated conditions. GAPDH mRNA levels were measured to control for the quantity and integrity of the input RNA. U > - * 4 of the D4Z4 subtelomeric repeat (a DNMT3b target sequence; CpG island; chromosome 4q35; %GC = 73.3) (Kondo et al. 2000), an Alu element in the p53 gene (repetitive sequence, CG-rich region; chromosome 17pl3; %GC = 55.7), and the M4-4 sequence (single copy sequence; CpG island; chromosome 16q22; %GC = 56.5) were all substantially affected in treated T24 cells, although D4Z4 was less affected (Figure 3.13). My results indicate that continuous zebularine treatment can effectively and globally demethylate various methylated regions throughout the human genome. Sequential treatm ent of T24 cells w ith 5-Aza-CdR followed by zebularine I next tested whether an initial treatment of 5-Aza-CdR followed by a continuous low dose of zebularine (5 x 10‘ 5 M) in T24 cells could maintain pl6 expression and hinder or prevent the remethylation rate of the pl6 5' region. Continuous zebularine treatment (5 x 10"5 M) resulted in a slow increase of pl6 induction, beginning at day 10 (Figure 3.14). Treatment with 1 C T 6 M 5-Aza-CdR caused a substantial p i 6 gene expression by day 3, which gradually diminished over time, consistent with the results as shown previously in my laboratory (Bender et al. 1999). Sequential treatment of 10'6 M 5-Aza-CdR followed continuously with 5 x 10‘ 5 M zebularine resulted in a robust pl6 gene expression by day 3, which was well maintained throughout the treatment, as opposed to that from 5-Aza-CdR alone (Figure 3.14). 138 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M4-4 E3 04Z4 C o 100 80 r o £ 60 40 O p53 Alu ____ 1 8 14 0 8 14 0 8 Days of Continuous Treatment 14 J Figure 3.13: Effects of continuous zebularine treatment on methylation status of various loci in T24 cells. Methylation levels of various hypermethylated loci in T24 cells before and after continuous zebularine treatment. M4-4 (%GC =56.5), D4Z4 (%GC = 73.3) and p53 Alu (%GC = 55.7) are hypermethylated loci found in T24 bladder cancer cells. The methylation status for each locus was quantitated by Ms-SNuPE analysis. The methylation percentage represents an average of two separate determinations from two independent experiments. Error bars, the SD of four determinations. o f th e copyright owner. Further reproduction prohibited without permission. 10-6 M 5-Aza-CdR-*- 5 x 10'5 M Zebularine Zebularine (5 x 10‘ 5 M) p l 6 GAPDH Figure 3.14: Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on p i6 mRNA expression. T24 cells were treated with either (1) 5 x 10'5 M zebularine continuously for 30 days, (2) 10~ 6 M 5-Aza-CdR for 24 h, or (3) 10-6 M 5-Aza-CdR for 24 h followed sequentially by 5 x 10'5 M zebularine continuously for up to 30 days. RNA was harvested at the indicated time points for RT-PCR analysis. The expression levels of p i 6 mRNA were determined for each of the three separate regimens at the indicated time points by RT-PCR analysis. GAPDH mRNA expression levels were measured to control for relative cDNA input. o The methylation of the pl6 5' region decreased slowly over a period of 30 days (97% to 85%) after continuous zebularine (5 x 10"5 M) (Figure 3.15). Treatment with K T6 M 5-Aza-CdR alone resulted in an appreciable drop of the methylation of pl6 5' region, followed by a gradual remethylation over time. In contrast, sequential treatment of KT6 M 5-Aza-CdR followed by 5 x KT5 M zebularine showed a drastic decline of the methylation of pl6 5' region, followed by minimal remethylation that was considerably slower than that of 5- Aza-CdR alone. Remethylation was therefore hindered by treatment of T24 cells with 5-Aza-CdR followed by continuous zebularine. These results indicated a potential clinical regimen combining both drugs, perhaps using 5-Aza-CdR as an initial loading drug, and the less toxic zebularine as a maintenance drug. I then analyzed the growth of the T24 cells under the various drug regimens and found that the cells treated with the sequential treatment of 5- Aza-CdR followed by zebularine were the most growth suppressed when compared to the control as well as the other drug regimens (Figure 3.16). Thus the sequential treatment which caused the most sustained expression of pl6 resulted in the slowest growth rate of the treated cells (Figures 3.14,3.16). 141 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 100 5 x 10'5 M Zebularine 10-6 M 5-Aza-CdR lO” 6 M 5-Aza-CdR- > 5 x 10'5 M Zebularine Days After Treatment Figure 3.15: Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on pl6 promoter/ 5' region methylation. T24 cells were treated with either (1) 5 x 10'5 M zebularine continuously for 30 days, (2) 10‘6 M 5-Aza- CdR for 24 h, or (3) 10"6 M 5-Aza-CdR for 24 h followed sequentially by 5 x 10"5 M zebularine continuously for up to 30 days. DNA was harvested at the indicated time points for methylation analysis. The methylation status of the pl6 prom oter/ 5' region was determined for each regimen at the indicated time points by Ms-SNuPE analysis. Methylation percentage represents the average of three individual CpG sites in the p i6 prom oter/ 5' £ region as measured by Ms-SNuPE from two independent experiments. Error bars, the SD of four determinations. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. 40-1 # Control F I 5 x 10-5 M Zebularine 3 0 - A 10-®M 5-Aza-CdR o 10'6 M 5-Aza-CdR—> 5 x 10‘ 5 M Zebularine 20* J 25 35 Days of Seeding Figure 3.16: Sequential treatment of 5-Aza-CdR followed by zebularine and its effect on cellular growth suppression. Cellular growths after treatment with the indicated drug regimens were plotted as population doublings against time. Cell counts were taken at the indicated time points to calculate population doublings. Initial drug treatment was started 24 h after seeding. 4^, DISCUSSION Zebularine is a novel inhibitor of DNA methylation, which is stable and minimally toxic (Barchi et al. 1992, Cheng et al. 2003, Kelley et al. 1986). Transient treatments with methylation inhibitors are commonly followed by re- silencing of genes, which is most likely due to the occurrence of remethylation (Bender et al. 1999, Flatau et al. 1984, Liang et al. 2002a, Velicescu et al. 2002). Zebularine's stability and minimal cytotoxicty allowed us to grow cells in the continuous presence of the drug, and this led to the induction and maintenance of p i6 expression in T24 cells, which thereby circumvented the problem of remethylation. The global demethylating effects of zebularine suggested that this methodology in applying the drug is effective, especially towards CpG- poor regions. Moreover, my previous work with nude mice showed that high doses of zebularine was were not highly toxic to the mice (Cheng et al. 2003). These observations suggest possible therapeutic strategies and clinical benefits in the continuous application of zebularine as a cancer therapy. Continuous treatment of T24 bladder cancer cells with zebularine resulted in a complete depletion of DNMT1, and surprisingly, these cells were still growing even after the depletion of DNMT1 throughout the 40-day time course. This appears to contrast a recent study, which showed that an intra-S- phase arrest can be triggered by the reduction in DNMT1 using an antisense oligonucleotide inhibitor (Milutinovic et al. 2003). The authors, however, also 144 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mentioned that depletion of DNMT1 by 5-Aza-CdR did not induce this arrest, presumably due to the fact that DNMT1 is trapped only after the replication fork has formed and the inability to prevent de novo synthesis of DNMT1. In our treatment of T24 cells with zebularine, which presumably has a mechanism akin to 5-Aza-CdR, I did not observe a distinct intra-S-phase arrest as demonstrated with the antisense oligonucleotide, suggesting different mechanisms of action between these inhibitors. Perhaps, continuous zebularine treatment can offer an alternative solution to study methylation effects in the absence of DNMT1 alone. This will be the focus of future studies. Interestingly, with the apparently complete depletion of DNMT1, T24 cells still retained substantial methylation of the p26 CpG islands (pl6 prom oter/ 5' region and exon 2), D4Z4, M4-4, and an Alu element in p53. However, DNMTSa and 3b3 were only partially affected by continuous zebularine treatment, suggesting that these enzymes may play important roles in the methylation of these four loci, especially of D4Z4 which is a specific DNMT3b target sequence. It is also intriguing to note that zebularine appear to preferentially target CpG-depleted regions (such as region 5 of pi 6 locus) over CpG-rich regions (such as region 8 of pl6 locus). Since zebularine selectively depleted DNMT1, this would suggest that the CpG-depleted regions are largely maintained by this enzyme, as was previously found with mouse embryonic stem cells in my laboratory (Liang et al. 2002a). DNMT1 may therefore not be 145 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the only enzyme required to maintain methylation of CpG-rich regions, as both DNMT3a and 3b3 were still largely present after drug treatment. As indicated by the hemimethylation data, DNMT3a and 3b, which are presumably targeted to CpG-rich regions, may function to randomly methylate hemimethylated molecules generated by zebularine (Lin et al. 2002), whereas in CpG-poor regions, there are greater amounts of hemimethylated sites present as compared to the fully methylated sites throughout the period of zebularine treatment, suggesting that perhaps these regions are basically maintained by DNMT1 alone. My observations also support recent findings that DNMT1 works cooperatively with DNMT3a and DNMT3b to maintain methylation of CpG- rich regions, such as CpG islands and repetitive elements (Liang et al. 2002a, Rhee et al. 2002). On the other hand, another recent study from Robert et al. (Robert et al. 2003) indicated that DNMT1 alone was necessary and sufficient to maintain global methylation and aberrant CpG island methylation in human cancer cells. The discrepancy between these results may arise from the differences in methodologies used to deplete cellular DNMT1 levels. Rhee et al. (Rhee et al. 2002) used a gene targeting approach and the study by Robert et al. used an antisense approach (Robert et al. 2003), while I used a mechanism- based inhibitor of DNA methylation. Each study may affect unknown variables and therefore include unaccounted biases. These models need to be 146 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. investigated further in order to elucidate the mechanism behind the maintenance of aberrant CpG islands in hum an cancer cells. My results strongly suggest that continuous zebularine treatment is an effective strategy in sustaining demethylation of various loci and preventing their remethylation. When given sequentially after 5-Aza-CdR in a continuous fashion, zebularine can substantially hinder the rate of remethylation, implicating a powerful combination in treating cancer caused by aberrant epigenetic mechanisms. These findings suggest clinical potential as a cancer therapy as well as combination therapy with other drugs, such as 5-Aza-CdR, histone deacetylase inhibitors (HDACs), and chemotherapies. 147 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 4 PREFERENTIAL D E M E T H Y L A T IO N OF CPG ISLANDS IN CANCER CELLS BY ZEBU LA R IN E INTRODUCTION Abnormal hypermethylation of the promoters of numerous cancer- related or tumor suppressor genes important in cellular proliferation is commonly found in primary neoplasms and tum or cell lines (Baylin et al. 2001, Baylin and Herman 2000, Jones and Baylin 2002, Jones and Laird 1999). Because epigenetic processes are potentially reversible, pharmacologic inhibitors of DNA methylation provide a conceptually attractive and rational approach to re-establish the anti-proliferative and other crucial cellular functions abnormally silenced by hypermethylation. The first described specific inhibitors of DNA methylation, 5- Azacytidine (5-Aza-CR) and its deoxy analog, 5-aza-2'-deoxycytidine (5-Aza- CdR), were both originally synthesized as cancer chemotherapeutic agents (Jones and Taylor 1980, Sorm et al. 1964). Both are pyrimidine ring analogs of cytidine and 2/-deoxycytidine, respectively, but have nitrogen atoms in place of the C-5 pyrimidine carbon atoms. 5-Aza-CR is primarily activated by uridine- cytidine kinase and can be incorporated into both RNA and DNA, whereas 5- 148 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Aza-CdR is activated by deoxycytidine kinase and is only incorporated into newly synthesized DNA (Bouchard and Momparler 1983, Taylor et al. 1984). Once incorporated into DNA, both compounds can form covalent complexes with DNA methyltransferases (DNMTs), leading to a depletion of active enzymes (Santi et al. 1983, Santi et al. 1984, Taylor et al. 1984). Clinically, these agents have already shown utility for the treatment of leukemia and myelodysplastic syndromes (Lubbert 2000). Nevertheless, these mechanism- based inhibitors of DNMTs have some drawbacks. Both drugs are quite toxic in vitro and in vivo, and they are unstable in aqueous solution, making them difficult to administer both experimentally and clinically (Beisler 1978). We recently characterized zebularine as a novel mechanism-based inhibitor of DNA methylation, exhibiting stability in acidic and neutral solutions and minimal toxicity both in vitro and in vivo (Cheng et al. 2003). As a cytidine analog containing a 2-(lH)-pyrimidinone ring, zebularine was initially developed as a cytidine deaminase inhibitor because it lacks the amino group on C-4 of the pyrimidine ring (Kim et ah 1986, Laliberte et al. 1992). Similar to 5- Aza-CR and 5-Aza-CdR, zebularine has been shown to form a tight, covalent complex with bacterial methyltransferases in vitro (Hurd et al. 1999, Zhou et al. 2002). However, a major concern with the usage of nucleoside analogs as inhibitors of DNA methylation is the potential non-specific effects towards normal and cancer cells. 149 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Zebularine can be stably administered to T24 cells in a continuous fashion to effectively sustain demethylation of the pi 6 5' region and other methylated loci (Cheng et al, submitted). The purpose of this study was to compare the effects of continuous zebularine treatment on normal fibroblasts and cancer cells. We studied the effects of zebularine on the growth, methylation as well as DNA methyltranferase (DNMT) protein levels in a panel of normal fibroblasts and cancer cell lines. Here we show that all cancer cells but not normal fibroblasts evaluated were growth suppressed by zebularine, as evidenced by an increase in the doubling time as well as the up-regulation of p21(WAFl) CDK inhibitor. Furthermore, normal fibroblasts were insensitive to the effects of zebularine in terms of demethylation and depletion of DNMT enzymes, whereas the 3 out of 5 cancer cells were sensitive to zebularine and induced DNA demethylation. Lastly, we performed a gene expression microarray chip on T24 bladder cancer cells and LD419 normal fibroblasts. We found that a larger number of genes were activated in T24 cells than in LD419 cells, and most of these genes activated belonged to a group of cancer-specific antigens. The augmentation of cancer-related antigen genes by zebularine suggests that this drug may have important clinical ramifications for its combination usage with immunotherapy. 150 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MATERIALS AND METHODS Cell lines T24 (bladder transitional carcinoma cells); HCT-15 {colon carcinoma cells), CEP AC-1 (pancreatic carcinoma cells) and CALU-1 (lung carcinoma cells) were obtained from the American Type Culture Collection (Rockville, MD). PCS (prostate carcinoma) cells were kindly provided by Dr. Gerry Coetzee. LD419, LD98 and T-l (human normal fibroblasts) were established in our laboratory. T24 cells were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 units/m l penicillin, and 100 pg/m l streptomycin (Gibco/Life Technologies, Inc., Palo Alto, CA). HCT-15 was cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin/streptomycin, and lx sodium pyruvate (Gibco/Life Techologies, Inc.). CFPAC-1 was cultured in IMDM medium supplemented with 10% FCS, penicillin/streptomycin, lx glutamine (Gibco/Life Techologies, Inc.). CALU-1 was cultured in McCoy's 5A medium supplemented with 10% FCS, penicillin/streptomycin, and lx glutamine. PCS was cultured in RPMI 1640 medium supplemented with 5% FCS and penicillin / streptomycin. LD419, LD98 and T-l were cultured in McCoy's 5A supplemented with 20% FCS and penicillin/streptomycin. All cultures were grown in a humidified incubator at 37°C in 5% C 0 2 . 151 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Drug treatments All normal and cancer cell lines were plated (3 x IQ5 cells /lQO-mm dish) and treated 24 h later with KT4 M zebularine for 8 continuous days. The medium was changed every 3 day thereafter along with fresh zebularine. DNA, RNA and protein lysates were harvested at the end of the treatment period for methylation, RT-PCR and Western blot analyses, respectively. Nucleic acid isolation RNA was collected and extracted from cultured cells using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's recommended protocol. DNA was collected as previously described (Gonzalez- Zulueta et al. 1995). RT-PCR analysis Total RNA (5 pg) extracted from cultured cells was reverse-transcribed using MMLV reverse transcriptase (Invitrogen, Carlsbad, CA) and random hexamers (Amersham-Pharmacia, Piscataway, NJ) in a total volume of 25 pi. The reverse transcription was performed as previously described (Gonzalez- Zulueta et al. 1995). cDNA was amplified with primers specific for either p i6, p21, p27 or GAPDH. The reverse transcription (RT)-PCR conditions, primers, and sequences for p i 6 and GAPDH are shown in Table 3.1 and for p21 and p27 152 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. are shown in Table 4.1. RT-PCR amplification reactions of each of the expressed genes were performed with 200 ng cDNA, 10% dimethylsulfoxide (DMSO), 100 pM dNTPs, Tacj DNA polymerase (Sigma), and 1 jxM primers. The RT-PCR conditions, primers, and sequences for DNMT1, 3a and 3b are performed as previously described (Robertson et al. 1999) (Table 3.1). All reactions were analyzed in the linear range of amplification. PCR products were resolved on 2% agarose gels. Oligonucleotide array analysis cRNA preparation. Total RNA (10 fig) was used as starting material for the cDNA preparation. The first and second strand cDNA synthesis was performed using the Superscript II System (hrvitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions, except using an oligo-dT primer containing a T7 RNA polymerase promoter site. Labelled cRNA was prepared using the BioArray High Yield RNA Transcript Labelling Kit (Enzo Life Sciences, Inc., Farmingdale, NY). Biotin- labelled CTP and UTP (Enzo) were used in the reaction, together with unlabeled triphosphates. Following the in vitro transcription reaction, the unincorporated nucleotides were removed using RNeasy columns (Qiagen). This experiment was performed by Dr. Torben F. Omtoft and Dr. Thomas Thykjaer (Aarhus University Hospital, Skejby, Denmark). Array hybridization, and scanning. Fifteen fig of cRNA was fragmented at 94°C for 35 rrdn in a buffer containing 40 mM Tris-acetate pH 8.1, 100 mM 153 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. /■Tab Table 4.1 Conditions and prim er sequences for RT-PCR Region Conditions Nucleotide Sequence p 2 l 94° 3 m in 94° 1 m in T j 57° 1 m in > 18-23X 72° 1 m in J 72° 5 m in 5'-AGGGTGACTTCGCCTGGGAGC-3' (sense) 5'-CAC AC AAACTG AG ACT A AGGC AG A AG ATGT-3' (antisense) p2? 94° 3 m in 94° 1 m in ” 4 57° 1 m in !■ 28X 72° 1 m in J 72° 5 m in 5'-GCGCCTTTAATTGGGGCTCCGGCTAA-3' (sense) 5'-GCTACATCCAACGCTTTTAGAGGCAGATCA-3' (antisense) 154 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. potassium acetate, 30 mM magnesium acetate. Prior to hybridization, the fragmented cENA in a 6x SSPE-T hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0,005% Triton) was heated to 95°C for 5 min and subsequently to 45°C for 5 min before loading onto the Affymetrix HG_U133A probe array cartridge. The probe array was then incubated for 16 h at 45°C at constant rotation (60 rpm). The washing and staining procedure was performed in the Affymetrix Fluidics Station. The probe array was exposed to 10 washes in 6x SSPE-T at 25°C, followed by 4 washes in 0.5x SSPE-T at 50°C. The biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate [final concentration of 2 m g/m l (Molecular Probes, Eugene, OR)] in 6x SSPE-T for 30 min at 25°C, followed by 10 washes in 6x SSPE-T at 25°C. An antibody amplification step was followed using normal goat IgG as blocking reagent [final concentration of 0.1 m g/m l, (Sigma)] and biotinylated anti-streptavidin goat antibody [final concentration of 3 m g/m l, (Vector Laboratories, Burlingame, CA)]. This was followed by a staining step with a streptavidin-phycoerythrin conjugate [final concentration of 2 m g/m l (Molecular Probes)] in 6x SSPE-T for 30 min at 25°C and 10 washes in 6x SSPE-T at 25°C. The probe arrays were scanned at 560 nm using a confocal laser-scanning microscope (Hewlett Packard GeneArray Scanner G2500A; Affymetrix, Inc., Santa Clara, CA). The readings from the quantitative scanning were analysed by the Affymetrix Gene Expression Analysis Software. In this analysis, genes upregulated or downregulated > 3- 155 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. fold after drug treatment were categorized into different groups. This experiment was performed by Dr. Torben F. Omtoft and Dr. Thomas Thykjaer (Aarhus University Hospital, Skejby, Denmark). W estern blot analysis of DNMT protein levels Cells were lysed by the addition of radioimiramoprecipitation (RIPA) buffer (PBS, 0.1% SDS, 0.5% norddet P-40 and 0.5% sodium deoxycholate) and protein extracts were prepared as previously described (Velicescu et al. 2002). Approximately 60 pg total protein extract was electrophoresed and transferred to PVDF membranes overnight at 4°C as previously described (Velicescu et al. 2002). The membranes were hybridized with antibodies against hum an DNMT1 (1:1000 dilution; New England Biolabs, Beverly, MA), hum an DNMT3b (T-16; 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and proliferating cell nuclear antigen (PCNA) (1:4000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) in Tris-Buffered Saline-Tween (TBS-T) buffer (0.1 M Tris, 1.5 M NaCl, and 1% Tween 20) with 5% non-fat dry milk overnight at 4°C. The human DNMT3a antibody was provided by Dr. Ye-Guang H u (Shanghai, China). The membranes were washed 3 times with TBS-T buffer at room temperature, and incubated with secondary antibodies as follows: anti-mouse-IgG-HRP (1:3000 dilution for PCNA; Santa Cruz); anti-rabbit-IgG-HRP (1:2000 dilution for DNMT1; Santa Cruz); anti-goat-IgG-HRP (1:10000 for DNMT3b; Calbiochem, San Diego, CA). All were incubated with the membrane for 1 hr at room 156 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. temperature. Afterwards, the membranes were washed 5 times with TBS-T at room temperature. Proteins were detected with the ECL chemiluminescent detection kit (Amersham-Pharmacia, Piscataway, NJ) and by exposure to Kodak X-OMAT AR film (Rochester, NY). Quantitation of DNA methylation levels by methylation-sensitive single- nucleotide prim er extension assay Genomic DNA (4 pg) with digested with EcdFJ (Roche, Indianapolis, IN) and treated with sodium bisulfite as previously described (Cheng et al. 2003). Methylation analysis was performed using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay (Cheng et al. 2003). The bisulfite-PCR and the qualitative methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay for pl6 5' region was performed as previously described (Cheng et al. 2003). The bisulifte-PCR primers and conditions for M4- 4 and D4Z4 are previously shown in Table 3.2. The Ms-SNuPE primers and conditions for M4-4 and D4Z4 are previously shown in Table 3.3. Determination of cell doubling time and population doublings All cultured cells (3 x 105 cells/100-mm dish) were plated and treated with K T * M zebularine 24 h later and continuously for 8 days, with fresh zebularine and media changes every 3 days. The cell num ber/dish was counted 157 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. with a Z1 Coulter Particle Counter (Beckman Coulter Corporation, Hialeh, FL) every 2 to 3 days. Untreated cells were analyzed under similar conditions as a control. The average cell number from two plates was determined, and the mean cell numbers were plotted to define the cell population doubling times. Initial drug treatment was started 24 h after seeding. 158 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Zebularine selectively inhibits the growth of cancer cells but not normal fibroblasts A major concern for the clinical application of nucleoside analogs is their non-specific effects towards both normal and cancer cells. Previously, I have demonstrated the growth suppression of T24 bladder cancer cells after continuous treatment with zebularine (Cheng et al, submitted). To further investigate the growth inhibitory effects of continuous zebularine treatment, I extended our study to a panel of 4 additional hum an cancer cell lines in addition to T24 cells (CALU-1, HCT15, CFPAC-1, and PCS), as well as including three normal hum an fibroblast cell lines (LD98, T-l, LD419). Interestingly, continuous treatment with zebularine retarded the growth of all hum an cancer cell lines but had little effect on normal human fibroblasts (Table 4.2). I next examined whether this growth inhibitory effect was associated with upregulation of the mRNA for pll(W AFl) an d /o r p27(KIPl), which are inhibitors of cyciin-CDK complexes involved in Gt and S phase progression (Gong et al. 2003, Gotz et al. 1996, Tam et al. 1994). Growth inhibition in cancer cells was found in association with a 2-4 fold induction of the p21 CDK inhibitor, whereas p27 mRNA levels remained relatively unchanged (Figures 4.1, 4.2). All of the cancer cells, but not the normal fibroblasts, were therefore 159 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.2 Effects of zebularine on growth suppression in normal and cancer cells Cell L in es D oubling Time (Hrs) Zebularine (-) (+) Percent Increase Normal Cells LD98 41 43 4 T-l 43 46 6 LD419 29 29 1 Cancer Cells T24 21 30 44 HCT15 26 37 41 CFPAC-1 38 51 32 PCS 39 59 53 CALU-1 32 42 33 Normal fibroblasts and cancer cells were either untreated or treated in the presence of K b4 M zebularine continuously for 8 days. The effects of zebularine on cell growth inhibition were analyzed in these cell lines by comparing the doubling times of these cells before and after drug treatment. The percent increase represents the increase in doubling time of cells after treatment as compared to the untreated control cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Zebularine p21 p27 GAPDH LD419 LD98 J Figure 4.1: Effects o f zebularine on growth regulatory genes in normal fibroblasts. A panel of normal fibroblasts (LD98, T-l, LD419) was either untreated or treated in the presence of 10"4 M zebularine continuously for 8 days. Expression levels of p21 and p27 mRNAs were determined by RT-PCR analysis. GAPDH mRNA expression levels were measured to control for relative cDNA input. Q \ Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. HCT15 CFPAC-1 - + PC3 CALU-1 — + — + Zebularine GAPDH Figure 4.2: Effects o f zebularine on growth regulatory genes in cancer cells. A panel of cancer cell lines (T24 bladder cancer, HCT15 colon cancer, CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells) was either untreated or treated in the presence of 10"4 M zebularine continuously for 8 days. Expression levels of p21 and p27 mRNAs were determined by RT-PCR analysis. GAPDH mRNA expression levels were measured to control for relative cDNA input. O n K3 responsive to the growth-suppressive effects of zebularine, and these effects are seemingly p22-dependent. Heterogeneous effects of zebularine on p l6 gene expression and dem ethylation in normal and cancer cells To further evaluate the selective growth inhibition of cancer cells by zebularine, I next analyzed the re-expression of the pi 6 gene, which is known to be abnormally silenced by methylation in all of these cancer cell lines. The p i 6 gene was expressed and unmethylated in the normal fibroblasts, and the mRNA expression was largely unaffected by zebularine treatment (Figure 4.3). On the other hand, the pl6 gene was induced in T24, HCT15 and CFPAC-1, but not in CALU-1 or PCS cancer cells (Figure 4.3). The methylation levels of the 5' region of the pl6 gene were decreased by zebularine treatment in the 3 cell lines which were inducible (Figure 4.3). These results suggested that the growth inhibitory effects of zebularine may be partially due to the up-regulation of p21 gene in all five cancer cell lines an d /o r the induction of pl6 in T24, HCT15 and CFP AC-1 cells. I then assayed the methylation levels of two other loci that are known to be methylated in both normal and cancer cell lines. M4-4 is a single copy sequence located in a CpG island that was previously characterized in our laboratory (Cheng et al, submitted), while D4Z4 is a subtelomeric repeat 163 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 43: Effects of zebularine on pl6 gene reactivation and p l6 5' region methylation in normal and cancer cells. A panel of normal fibroblasts (LD98, T-l, LD419) and cancer cell lines (T24 bladder cancer, HCT15 colon cancer, CFPAC- 1 pancreatic cancer, PCS prostate cancer and CALU-1 lung cancer cells) were either untreated or treated in the presence of Iff4 M zebularine continuously for 8 days. (Top) Methylation status of the p!6 5' region was quantitated by Ms- SNuPE analysis (as described in "M aterials. and Methods"). Methylation percentage represents the average of three individual CpG sites in each region as assayed from two independent experiments. Error bars represent ranges of two determinations. (Bottom) The expression level of p i6 mRNA was determined by RT-PCR analysis. The GAPDH mRNA expression levels were the same ones shown above in Figures 4.1 and 4.2. 164 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o o o 00 o O O M %) u o iiF fii|i3 |^ u oiS au ,g 91^ w * ttj 1^9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequence (Kondo et al. 2000). Both M4-4 and D4Z4 were hypermethylated in all cell lines, and demethylation was observed only in T24, HCT15, and CFP AC-1 cells, while minimal demethylating effects were observed in normal fibroblasts, CALU-1 and PCS cancer cells (Figures 4.4, 4.5). Consistent with the finding for the methylation of the p i 6 5' region, these results confirmed that the methylation of all normal fibroblasts was unaffected by zebularine, as well as two cancer cell lines that were refractory to the induction of the pl6 gene by zebularine. Differential cellular responses to depletion of DNMT levels by zebularine. The mechanism by which zebularine demethylates almost certainly requires the incorporation of the drug into the DNA and the formation of covalent complexes with DNMT(s) to deplete their enzymatic activities, as is the case with both 5-Aza-CR and 5-Aza-CdR (Bouchard and Momparler 1983, H urd et al. 1999, Santi et al. 1984, Taylor et al. 1984, Zhou et al. 2002). Different assays have been developed to track the levels of extractable DNMTs and individual DNMTs bound to genomic DNA using antibodies specific for each DNMT (Liu et al. 2003, Velicescu et al. 2002). I had previously shown the effects of continuous treatment with zebularine on the protein levels of DNMTs in T24 bladder cancer cells using Western blot analysis (Cheng et al, submitted). It was of obvious interest to analyze the protein levels of DNMT1, 3a and 3b in 166 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. Zebularine (10"4 M) Control CFPAC-1 C A L U LD98 LD419 o\ Figure 4.4: Effects of zebularine on methylation of M4-4. A panel of normal fibroblasts (LD98, T-l, LD419) and cancer cell lines (T24 bladder cancer, HCT15 colon cancer, CFPAC-1 pancreatic cancer, PCS prostate cancer and CALU-1 lung cancer cells) were either untreated or treated in the presence of lO- 4 M zebularine continuously for 8 days. Methylation status of the M4-4 was quantitated by Ms-SNuPE analysis (as described in "Materials and Methods"). Methylation percentage represents the average of three individual CpG sites in each region as assayed from two independent experiments. Error bars represent ranges of two separate determinations. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. d o 100 n 80 *S 6 0 a Q m Control O Zebularine (10 4 M) 3 40 - 20 - 0 LD98 T -l LD419 T24 HCT15 CFPAC-1 PCS CALU-1 oo Figure 4.5: Effects of zebularine on methylation of D4Z4. A panel of normal fibroblasts (LD98, T-l, LD419) and cancer cell lines (T24 bladder cancer, HCT15 colon cancer, CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells) were either untreated or treated in the presence of 1 C H M zebularine continuously for 8 days. Methylation status of the D4Z4 was quantitated by Ms-SNuPE analysis (as described in "Materials and Methods"). Methylation percentage represents the average of three individual CpG sites in each region as assayed from two independent experiments. Error bars represent ranges of two separate determinations. the normal and cancer cells before and after continuous zebularine treatment. Zebularine barely affected the levels of extractable DNMT1, 3a and 3b2/3 in all normal fibroblasts, as well as PCS and CALU-1 cancer cells (Figures 4.6, 4.7). On the contrary, zebularine completely depleted DNMT1 in T24, CFPAC-1 and HCT-15 cancer cells, while only partially affecting DNMT3a and 3b2/3 in these cells (Figure 4.7). The mRNA levels of each DNMT, as measured by semiquantitative RT-PCR, remained unchanged after zebularine treatment (data not shown), suggesting that the depletion of DNMT proteins was due to trapping of the enzymes to the zebularine-incorporated DNA rather than an inhibition of transcription or cell proliferation. Cells that were refractory to the demethylating effects of zebularine showed minimal effects on the depletion of DNMT1, 3a and 3b. For instance, PCS cells showed only a partial depletion of DNMT1, suggesting that some of the drug may be incorporated into the DNA yet at a level that was insufficient to cause measurable demethylation. These results support the idea that demethylating effects of zebularine most likely require its incorporation into DNA followed by the complete depletion and inactivation of the methyltransferase enzyme(s). Zebularine substantially alters gene expression in T24 bladder cancer cells I also wanted to investigate the effects on continuous zebularine treatment on global gene expression profiles in normal and cancer cells. A high- 169 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. LD419 LD98 T-l Zebularine DNMT1 > ' DNMT3a > DNMT3b3 > 1 Figure 4.6: Effects of zebularine on D N M T protein levels in normal cells. Western blot analysis of DNMT1, DNMT 3a and 3b3 protein levels after continuous zebularine treatment (10"4 M) for 8 days in a panel of normal fibroblasts (LD98, T-l, LD419). DNMT3b3 represents the predominant isoform in that specific cell line. Cell lysates were obtained from treated and untreated control cells (as described in "Materials and Methods") and were analyzed by Western blot analysis with specific antibodies for DNMT1, DNMT3A, and DNMT3b proteins. o Reproduced w ith permission o f th e copyright owner. Further reproduction prohibited without permission. HCT15 CFPAC-1 PC3 CALU-1 Zebularine DNMT1 DNMTSa DNMT3b2 DNMT3B3 PCNA Figure 4.7: Effects o f zebularine on DNMT protein levels in cancer cells. Western blot analysis of DNMT 1, DNMT 3a and 3b2/3 protein levels after continuous zebularine treatment (10'4 M) for 8 days in a panel of cancer cell lines (T24 bladder cancer, HCT15 colon cancer, CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells). Either DNMT3b2 or 3b3 represents the predominant isoform in that specific cell line. Cell lysates were obtained from treated and untreated control cells (as described in “Materials and Methods”) and were analyzed by Western blot analysis with specific antibodies for DNMT1, DNMT3A, and DNMT3b proteins. density oligonucleotide gene expression microarray analysis on LD419 and T24 cells showed that a considerable number of genes were up- or downregulated by > 3-fold after 8 days of continuous drug treatment in T24, and rather few in LD419 cells (Table 43-4.6). A total of 35 genes were found to be overexpressed (> 3-fold) in T24 cells after 8 days of continuous zebularine treatment, and interestingly, 9 of these (26%) were cancer-related antigens (Table 4.3). The level of induced expression varied from 3-fold for GAGE2 to a maximum of 15-fold for SPANXA1. A total of 39 out of 13,300 genes were found to be downregulated in T24 cells by continuous zebularine treatment (Table 4.4). In contrast to T24 cells, only 4 genes were upregulated and 8 genes were downregulated in LD419 fibroblasts (Table 4.5, 4.6). This microarray analysis is also consistent with earlier work from my laboratory on the global effects of gene expression by 5-Aza-CdR in both cell lines (Liang et al. 2002). None of the genes that were up or downregulated overlapped for both cell lines. The microarray data demonstrated that a large cluster of cancer / testis-specific antigens were activated in cancer cells and not in normal cells, suggesting the potential utilization of zebularine in the upregulation of these genes which constitute promising targets for immunotherapy (Gillespie and Coleman 1999, Weber et al. 1994). This experiment was performed by Dr. Torben F. Omtoft and Dr. Thomas Thykjaer (Aarhus University Hospital, Skejby, Denmark). 172 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.3* Genes induced > 3 fold in T24 cells 8 days after continous zebularine treatment Probe Id Unigene Gene Symbol Fold Change Map Location 207739_s_at Hs.278444 G A G E 2 3 .0 xpll.4-pll.2 205130_at Hs.104119 R A G E 4.0 14q32 220922_s_at Hs.334464 S P A N X A 1 15.3 xq27.1 220217_x_at Hs.343879 SPANXC 6.7 xq27.1 209942_x_at Hs.36978 MAGEA3 11.7 xq28 214612_x_at H s.278460 MAGEA6 7 .4 xq28 210467_x_at Hs.169246 MAGEA12 4.9 xq28 211674_x_at Hs.167379 C T A G 1 4.3 xq28 215733_x_at Hs.87225 C T A G 2 3 .6 xq28 Interferon related 206924_at Hs.1721 IL 1 1 5.9 19ql3.3-ql3.4 206172_at Hs.25954 IL13RA2 3.8 xql3.1-q28 D ther 212003_at Hs.226770 D K FZP566C 0424 3.0 lp36.13 201761_at Hs.154672 MTHFD2 3.5 2pl2 205083_at Hs.174151 AOX1 3.4 2q33 204421_s_at Hs.284244 FGF2 4.2 4q26-q27 200730_s_at Hs.227777 PTP4A1 3.4 6ql2 202627_s_at Hs.82085 S E R P IN E 1 3.9 7q21.3-q22 205047_s_at Hs.75692 A S M S 4.0 7q21-q21 201616_s_at Hs.325474 C A L D 1 4.7 7q33 220892_s_at Hs.286049 P SA 4.4 9q21.2 201426_s_at Hs.297753 VIM 8 .1 10pl3 201490_s_at Hs.173125 PPIF 3 .2 10q22-q23 218566_s_at Hs.22857 CHORDC1 3 .0 llql4.2 205547_s_at Hs.75777 TAGLN 5.3 llq23.2 214452_at Hs.157205 BCAT1 3 .2 12pter-ql2 204222_s_at Hs.64639 G L IP R 1 3.8 12ql5 205660_at Hs.1 1 8 6 3 3 OASL 3 .2 12q24.2 219477_s_at Hs.325667 T H S D 1 3.5 13ql4.13 208290_s_at Hs.334810 E IF 5 3 .3 14q32.33 217370_x_at Hs.99969 FUS 3 .0 16pll.2 202397_at Hs.151734 NUTF2 3 .3 16q22.1 201195_s_at Hs.184601 SLC7A5 3.0 16q24.3 218738_s_at Hs.180403 S T R E M 3.0 18qll.2 204614_at Hs.75716 S E R P IN B 2 3.6 18q21.3 / 203887_s_at Hs.2030 THBD 3 .9 20pl2-cen / * ixperim ent p erfo rm ed by Drs. T. Qmtoft and T. Thykjaer (A arh u s University H o sp ital, Skejby, Denmark) 173 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.4* Genes downregulated > 3 fold in T24 cells 8 days after continous zebularine treatment Probe Id Urtigene Gene Symbol Fold Change Map location Other 202286_s_at Hs.23582 TACSTD2 -6.7 Ip32-p31 209114_at Hs.38972 TSPAN-1 -4.6 lpter~p32.1 201730_s_at Hs.169750 TPR -3.7 lq25 210827_s_at Hs.166096 E L F 3 -5.3 lq32.2 204622_x_at Hs.82120 NR4A2 -3.9 2q22-q23 208993_s_at Hs.77965 PPIG -3.2 2q31.1 203706_s_at Hs.173859 FZD7 -3.9 2q33 204035_at Hs.75426 S C G 2 -3.4 2q35-q36 208510_s_at Hs.100724 PPA R G -3.8 3p25 202688_at Hs.83429 TNFSF10 -11.0 3q26 205014_at Hs.1690 HBP17 -6.5 4pl6-pl5 207441_at Hs.2207 PROL3 -6.2 4ql3.3 201416_at Hs.83484 S O X 4 -5.4 6p22.3 209173_at H s.91011 A G R 2 -4.3 7p21.3 210029_at Hs.840 INDO -6 .1 8pl2-pll 203980_at Hs.83213 FABP4 -5.1 8q21 200696_s_at Hs.290070 GSN -3.2 9q33 209604_s_at Hs.169946 GATA3 -5.2 10pl5 204151_x_at Hs.306098 AKR1C1 -4.3 10pl5-pl4 211653_x_at Hs.201967 AKR1C2 -5.7 10pl5-pl4 200832_s_at Hs.119597 S C D -5.4 10q23-q24 213293_s_at Hs.318501 TRIM22 -8.4 llpl5 204070_at Hs.17466 R A R R E S 3 -4.3 Hq23 202291_s_at Hs.279009 MGP -9.3 12pl3.1-pl2.3 203824_at Hs.84072 TM4SF3 -9.6 12ql4.1-q21.1 212794_s_at Hs.12144 ■ K IA A 1033 -3.8 12q24.11 218175_at Hs.288909 F L J2 2 4 7 1 -3.0 12q24.31 218723_s_at Hs.76640 RGC32 -5.4 13ql3.3 212192_at Hs.109438 LOCI 15207 -3.6 13q22.1 203570_at Hs.65436 LOXL1 -3.0 15q22 203407_at Hs.74304 PPL -3.3 16pl3.3 208610_s_at Hs.197114 S R R M 2 -3.5 16pl3.3 202659_at H s.9661 PSMB10 -3.8 16q22.1 202054_s_at Hs.159608 A LD H 3A 2 -3.3 17pll.2 205623_at Hs.575 A L D H 3A 1 -7.2 17pll.2 204990_s_at Hs.85266 ITGB4 -3.2 17qll-qter 204734_at Hs.80342 KRT15 -4.4 17q21 207186_s_at Hs.99872 FALZ -3 .1 17q24 208663_s_at Hs.118174 TTC3 -3 .1 21q22.2 * E xperim ent p e rfo rm e d by Drs. T. Gmtoft and T. T hykjaer (Aarhus U n iv ersity Hospital, Skejfoy, Denmark) 174 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 45* Genes upregulated > 3 fold in LP419 8 days after continous zebularine treatment Probe Id Unigene Gene Symbol Fold Change Map Location 202437_s_at H s.154654 CYP1B1 4.7 2p21 209821_at Hs.348390 DVS27 3.0 9p24.2 204273_at Hs.82002 EDNRB 3.6 13q22 205870_at Hs.250882 BDKRB2 3.4 14q32.1-q32.2 — — — — — — — —^ * Experiment performed by Dr s. T. Omtoft and T. Thykjaer (Aarhus University Hospital, Skejby, Denmark) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. / T a b l e d Genes downregulated > 3 fold in LD419 8 days after continotts zebularine treatment Probe Id Unigene Gene Symbol Fold Change Map Location 217294_s_.at Hs.381173 ENOl -4.2 Ip36.3-p36.2 214336_s_.at Hs.75887 COPA -3.5 Iq23-q25 2 1 2 1 0 7 _ s_ .at Hs.74578 D D X 9 -3.9 lq25 214908_s_.at Hs.203952 T R R A P -3.0 7q21.2-q22.1 212009_s..at Hs.75612 S T IP 1 -3.0 Hql3 210338_s_.at Hs.180414 HSPA8 -3.5 llq24.1 211300_s..at Hs.1846 T P 53 -3.6 17pl3.1 202067_s_.at Hs.213289 LDLR -3.9 19pl3.3 * Experiment performed by Drs. T. Orntoft and T. Thykjaer (Aarhus University Hospital, Skejby, Denmark) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION An important concern for the clinical application of DNA methylation inhibitors is whether they will non-selectively affect normal as well as cancer cells. Here I have shown that zebularine can selectively target cancer cells but not normal fibroblasts, in terms of growth inhibition, demethylation, and depletion of DNA methyltransferases (DNMTs). My laboratory is currently testing the generality of these findings by testing the response of normal epithelially-derived cells to zebularine. While it is possible that they will not react in the same way as fibroblasts, my earlier studies using mice given daily doses of zebularine showed little evidence of toxicity. Zebularine may therefore be quite selective toward cancer cells and have therapeutic potential as an anticancer therapy. Although three of the five cancer cells treated with zebularine showed pl6 induction, all of them showed an up-regulation of the p21 but not the p27 gene. These CDK inhibitors are known to be differentially regulated (Gong et al. 2003, Ilyin et al. 2003), and DNA methylation is unlikely to play a role in the modulation of their expression because both genes were expressed even in the untreated cells. The negative effects of zebularine on the growth of all five cells might therefore be due to an induction of p21 an d /o r the pl6 gene. The p21 protein can directly arrest DNA replication in response to DNA damage by 177 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. binding to proliferating cell nuclear antigen (PCNA) (Waga et al. 1994). The increased p21 expression points to a novel mechanism for zebularine in addition to its effects on DNA methylation. The specificity of zebularine for cancer cells is probably due to differential metabolism compared to normal cells. The drug requires activation by phosphorylation before it can be incorporated into nucleic acids, and the conversion of the nucleoside to the monophosphate is the first step in this process. My laboratory is therefore currently determining whether cancer cells have higher levels of the uridine/cytidine kinase enzyme, which is the first enzyme in this process. Once phosphorylated, the 2-( 1 H)-pyrimidinone ring can be incorporated into RNA and into DNA after the reduction by ribonucleotide reductase of the ribose moiety to deoxyribose. The incorporation of the 2-(lH)- pyrimidinone ring into DNA is almost certainly required for the depletion of DNMT1 and the inhibition of DNA methylation that I observed in three of the five cancer cells. Indeed, preliminary experiments have shown that the 2-(lH)- pyrimidinone ring can be found in the DNA of treated T24 cells, although levels at ten times less than in RNA (Marquez et al, unpublished). The incorporation of 5-Aza-CR into RNA is known to produce disassembly of polyribosomes, co acceptor function of tRNA and marked inhibition of protein synthesis (Li et al. 1970, Momparler et al. 1976, Reichman and Penman 1973). The incorporation of zebularine into RNA could thereby conceivably explain the growth inhibitory 178 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. properties of the drug in the CALU-1 and PCS cells, even though no detectable DNA demethylation was observed. It was not altogether surprising to find cell lines refractory to the demethylating effects of zebularine, since various other cell lines have been to shown to be refractory to 5-Aza-CdR (Bender et al. 1998, Flatau et al. 1984, Michalowsky and Jones 1986, Michalowsky and Jones 1987). Zebularine might therefore be multi-factorial in its effects, but the specificity for cancer cells makes it an exciting drug. The differential depletion of DNMTs by zebularine in the normal and cancer cell lines is quite interesting. The levels of DNMT1, DNMTSa and DNMT3b2/3 were relatively unaffected in normal fibroblasts after zebularine treatment, suggesting that little zebularine is incorporated into their DNA. However, the partial depletion observed for DNMT1 in LD419 fibroblasts suggests that some drug may have been incorporated into the normal cellular DNA. The cancer cells that responded to zebularine in terms of demethylation showed a complete depletion of DNMT1 and partial depletion of DNMTSa and DNMT3b2/3. We had previously shown that zebularine preferentially depletes DNMT1 over the other DNMTs in T24 cells (Cheng et al, submitted), implicating a more specific and higher affinity of this drug for DNMT1. We have also previously shown that both 5-Aza-CdR and zebularine display differential depletion of DNMTs in T24 cells using Western blot analysis (Cheng et al, submitted; (Velicescu et al. 2002). The complete depletion of ■ 179 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DNMT1 appeared to correlate with the demethylation of various loci in these cell lines, suggesting that DNMT1 depletion may be an important indicator of the demethylating ability of zebularine. The reasons for the apparent specificity of the 2-{lH)-pyrimidinone ring for the DNMT1 enzyme is unexpected since all known cytosine-5 DNA methyltransferases appear to utilize the same mechanism of action (Cheng and Roberts 2001). Perhaps, zebularine exhibits a greater enzymatic binding affinity for DNMT1 than the other DNMTs. However, this is still unclear and needs to be clarified. Nevertheless, it was recently shown that DNMTSbS isoform has a reduced catalytic activity (Okano et al. 1999, Soejima et al. 2003), which might compromise its ability to complex with zebularine, thereby explaining the partial depletion of this enzyme by the drug. In addition, the partial depletion of DNMTSa by zebularine can potentially be due to the fact that DNMTSa protein is expressed throughout the cell cycle, as opposed to DNMT1 and DNMTSb which are cell-cycle regulated (Robertson et al. 2000), and the depletion of DNMTSa can therefore only occur exclusively during the S-phase in which the 2-(lH)-pyrimidinone ring is incorporated into the DNA. Our microarray data suggest that a combinatorial approach for the treatment of hum an cancers could involve the use of demethylating agents to augment the presentation of specific cell surface antigens, which could then be targeted by immunotherapy. The activation of cancer-related or tumor antigens 180 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the MAGE family has been observed in a number of hum an cancers, and this was shown to correlate with genomic DNA hypomethylation (De Smet et al. 1996). Based on these observations, therapeutic strategies have been recently developed that strive to direct cellular immunity towards tumors that express MAGE antigens (Gillespie and Coleman 1999). A caveat of this strategy is that the majority of hum an tumors do not express MAGE antigens, however, treatment of tumor cells with 5-Aza-CdR induces their expression (Liang et al. 2002b, Weber et al. 1994). Our finding show that MAGE genes and other cancer / testis-specific antigens represent a significant proportion of the genes upregulated by zebularine, and this appears specific to cancer/testis but not normal fibroblasts. Our microarray data also showed that a number of genes were downregulated in T24 bladder cancer cells after continuous zebularine treatment. These downregulated genes may be directly affected by the drug itself or due to the indirect induction of negative regulatory factors, as was postulated for 5-Aza-CdR (Liang et al. 2002b). Nevertheless, none of the down regulated genes belonged to the group of cancer-specific antigens. The combination regimen of zebularine followed by immunotherapy in the treatment of various human cancers will be the focus of future studies. In addition to its stability, minimal toxicity and ability to sustain demethylation through continuous administration, zebularine is also very selective towards cancer cells and thus represents a promising candidate for epigenetic therapy. 181 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER 5 SUMMARY AND CONCLUSIONS There is a growing realization that important control pathways in hum an cancers are commonly inactivated by epigenetic as well as genetic mechanisms. Transcriptional silencing by abnormal methylation of CpG islands is a prevalent epigenetic mechanism of tumor suppressor gene inactivation during tumorigenesis (Esteller and Herman 2002, Jones and Baylin 2002, Rountree et al. 2001), making them targets for reactivation by methylation inhibitors such as aza and other nucleoside analogs. Unfortunately, the mutagenic, cytotoxic, and chemically unstable properties of nucleoside analogs have discouraged and limited their widespread clinical use. Over the past 20 years, no new nucleoside analogs have been found which can effectively inhibit DNA methylation without having these undesirable properties associated with them. The research described in this thesis characterizes the role of a novel inhibitor of DNA methylation, zebularine, and potential application in utilizing the drug as a chemotherapeutic agent for reversing abnormal methylation in cancer. In Chapter 2, my studies focused on examining the effectiveness of zebularine as a novel inhibitor of DNA methylation in Neurospom crassa, cultured mammalian cells and tumor cells grown in the nude mice. The ability of zebularine to reactivate the hph gene, a gene silenced by methylation in 182 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Neurospora crassa, was demonstrated using a hygromycin gene reactivation assay. Zebularine was shown to inhibit DNA methylation and reactivate the hph gene. I then analyzed the inhibition of DNA methylation by zebularine in mammalian cells, C3H 10T1/2 C18 (1011/2) mouse embryo cells and 124 hum an bladder carcinoma cells, using the methylation-sensitive single nucleotide single primer extension (Ms-SNuPE) assay. Furthermore, the effects of zebularine on tumor growth and reactivation of a silenced p i 6 gene in the hum an bladder carcinoma cells grown in BALB/c nu/nu mice were also evaluated. Ih e results demonstrate that zebularine was capable of inducing the myogenic phenotype in 1011/2 cells, reactivating a silenced pl6 gene and demethylating its prom oter/5' region in 124 bladder carcinoma cells in vitro and in tumors grown in nude mice. Zebularine was also minimally cytotoxic to 124 cells in vitro and to tumor-bearing mice as assessed by minimal weight changes. Compared with tumor volumes in control mice, tumor volumes were significantly reduced in mice treated with high dose zebularine administered by intraperitoneal injection (Pc.001) or by oral gavage feeding (Pc.OQl). Ihese results suggest zebularine is a stable, effective inhibitor of DNA methylation and the first drug in its class able to reactivate an epigenetically silenced gene by oral adminstration. Motivated by my findings in Chapter 2 which demonstrated that zebularine was less potent than either 5-Aza-CR or 5-Aza-CdR in the animal 183 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cell in vitro systems and that the drug necessitated relatively large doses to achieve its inhibitory effects, I proceeded to find a more effective method in potentiating the effects of zebularine in Chapter 3, I examined the effects of continuous treatment with zebularine on the reactivation of the p l6 gene and demethylation of pl6 5' region in T24 bladder cancer cells. I show that continuous application of zebularine to T24 cells induces and maintains pl6 gene expression and sustains demethylation of the 5' region for over 40 days, preventing remethylation from occurring. In addition, continuous zebularine treatment effectively and globally demethylated various hypermethylated regions, especially CpG-poor regions. Also, zebularine selectively caused a complete depletion of extractable DNA methyltransferase 1 (DNMT1), whereas only partial depletions of DNMT3a and DNMT3b3. Lastly, sequential treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR) followed by zebularine was shown to hinder the remethylation rate of p l6 5' region and gene re-silencing, suggesting clinical promise in the combination application of both drugs for cancer therapy. In Chapter 4, I sought to compare the effects of zebularine on normal hum an fibroblasts and hum an cancer cells. Using a panel of 3 normal fibroblasts (LD98 and LD419 bladder fibroblasts and T-l skin fibroblasts) and 5 cancer cells (CALU-1 lung cancer, PCS prostate cancer, CFPAC-1 pancreatic cancer, T24 bladder cancer and HCT15 colon cancer), I investigated the effects 184 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of continuous zebularine treatment on cell growth, pl6 gene activation, methylation of pi 6 5' region, M4-4 and D4Z4, and DNMT protein levels. I show that zebularine selectively suppressed the cell growth in all cancer cells but not in normal fibroblasts. Zebularine preferentially upregulated p2t in the cancer cell lines but not normal fibroblasts, suggesting the mechanism for growth suppression was p22-dependent. The results also show that zebularine demethylated various methylated genes and depleted DNA methyltransferase 1 (DNMT1) in 3 out of 5 cancer cell lines but not normal fibroblasts. Finally, to compare the global gene expression profile before and after continuous zebularine treatment, T24 bladder cancer cells and LD419 normal fibroblasts were analyzed using gene expression microarray chips. Zebularine strongly altered gene expression in T24 cells by activating tumor suppressor and cancer- related antigen genes but induced few changes in LD419 normal fibroblasts. Thus, zebularine is selective towards cancer cells and may represent a strong candidate for epigenetic therapy. My studies described in this thesis characterized the role of a novel demethylating agent, zebularine and the potential clinical utilization of this drug as a chemotherapeutic agent to possibly reactivate dormant growth regulatory genes silenced by aberrant de novo methylation. Zebularine, a stable cytidine analog, is not only minimally toxic in vitro and in vivo but also active when administered orally in vivo. In addition, this drug can prevent 185 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. remethylation when administered continuously to cultured cells and is apparently selective towards cancer cells. These properties give the drug significant potential as a useful pharmacologic inhibitor of methylation in biological systems and make it a promising candidate as an antitumor agent for the treatment of cancers which arise as a result of methylation errors. 186 Reproduced with permission of the copyright owner. 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Creator Cheng, Jonathan Chi-Hong (author)

Core Title Characterization of zebularine: A novel inhibitor of DNA methylation with clinical potential

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, molecular,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Jones, Peter A. (committee chair), Coetzee, Gerhard (committee member), Laird, Peter W. (committee member)

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