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

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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.
Publié le : jeudi 1 janvier 2009
Lecture(s) : 21
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Source : UB-ED.UB.UNI-GREIFSWALD.DE/OPUS/VOLLTEXTE/2009/646/PDF/090629_THUY_DISSERTATION.PDF
Nombre de pages : 165
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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
approaches that can increase survival and reduce long term side effects of patients.
Histone deacetylase inhibitors (HDIs) have emerged as a promising new class of
antineoplastic agents in cancer therapy. Among them, suberoylanilide hydroxamic acid
®(SAHA, vorinostat, Zolinza ) is a highly potential HDI which has been approved for
treatment of cutaneous T-cell lymphoma and is currently used for treatment of various tumor
cell types both in vivo and in vitro. However, only little information has been reported on the
effects of SAHA on primary central nervous system (CNS) tumors including
medulloblastoma.
The DAOY cell line represents the most widely used model cell line for investigation
of medulloblastoma. In a recent study, it was reported that SAHA induces apoptosis and cell
cycle arrest of DAOY cells (Sonnemann et al., 2006). However, the molecular mechanisms
underlying this antitumor activity are still not clear. Therefore, in this study, effects of SAHA
on DAOY cells were analysed at the protein level by using both gel-based and gel-free
proteomic approaches.
A 2D proteome reference map of DAOY cells in pH range of 4-7 was created from
control and 10 µM SAHA treated cells via a combined analysis using 2D electrophoresis and
MALDI-TOF/TOF-MS. This reference map covers 1196 identified protein spots of more than
770 distinct proteins. This is the first report of a 2D proteome map of SAHA treated DAOY
cells. Moreover, the number of covered proteins was increased with the aid of a 1D-RP-LC-
ESI-MS/MS analysis. Both methods together gave rise to a total of over 1200 distinct protein
species, which is the largest catalogue of proteins identified in DAOY cells so far.
In SAHA treated cells, a series of proteins were found to be subjected to protein
degradation after treatment with the drug, including mainly cytoskeleton proteins (e.g. β-
tubulin, β/γ-actin, vimentin, filamin interacting protein 1), heat shock protein HS90B and a
component of the FACT chromosomal remodelling complex (SSRP1). Most of those proteins
are known substrates for caspases. Interestingly, several of these protein degradations are
reported as typical apoptotic events in brain cells such as fragmentations of lamin A/C, α-
spectrin, myosin-9 and SSRP1.
i
The 2D reference map was then used as an annotated database for further investigation
of changes in protein expression and protein modification profiles of DAOY cells following
SAHA treatment. By using the 2D-DIGE technique, SAHA was found to induce significant
changes in protein levels of DAOY cells, especially at the concentration of 10 µM while
considerably fewer changes in the protein pattern were observed after treatment with the
lower dose of 2 µM.
Quantitative analysis of total protein extracts using the 2D-DIGE technique
(employing pH range of 4-7) and spectral counting (employing a 1D-RP-LC separation)
resulted in the identification of 213 differentially expressed proteins after treatment with 10
µM SAHA. Most of the targeted proteins belong to the groups of cytoskeleton proteins (e.g.
lamin B1, calreticulin, dynexin), heat shock proteins (e.g. HSP71, HSP7C, CH60, GRP78)
and brain signal transductors (e.g. 14-3-3E, 14-3-3T, CRK, MARCS). Other proteins that
changed in levels after SAHA treatment include proteins involved in chromatin remodelling
(e.g. RUBV1, RUBV2), transcription regulation (e.g. YBOX, CBX5), redox regulation (e.g.
TXND4, TXND5, BIEA), metabolism (e.g. G6PI, K6PP, LDHB) and RNA processing
(HNRP K). In addition, cathepsin D, one of autophagic executors, was increased by SAHA
treatment while different subunits of the 26S proteasome complex were decreased in levels
after addition of SAHA. Interestingly, we found alterations of mitochondrial proteins
indicating the perturbation of mitochondrial function.
VDACs are pore forming proteins located on the outer mitochondrial membrane
which is known to play an important role in the release of apoptogenic proteins such as
cytochrome-c from mitochondria to cytoplasm and induction of apoptosis. In this study,
VDAC1 and VDAC3 were found to be overexpressed after incubation with SAHA, which
might lead to an extensive release of apoptogenic proteins. This result is consistent with the
study of Sonnemann and co-workers showing that SAHA induced the mitochondrial apoptotic
pathway of DAOY cells (Sonnemann et al., 2006).
Furthermore, these results are also in agreement with the previously known antitumor
activities of SAHA reported for other cancer cell lines, e.g. the up-regulation of heat shock
proteins, prostaglandin synthase 3, ubiquinol cytochrome c reductase or the down-regulation
of MARCS proteins.
In the second part of the work reported here, changes in protein modification profiles
of DAOY cells were analysed by 2D western blot analysis (for acetylation) and direct staining
with Pro-Q Diamond (for phosphorylation). By 2D Western blot analysis employing a
ii
specific antibody directed against the ε-acetylated lysine residues of proteins, SAHA was
shown to induce a strong accumulation of acetylated proteins. Besides histones as the primary
target, 43 protein spots were found to display increased acetylation levels after addition of
SAHA as compared to control cells. Among them, there are different isoforms of α-tubulin,
moesin, heat shock proteins (HSP71 and HSP7C), FK506-binding protein 4 (FKBP4) and
glutathione S-transferase Mu 3 (GSTM3), which might be important targets for further
investigation.
On the other hand, using the Pro-Q diamond stain, ~25% of expressed proteins in
DAOY cells were reported to be potential phosphoproteins with most of them being low
abundant proteins. However, treatment with SAHA induced only minor changes in
phosphorylation status of these proteins. Among them, we found different isoforms of
Sequestosome-1 (p62) protein. However, the changes were indirect due to the decrease in the
level of this highly phosphorylated protein but not the change in the phosphorylation status of
isoforms.
The study demonstrates the usefulness of complementary LC-based and 2D gel-based
strategies in the proteomic analysis of DAOY cells. Overexpression of important proteins
such as VDACs and cathepsin D should be further investigated by other methods such as
Western blotting. Moreover, in the LC-based strategy, prefractionation methods could be
employed to cover more hydrophobic proteins.


iii
Introduction Dissertation
CHAPTER 1: INTRODUCTION 
1.1. Childhood brain tumors
1.1.1. Overview of pediatric primary central nervous system tumors
Tumors of the central nervous system (CNS) are a heterogeneous group of neoplasms
which vary widely by origin, morphologic features, genetic alteration, growth potential, extent
of invasiveness, tendency for progression and recurrence, and treatment response (Gurney &
Kadan-Lottick, 2001; Louis et al., 2007). Like any other tumor that can develop in the human
body, CNS tumors are either invasive (malignant) or not invasive (benign). Furthermore,
considering the site of origin, these tumors can be categorized as primary or secondary
tumors. The primary CNS tumors are those which originate in the brain whereas the
secondary CNS tumors spread to the brain after developing in another part of the body.
Primary CNS tumors are the most common solid tumor and the second most
frequent malignancy of childhood (Gururangan & Friedman, 2004), accounting for 20% of all
pediatric cancers (Rickert, 2004). In the world, approximately 30.000-40.000 children develop
primary CNS tumors each year (Bleyer et al., 1999), while in the United State, an estimated
3.750 new cases of childhood CNS tumors were expected to be diagnosed in 2007 (Central
Brain Tumor Registry of the United State, Statistical report, 2007-2008). Due to the high
incidence rate (about 3/100.000 children) and the relatively poor survival rate, CNS tumors
are one of the greatest challenges of pediatric oncology (Kaatsch et al., 2001).
For simplicity, primary CNS tumors can be classified as glial tumors (gliomas) and
nonglial tumors (nongliomas) (Buckner et al., 2007). Gliomas develop from glial cells
(supporting cells of the nervous system) and can be divided into more specific subtypes such
as astrocytoma, oligodendroglioma, and ependymoma. On the other hand, nongliomas are
CNS tumors that arise from areas other than glial tissues such as the nerves, glands, or blood
vessels. They consist of benign tumors like meningiomas and pituitary adenomas, as well as
malignant tumors, for example, medulloblastomas, primary CNS lymphomas, and the rarely
occurring CNS germ cell tumors.

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