DNA hypomethylation and gene expression in bladder cancer [Elektronische Ressource] / vorgelegt von Olusola Yakub Dokun

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• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2009
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Forschungslabor der Urologischen Klinik
des Universitätsklinikum Düsseldorf
Direktor: Prof. Dr. med. P. Albers









DNA Hypomethylation and Gene Expression in
Bladder Cancer







Inaugural-Dissertation



zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf


vorgelegt von

Olusola Yakub Dokun
aus Osun state, Nigeria



Düsseldorf, Juni 2009
TABLE OF CONTENTS
1.0 Introduction 1
1.1 DNA methylation: an overview 1
1.2 The multiple roles of DNA methylation in cancer 5
1.3 DNA hypermethylation 5
1.3.1 Causes of DNA hypermethylation 5
1.3.2 Consequences of DNA hypermethylation 7
1.4 DNA hypomethylation 8
1.4.1 Causes of DNA hypomethylation 8
1.4.2 Consequences of DNA hypomethylation 9
1.5 Bladder cancer 10
1.5.1 Pathogenesis of bladder cancer 11
1.5.2 Biomarkers in bladder cancer 13
1.6 Selected genes from microarray studies 15
1.6.1 S100A4 and S100A9 15
1.6.2 SNCG 17
1.6.3 LCN2 18
1.7 Aim of study 19

2.0 Materials and Methods 21
2.1. Tissues, Cells and Materials 21
2.1.1 Bladder and Prostate cancer cell lines 21
2.1.2 Bladder Tissue samples 22
2.2 Chemicals and Regents 24
2.3 Enzymes and antibodies 25
2.4 Kits 25
2.5 Growth media, buffers and solutions 25
2.6 Oligonucleotide primers and PCR assays 29
2.6.1 Oligonucleotides primers 29
2.6.2 PCR reagents 29
2.7 Equipments and materials 31
2.8 Softwares and databases 31
2.9 Cultivation of human cells 32
2.9.1 Culture of cancer cell lines and fibroblasts 32
2.9.2 Preparation of primary urothelial cells from human ureters 32
2.9.3 Treatment of cultured cells with demethylating agent 33
2.10 Preparation of nucleic acids from human cells 34
2.10.1 RNA isolation from cultured cells and frozen tissues 34
2.10.2 Genomic DNA isolation from cultured cells 34
2.11 Cloning of PCR products 35
2.11.1 Ligation 35
2.11.2 Transformation 35
2.11.3 Plasmid purification 35
2.12 RT PCR 36
2.12.1 Reverse transcription 36
2.12.2 Quantitative PCR 37
2.13 Analysis of modified DNA 41
2.13.1 Bisulfite treatment of DNA 41
2.13.2 PCR analysis of bisulfite treated DNA 42


2.14 Microarray experiments 43
2.14.1 Microarray I 43
2.14.2 Microarray II 43

3.0 Results 46
3.1 Results of microarray I 46
3.1.1 Expression analysis of hypomethylation candidate genes 49
3.1.2 Expression analysis of SNCG 49
3.1.3 Expression analysis of S100A4 51
3.1.4 Expression analysis of S100A9 52
3.1.5 Expression analysis of LCN2 53
3.1.6 Expression analysis of SNCG, S100A4, S100A9 and LCN2
in normal bladder and tumor tissue samples 55
3.1.7 Methylation analysis of the regulatory regions of SNCG,
S100A4, S100A9, LCN2 and an intronic regulatory region of S100A4 57
3.1.8 Methylation analysis of SNCG 58
3.1.9 Methylation analysis of S100A4 60
3.1.10 Methylation analysis of S100A9 62
3.1.11 Methylation analysis of LCN2 63
3.2 Results of microarray II 64
3.2.1 Design and general evaluation of microarray II 64
3.2.2 Bioinformatic analysis of the candidate list of genes 66
3.2.3 Expression analysis of H2AFY 76
3.2.4 Expression analysis of PCAF 77
3.2.5 Expression analysis of MYST4 78
3.2.6 Expression analysis of JMJD1A 79
3.2.7 Expression analysis of MYST4, JMJD1A, H2AFY,
PCAF and CBX7 in normal bladder and tumor tissue samples 80
3.2.8 Expression analysis of DDX58 82
3.2.9 Expression analysis of KLF4 83
3.2.10 Expression analysis of SIRT7 84
3.2.11 Expression analysis of LOXL2 85
3.2.12 Expression analysis of SIRT1 86
3.2.13 Expression analysis of DEPDC1 87

4.0 Discussion 88
4.1 DNA methylation and expression of SNCG, S100A4,
S100A9 and LCN2 in bladder cancer 88
4.1.1 Expression of SNCG, S100A4, S100A9 and LCN2 in human cancers 88
4.1.2 Relationship between expression and methylation
of SNCG, S100A4, S100A9 and LCN2 in bladder cancer 91
4.2 Analysis of further hypomethylation candidate genes 97
4.2.1 Searching further hypomethylation candidate genes by
microarray expression analysis of differential response to 5;aza;dC 97
4.2.2 Expression analysis of candidate genes from microarray II
in urothelial carcinoma 103

5 Summary 115

6 References 119

7 Appendix 133

8 List of Abbreviations 148

9 Acknowledgement 149
1 Introduction 1

INTRODUCTION
1.1 DNA methylation: an overview
DNA methylation is a reversible modification of DNA, characterized in mammalian cells by
the addition of a methyl group from S;adenosylmethionine to the carbon 5 position of
selected cytosine residues that precede a guanine residue. This reaction is catalysed by DNA
methyltransferases, which include DNMT1, DNMT3A and DNMT3B. DNMT1 is a
maintenance methyltransferase that preferentially transfers methyl groups to hemimethylated
DNA subsequent to replication. DNMT3A and DNMT3B are de novo methyltransferases
capable of transferring methyl groups to CpG dinucleotides of unmethylated DNA [Goll et al,
2005]. DNMT1 and DNMT3B act cooperatively in many instances during development [Kim
et al, 2002; Reik 2007], but also to repress genes in human cancer [Rhee et al, 2002]. The
DNMT3A plays an active role in paternal and maternal imprinting [Kaneda et al, 2004]. In
male germ cell development, it forms heterotetramers with a further member of the DNMT
family, DNMT3L, which lacks important catalytic amino acids and acts as a regulatory
subunit [Cheng and Blumenthal, 2008].
Because methylated cytosines tend to mutate towards thymines in the course of evolution,
there are fewer than expected CpG dinucleotides in the mammalian genome. The majority of
these are nearly always methylated. However, CpG dinucleotides clustered in stretches of
DNA known as CpG islands are nearly always unmethylated (>95%). CpG islands are now
defined as having minimum G:C content of 55% and a CpG to GpC ratio of at least 0.65
[Laird et al, 2003]. They vary in size from 0.5 to 5 kb and are associated with about 50% of
mammalian genes. These CpG islands are predominantly located around the transcriptional
start site including the basal promoter region of human genes. Methylation within these
islands is associated with repression of the corresponding gene [Esteller 2008; Jone and
Baylin 2007]. The fraction of CpG;islands at the 5’;end of genes that are methylated in
various normal somatic cells is estimated as around 5% [Weber et al, 2007], but the fraction
increases in certain pathological states (see below).
DNA methylation plays vital roles in the regulation of gene expression, cellular
differentiation and development, genomic imprinting, X;chromosome inactivation, repression
of retrotransposons, maintenance of chromosome integrity, brain function and development
of the immune system [Miranda and Jones, 2007; Schulz and Dokun, 2009]. DNA
methylation is part of the complex epigenetic network that regulates gene expression and
genomic structure. DNA methylation and DNA methyltransferases interact mutually with
1 Introduction 2

proteins that function in the modification of histones and remodelling of chromatin. This
interaction regulates the DNA methylation patterns throughout the genome and at specific
genes under normal states as well as in disease state.
Histones in transcriptionally active chromatin regions and in particular at CpG;islands and
other active regulatory regions are acetylated at various sites. Sequences methylated at CpG
sites by DNA methyltransferases (DNMT1, DNMT3A or DNMT3B) are targeted by methyl;
binding domain (MBD) proteins like MBD2 and MeCP2. The presence of MBDs attracts
histone deacetylases (HDAC1 and HDAC2) and chromatin remodeling activities resulting in
the stable transformation of the chromatin structure from an open to a closed conformation
that prevents transcriptional activation. This inactive epigenetic state is characterized by
deacetylated histones.






















Figure 1.1 The effect of DNA methylation on chromatin structure as depicted by Robertson and Wolffe,