Genome-wide analysis of aberrant CpG island methylation in human tumors [Elektronische Ressource] / vorgelegt von Claudia Gebhard

Claudia Berger

Sujets

• Gesundheit

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 19
• Langue: Deutsch
• Poids de l'ouvrage: 25 Mo

Extrait

Genome-wwwiiidddeee aaannnaaalllyyysssiiisss ooofff aaabbbeeerrrrrraaannnttt CCCpppGGG iiisland
methylation in human tumors


Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen
Fakultät III BBiioologie und vorklinische Medizin ddeerr UUnniivveerrssiittäätt
Regensburg







vorgelegt von
Claudia Gebhard
aus Rötz
Januar 2010




The work presented in this thesis was carried out in the Department of Hematology and
Oncology at the University Hospital Regensburg from July 2005 to January 2010.


Die vorliegende Arbeit entstand in der Zeit von Juli 2005 bis Januar 2010 in der Abteilung für
Hämatologie und internistische Onkologie des Klinikums der Universität Regensburg.



















Promotionsgesuch eingereicht am: 20. Januar 2010
Die Arbeit wurde angeleitet von: PD Dr. Michael Rehli, Prof. Dr. Stephan Schneuwly



Prüfungsausschuss:
Vorsitzender: Prof. Dr. Richard Warth
1. Prüfer (Erstgutachten): Prof. Dr. Stephan Schneuwly
2. Prüfer (Zweitgutachten): Prof. Dr. Michael Rehli
3. Prüfer: Prof. Dr. Armin Kurtz














Real success
is finding your lifework
in the work that you love.

David McCullough










Table of Contents
1 INTRODUCTION ........................................................................................... - 1 -
1.1 Characterization of specific tumor types ............................................................................ - 1 -
1.1.1 Leukemia ......................................................................................................................... 1
1.1.1.1 Normal hematopoiesis and leukemia development .................................................... 1
1.1.1.2 Acute myeloid leukemia (AML) .................................................................................... 2
1.1.2 Colorectal cancer ............................................................................................................. 4
1.2 The concept of epigenetics .................................................................................................. - 5 -
1.3 DNA methylation .................................................................................................................... - 5 -
1.4 Biological functions and consequences of DNA methylation .......................................... - 7 -
1.5 Regulation of DNA methylation ........................................................................................... - 8 -
1.6 Epigenetics and gene regulation ......................................................................................... - 9 -
1.6.1 Mechanisms of methylationmediated gene silencing ..................................................... 9
1.6.2 Cooperation between DNA methylation and chromatin modifications .......................... 10
1.6.3 The histone code ........................................................................................................... 13
1.6.3.1 Histone acetylation .................................................................................................... 14
1.6.3.2 Histone methylation ................................................................................................... 15
1.6.3.3 Recognition of chromatin modifications and the translation of the histone code ...... 15
1.6.4 Noncoding RNA ............................................................................................................ 18
1.7 Epigenetic alterations during tumorigenesis ................................................................... - 18 -
1.7.1 Global hypomethylation ................................................................................................. 19
1.7.2 Regional hypermethylation ............................................................................................ 20
1.7.3 Differential DNA methylation patterns in AML and colorectal cancer ........................... 21
1.7.4 Differential histone modifications in tumors ................................................................... 22
1.7.5 Therapeutic strategies targeting epigenetic aberrations ............................................... 24
2 RESEARCH OBJECTIVES ......................................................................... - 26 -
3 MATERIAL AND EQUIPMENT ................................................................... - 27 -
3.1 Equipment ............................................................................................................................ - 27 -
3.2 Consumables ....................................................................................................................... - 28 -
3.3 Chemicals ............................................................................................................................. - 29 -
3.4 Enzymes and kits ................................................................................................................ - 29 -
3.5 Molecular weight standards ............................................................................................... - 30 -
3.6 Oligonucleotides ................................................................................................................. - 30 -
3.6.1 Sequencing primers ....................................................................................................... 30
i 3.6.2 Realtime PCR primers for MCIp ................................................................................... 31
3.6.3 Realtime PCR primers for ChIPonchip ...................................................................... 32
3.6.4 Realtime RTPCR primer .............................................................................................. 33
3.6.5 LMPCR oligonucleotides .............................................................................................. 33
3.6.6 Bisulfite amplicon generation (Nested PCR) ................................................................. 33
3.6.7 MassARRAY QGE ......................................................................................................... 34
3.6.7.1 Oligonucleotides ........................................................................................................ 34
3.6.7.2 Competitors ................................................................................................................ 34
3.6.8 Bisulfite amplicon generation (MassARRAY) ................................................................ 35
3.7 Antibodies ............................................................................................................................ - 35 -
3.8 Antibiotics ............................................................................................................................ - 35 -
3.9 Plasmids ............................................................................................................................... - 35 -
3.10 E.coli strains ........................................................................................................................ - 35 -
3.11 Cell lines ............................................................................................................................... - 36 -
3.12 Databases and software ...................................................................................................... - 36 -
3.13 Statistical testing ................................................................................................................. - 37 -
4 METHODS ................................................................................................... - 38 -
4.1 General cell culture methods ............................................................................................. - 38 -
4.1.1 Cell line culture conditions and passaging .................................................................... 38
4.1.2 Culturing of stably transfected Drosophila S2 cells and expression of the
methyl binding polypeptide MBDFc .............................................................................. 38
4.1.3 Assessing cell number and vitality ................................................................................. 39
4.1.4 Freezing and thawing cells ............................................................................................ 39
4.1.5 Mycoplasma assay ........................................................................................................ 40
4.1.6 Isolation of human monocytes ....................................................................................... 40
4.2 General protein biochemical methods .............................................................................. - 41 -
4.2.1 Purification of the recombinant protein MBDFc ............................................................ 41
4.2.1.1 Dialysis ....................................................................................