Molecular analysis of small Ras-related GTPase genes with potential tumor suppressor function in human gliomas [Elektronische Ressource] / Natalie Schmidt. Gutachter: Guido Reifenberger ; Dieter Willbold

Schmidt Francis, Natalie

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2011
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Molecular analysis of small Ras-related GTPase genes with
potential tumor suppressor function in human gliomas

Inaugural-Dissertation

zur Erlangung des Doktorgrades

der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf

vorgelegt von

Natalie Schmidt
aus Berkeley/CA/USA


Düsseldorf, Mai 2011 Aus dem Institut für Neuropathologie
der Heinrich-Heine-Universität Düsseldorf
(Direktor: Prof. Dr. Guido Reifenberger)

















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf

Referent: Prof. Dr. G. Reifenberger
Korreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 05.07.2011



















Results of this doctoral thesis are going to be published in the following original paper:
Schmidt N, Windmann S, Reifenberger G, Riemenschneider MJ. DNA
hypermethylation and histone modifications downregulate the candidate tumor
suppressor gene RRP22 on 22q12 in human gliomas. Brain Pathol 2011 (in press)
Contents I
Contents
1 Introduction ..................................................................................................... 1
1.1 Gliomas: Incidence, epidemiology, clinical behavior ........ 1
1.1.1 WHO classification of gliomas ......... 2
1.1.2 Molecular pathology of gliomas ....... 3
1.2 Small GTPase genes of the Ras-family ........................................................... 6
1.2.1 Ras-related protein on chromosome 22 (RRP22) ............ 8
1.2.2 Ras-homolog gene family member B (RHOB) ................. 9
1.2.3 Ras-like family 11 member A (RASL11A) ...................... 11
1.2.4 Ras-like, estrogen-regulated, growth inhibitor (RERG) .................................. 12
1.2.5 Aplasia ras homologue member I (ARHI) ................................ 13
1.3 Goals and experimental approach of this study ............. 15
2 Materials ....................................................................................................... 16
2.1 Cell lines and patients ................... 16
2.1.1 Cell lines ....... 16
2.1.2 Patients ......... 16
2.2 Laboratory equipment ................................................................................... 17
2.3 Consumables ................................ 19
2.4 Chemicals, enzymes and antibodies ............................. 20
2.5 Kits, reagents and assays ............................................................................. 23
2.6 Solutions, buffers and gels ............ 24
2.7 sh RNAs ........................................ 30
2.8 Oligonucleotides............................................................ 31
3 Methods ........................................ 32
3.1 Molecular biological methods ........................................ 32
3.1.1 Extraction of nucleic acids ............. 32
3.1.2 PCR analysis ................................................................ 33
3.1.3 DNA methylation analysis ............................................. 35
3.1.4 Mutation analysis .......................................................... 36
3.1.5 Microsatellite analyses .................. 38
3.1.6 Chromatin immunoprecipitation assay ........................... 40
3.2 Protein based methods ................................................................................. 42
3.2.1 Extraction of proteins from cultured cells ....................... 42
3.2.2 Protein quantification ..................... 42
3.2.3 Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis (SDS-PAGE) 43
3.2.4 Western blot analysis .................................................................................... 43
3.3 Cell based methods ...................... 45
3.3.1 Cultivation of glioma cells .............. 45 Contents II
3.3.2 Treatment of glioma cells with 5-aza-2‟-deoxycytidine or histone deacetylase
inhibitor trichostatin A .................................................................................... 45
3.3.3 Generation of stably ARHI-depleted glioblastoma cell lines ........................... 46
3.3.4 Functional assays ......................... 47
3.4 Statistical methods ........................ 49
4 Results .......................................................................................................... 50
4.1 Molecular characterization of small GTPase genes ....... 50
4.1.1 mRNA expression analysis in human gliomas and glioblastoma cell lines ..... 51
4.1.2 DNA methylation analysis ............. 55
4.1.3 Results of mutational analyses ...................................................................... 63
4.1.4 Microsatellite analyses for allelic losses on chromosome 22q12 ................... 64
4.1.5 Chromatin immunoprecipitation (ChIP) analysis in glioblastoma cell lines ..... 65
4.2 Functional analyses of ARHI in glioblastoma cell lines .. 72
4.2.1 Generation of ARHI-depleted glioblastoma cell lines ..................................... 73
4.3 Results of functional assays .......................................... 74
4.3.1 Influence of ARHI knock-down on glioma cell proliferation ............................ 74
4.3.2 Influence of ARHI knock-down on apoptotic activity ...... 75
5 Discussion ..................................................................................................... 78
5.1 Molecular analyses of RRP22 in human gliomas ........... 79
5.2 Molecular analyses of RHOB in human gliomas ............ 81
5.3 Molecular analyses of RASL11A and RERG in human gliomas .................... 82
5.4 Functional analysis of ARHI in human glioblastoma cell lines ........................ 83
6 Abstract ......................................................................................................... 86
7 Zusammenfassung ........................................................................................ 87
8 References .................................... 88
9 Danksagung .................................................................. 98
10 Ehrenwörtliche Erklärung .............................................. 99

Abbreviations III
Abbreviations
A diffuse astrocytoma
AA anaplastic astrocytoma
AO anaplastic oligodendroglioma
AOA anaplastic oligoastrocytoma
APS ammoniumpersulfat
ATCC American Tissue Culture Collection
BSA bovine serum albumine
bp base pairs
°C degree Celsius
cDNA complementary DNA
CpG Cytosin-phosphatidyl-Guanin
DEPC diethylpyrocarbonate
DPBS Dulbecco‟s Phosphate Buffered Saline
DMEM Dulbecco‟s Modified Eagle Medium
DMSO dimethylsulfoxid
DNA desoxyribonucleic acid
dNTP desoxyribonucleoside-5‟-triphosphat
DTT dithiothreitol
EDTA ethylendiamintetraacetat
ELISA enzyme linked immunosorbent assay
FACS fluorescence activated cell sorting
FCS fetal calf serum
GFP green fluorescent protein
GBM glioblastoma (multiforme) Abbreviations IV
GITC guanidinium isothiocyanate
h hour
HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
IgG Immunoglobulin G
kb kilobase
kDa kilo-Dalton
LOH loss of heterozygosity
M molar
NB non-neoplastic brain tissue
mA milli-ampere
min minute
ml milliliter
mRNA messenger ribonucleic acid
µl microliter
O oligodendroglioma
OA oligoastrocytoma
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
PI propidium iodide
PMSF phenylmethanesulfonyl fluoride
RNA ribonucleic acid
rpm rotation per minute
RT room temperature
RT-PCR reverse transcription - polymerase chain reaction Abbreviations V
SD standard deviation
SDS sodium dodecyl sulfate
sec second
shRNA small hairpin RNA
SSCP single strand conformation polymorphism
T tumor
Taq Thermus aquaticus
TBE Tris-borate-EDTA
TBS-T Tris-buffered saline with Tween 20
TE Tris-EDTA
TEMED N-N-N-N-Tetraethylmethyldiamine
Tris Tris(hydroxymethyl)aminomethane
U unit
v/v volume in volume
V voltage
W watt
w/v weight in volume
WHO World Health Organization
Introduction 1
1 Introduction
1.1 Gliomas: Incidence, epidemiology, clinical behavior
Primary tumors of the central nervous system (CNS) account for about 2-3 % of all
cancers. In pediatric patients, primary CNS tumors represent the second most common
tumor type after the acute leukemias and the most common cause of cancer-related
death (Riemenschneider and Reifenberger 2009b).
Gliomas are the most common primary brain tumors (around 50 %). Gliomas are
histologically classified according to the WHO (World Health Organisation)
classification of tumors of the central nervous system in its latest edition from 2007
(Louis et al 2007a). Classification is ma