Investigation of the effects of the histone deacetylase inhibitor SAHA on the medulloblastoma cell line DAOY [Elektronische Ressource] / vorgelegt von Pham Thu Thuy

Gutjahr

Sujets

• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Investigation of the effects of the histone 
deacetylase inhibitor SAHA on the 
medulloblastoma cell line DAOY 
Inauguraldissertation
zur
Erlangung des akademischen Grades
Doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald


Vorgelegt von Pham Thu Thuy
Geboren am 24.12.1980
in Namdinh, Vietnam



Greifswald, 06/2009
Dekan: Prof. Dr. Klaus Fesser
1. Gutachter: Prof. Dr. Uwe Völker
2. Gutachter: Prof. Dr. James F. Beck
Tag der Promotion: 29/06/2009
Erklärung

Hiermit erkläre ich, daß diese Arbeit bisher von mir weder an der Mathematisch-
Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch an
einer anderen wissenschaftlichen Einrichtung zum Zwecke der Promotion eingereicht wurde.
Ferner erkläre ich, daß ich diese Arbeit selbstständig verfasst und keine anderen als die darin
angegebenen Hilfsmittel benutzt habe.

Greifswald, Juni 2009 Unterschrift


Pham Thu Thuy CONTENTS
Abbreviations
Summary
Chapter 1: Introduction ......................................................................................................... 1
1.1. Childhood brain tumors ................................................................................................ 1
1.1.1. Overview of pediatric primary central nervous system tumors .............................. 1
1.1.2. Medulloblastoma ..................................................................................................... 2
1.1.3. Current treatment of medulloblastoma .................................................................... 4
1.1.4. DAOY cell line ....................................................................................................... 5
1.2. Epigenetic mechanisms of cancer .................................................................................. 5
1.2.1. DNA methylation .................................................................................................... 6
1.2.2. RNA associated mechanisms 6
1.2.3. Histone modification ............................................................................................... 7
1.3. Role of histone acetylation in the regulation of gene expression .................................. 7
1.3.1. Histone acetyltransferases (HATs) ......................................................................... 8
1.3.2. Histone deacetylases (HDACs) ............................................................................... 8
1.3.3. Expression of HATs and HDACs in cancer cells .................................................... 9
1.4. Histone deacetylase inhibitors (HDIs) in cancer treatment ......................................... 10
1.4.1. Classification of HDIs ............................................................................................ 11
1.4.2. Molecular mechanism of action of HDIs .............................................................. 11
1.4.3. SAHA - a potential drug for cancer treatment ...................................................... 14
1.5. Gel-based and gel-free proteomic approaches ............................................................. 15
1.6. Aims of the dissertation ................................................................................................ 18
Chapter 2: Materials and methods ..................................................................................... 20
2.1. Materials ....................................................................................................................... 20
2.1.1. Cell line ................................................................................................................. 20
2.1.2. Chemicals . 21
2.1.3. Instruments ............................................................................................................ 23
2.2. Methods ........................................................................................................................ 23
2.2.1. Cell harvesting by trypsinization .......................................................................... 23
2.2.2. Preparation of protein extracts by freezing and thawing ...................................... 24
2.2.3. Protein quantification by Bradford assay .............................................................. 24
2.2.4. Gel-based proteomic approaches 24
2.2.4.1. 2D-DIGE technique ........................................................................................... 25 2.2.4.2. Analysis of protein post-translational modifications ......................................... 29
2.2.4.3. Protein identification by mass spectrometry ...................................................... 32
2.2.5. Gel-free proteomic approaches ............................................................................. 35
2.2.5.1. Preparation of samples for LC-MS/MS analysis ............................................... 35
2.2.5.2. Analysis of peptide mixtures by 1D-RP-LC-MS/MS ........................................ 35
2.2.5.3. Database searching and data analysis for gel-free protein identification and
relative quantitation ......................................................................................................... 35
2.2.6. Functional classification of proteins ...................................................................... 36
Chapter 3: Results ................................................................................................................ 38
3.1. 2D gel-based proteomic approaches ........................................................................... 39
3.1.1. 2D proteome reference map of DAOY cells .......................................................... 39
3.1.2. Quantitative analysis by 2D-DIGE ....................................................................... 47
3.1.3. Analysis of acetylated proteins by 2D Western blot analysis ................................ 62
3.1.4. Quantitative analysis of phosphoproteins .............................................................. 68
3.2 Gel-free proteomic approaches ..................................................................................... 72
3.2.1. Protein identification by 1D-RP-LC-MS/MS ....................................................... 72
3.2.2. Protein quantification by spectral counting ........................................................... 73
3.3. Comparison of results from 2D gel-based and gel-free approaches ............................ 79
3.3.1. Protein identification by 2D-PAGE-MALDI-MS and 1D-RP-LC-MS ................ 79
3.3.2. Protein quantification by 2D-DIGE and spectral counting ................................... 80
Chapter 4: Discussion ........................................................................................................... 86
4.1. 2D proteome reference map of DAOY cells and protein identification ..................... 86
4.2. Effects of SAHA on protein expression profile of DAOY cells ................................. 87
4.3. Effects of SAHA on protein post-translational modifications .................................. 100
Conclusion ........................................................................................................................... 103
References ......... 104
Acknowledgements
Curriculum vitae
Appendix
Abbreviations
2-DE : Two dimensional electrophoresis
ACN : Acetonitrile
AP Alkaline phosphatase :
APS : Ammonium persulphate
BCIP : 5-brom-4-chlor-3-indoxylphosphate
BSA : Bovine serum albumin
CHAPS : 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
CNS Central nervous system :
DMEM : Dulbecco's Modified Eagle Medium
DMF : Dimethylformamide
DTT : Dithiothreitol
HAT : Histone acetyltransferase
HDAC Histone deacetylase :
HDI : Histone deacetylase inhibitor
IAA : Indole-3-acetic acid
IEF : Isoelectric focusing
IPA : Ingenuity pathway analysis
IR Ionizing radiation :
NBT : Nitro blue tetrazolium chloride
PANTHER : Protein analysis through evolutionary relationships
PBS : Phosphate buffered saline
PTM : Post translational modification
PVDF Polyvinylidene difluoride :
SAHA : Suberoyl anilide hydroxamic acid
SDS : Sodium dodecyl sulphate
SILAC : Stable isotope labelling by amino acids in cell culture
TBS : Tris buffered saline
TEMED N,N,N',N'-tetramethylethylenediamine :
TFA : Trifluoroacetic acid
TRAIL : Tumor necrosis factor related apoptosis inducing ligand
TrEMBL : Translated EMBL nucleotide sequence data library
TRIS : Trishydroxymethyl aminomethane
WHO The world health organization :
min : Minute
rpm : Rounds per minute
sec : Second


SUMMARY
Medulloblastoma is one of the most common malignant childhood brain tumors.
Although advances in multimodal treatment have significantly improved the survival rate, the
outcome of children is still very poor. Therefore, there is an urgent need to develop novel
approa