Kruppel-like factor 4 is a tumor suppressor in B-cell non-Hodgkin lymphoma and in classical Hodgkin lymphoma [Elektronische Ressource] / presented by Hanfeng Guan

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• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2011
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Universität Ulm
Institut fuer Physiologische Chemie

Direktor: Prof. Thomas Wirth

Kruppel-like factor 4 is a tumor suppressor in B-cell non-Hodgkin
lymphoma and in classical Hodgkin lymphoma


Dissertation
for the attainment of the
Doctoral Degree of Medicine (Dr. med.)
at the Faculty of Medicine, University of Ulm, Ulm, Germany


Presented by
Hanfeng Guan
born in Zaoyang, Hubei Province, P. R. China


2011

































Amtierender Dekan: Prof. Dr. rer. Nat Thomas Wirth
1. Berichterstatter: Prof. Dr. rer. Nat Thomas Wirth
2. Berichterstatter: Prof. Dr. Peter Möller



Tag der Promotion: 14.01.2011
- II -

Contents
Abbreviations....................................................................................................................... IV  
1 Introduction........................ 1  
1.1 B-cell lymphoma ......................................................................................................... 1  
1.1.1 Classical Hodgkin Lymphoma............. 3  
1.1.2 Hodgkin and Reed-Sternberg cells....... 3  
1.1.3 Burkitt's lymphoma (BL)...................................................................................... 5  
1.2 Epigenetic mechanisms in cHL lymphogenesis.......................... 5  
1.3 Krüppel-like factor 4................................... 6  
1.4 Aims of the Study........................................................................ 8  
2 Materials and Methods....... 9  
2.1 Cell lines and Cell culture........................... 9  
2.1.1 Cell lines used in this study .................................................................................. 9  
2.1.2 Culture conditions................................. 9  
2.1.3 5-aza-2'-deoxycytidine treatments...... 10  
2.2 Transfection with Amaxa nucleofector ..................................... 10  
2.3 RNA isolation and Quantitative PCR (Q-PCR)........................ 10  
2.4 Human material ......................................................................... 14  
2.5 B cell isolation........... 14  
2.6 Quantitative analysis of promoter methylation ......................................................... 16  
2.7 Immunoblot............................................... 17  
2.8 Cell proliferation assay, apoptosis and cell cycle analysis........................................ 19  
2.9 ShRNA knock-down of BAK1.................. 20  
2.10 Gene expression profiling using Affymetrix GeneChip.......... 21  
2.10.1 Sample preparation........................................................................................... 21  
2.10.2 Affymetrix Gene Chip...................... 21  
2.10.3 Data analysis..................................... 21  
2.11 Specific materials .................................................................... 22  
2.11.1 Chemicals......... 22  
2.11.2 Reagents and materials ..................................................................................... 23  
2.11.3 Cell culture....... 24  
2.11.4 Buffers .............................................. 24  
3 Results .............................................................................................................................. 26  
3.1 KLF4 is epigenetically silenced in B-cell lymphomas.............................................. 26  
3.1.1 KLF4 is silenced in B-cell lymphoma cell lines................. 26  
3.1.2 Inhibition of DNA methylation activates KLF4 expression. 28  
3.1.3 Qutification of KLF4 promoter methylation in B-cell lymphomas.................... 28  
3.2 KLF4 induces cell cycle arrest in BL cell lines......................................................... 33  
3.3 KLF4 causes apoptosis in cHL cell lines................................... 36  
3.4 KLF4 activates genes involved in regulation of cell cycle and apoptosis................. 40  
3.5 BAK1 is responsible for KLF4-induced cell death................... 42  
3.6 Gene expression profiling reveals novel target genes of KLF4 45  
3.7 KLF4 regulates expression of MSC .......................................................................... 49  
4 Discussion......................................................... 51  
5 SUMMARY...................... 56  
6 References........................................................................................ 57  
Acknowledgements ............................................. 66  
CURRICULUM VITAE...................................................................... 68  

- III -

Abbreviations
4-OHT 4-hydroxytamoxifen
5-aza-dC 5-aza-2'-deoxycytidine
aa amino acid
ABF-1 activated B-cell factor-1 (musculin)
ACTB actin beta
Apaf-1 apoptosis activating factor
APS ammonium persulfat
BAK1 BCL2-antagonist/killer 1
BAX BCL2–associated X protein
BCL2 B-cell CLL/lymphoma
BCL2L1 BCL2-like 1 (BCL L) x
BCR B cell receptor
BL Burkitt´ lymphoma
bp base pair
BSA bovine serum albumin
CCND2 Cyclin D2
CD cluster of differentiation
CDK cyclin-dependent kinase
CDKN1A cyclin-dependent kinase inhibitor 1A (p21, Cip1)
CDKN1C cyclin-dependent kinase inhibitor 1C (p57, Kip2)
cDNA complementary DNA
CFLAR caspase-8 and FADD-like apoptosis regulator (c-FLIP)
cHL Classical Hodgkin´s lymphoma
DLBCL diffuse large B-cell lymphoma
DMEM Dulbecco's modified eagle medium
DMSO Dimethyl sulfoxide
DNA deoxyribonucleic acid
- IV -


DNMT DNA (cytosine-5-)-methyltransferase
dNTP deoxyribonucleotide
Dox doxycycline
EBF1 early B-cell factor 1
eBL endemic BL
EBV Epstein-Barr virus
EDTA ethylenediaminetetraacetic acid
ER estrogen receptor
FACS Fluorescence-activated cell sorting
FL Follicular lymphoma
GC germinal center
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HIV human immunodeficiency virus
HL Hodgkin´s lymphoma
HLH helix-loop-helix
HRS Hodgkin and Reed-Sternberg
HSP90 heat shock protein 90
Ig immunoglobulin
kb kilo base pairs
kDa kilodalton
LB Luria-Bertani medium
KLF4 Kruppel-like factor 4
MACS Magnetic-activated cell sorting
min minutes
MM Multiple Myeloma
mRNA messenger RNA
MYC v-myc myelocytomatosis viral oncogene homolog
NF-B nuclear factor of kappa light polypeptide gene enhancer in B-cells
- V -

κ
NHL non Hodgkin Lymphoma
NLPHL nodular lymphocyte-predominant Hodgkin's lymphoma
NMZL Nodal marginal zone B cell lymphoma
NOTCH1 notch 1
nt nucleotide
PBS phosphate-buffered saline
PCR polymerase chain reaction
PI propidium iodide
PMBL Primary Mediastinal B-cell lymphoma
POU2AF1 POU class 2 associating factor 1
POU2F2 POU class 2 homeobox 2
PU.1 spleen focus forming virus (SFFV) proviral integration oncogene spi1
RNA ribonucleic acid
RPMI Roswell Park Memorial Institute medium
RT-PCR reverse transcriptase-PCR
SDS sodium dodecyl sulfate
shRNA short hairpin RNA
TAE Tris acetate EDTA
Taq Thermus aquaticus polymerase
TCF3 transcription factor 3
TE Tris EDTA
TEMED tetramethylethylenediamine
TNF tumor necrosis factor
TNFR TNF-receptor
TRAF TNF receptor-associated factor
TRAILR Trail receptor

zVAD-fmk carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone

- VI -


1 Introduction
1.1 B-cell lymphoma
Lymphoma is the most common form of hematological malignancy in the developed world
with about 20 new cases each year per 100,000 people. According to its cellular origin, it
can be divided into B-cell or T cell lymphoma, although the latter only represents about
5%, with the majority of lymphomas are of B-cell origin (Küppers 2005). The current
World Health Organization lymphoma classification distinguishes B-cell lymphoma into
about 15 types (Elaine et al. 2001).
B-cell lymphomas are often derived from germinal centre B cells or from B cells that have
passed through GC, e.g. Follicular lymphoma (20% of all lymphomas), Burkitt's
lymphoma (2%), Diffuse large B cell lymphoma (30-40%, GC or post-GC B cells) and
Hodgkin's lymphoma (10%). We focus our study on Burkitt’s lymphoma and Hodgkin’s
lymphoma. Although both lymphomas have the same origin, they are unique models and
differ from each other in a lot of ways. BL represents most of the non-Hodgkin lymphomas
that preserve the B-cell phenotype whereas Hodgkin’s lymphoma has almost lost its B cell
identity and obtained phenotypes of other lineages. On the other hand, as an indolent
tumor, HL responds well to therapy and has a good prognosis, whereas BL is very
aggressive.



1
World Health Organization Classification of B-