Investigation of the function of the IKK2/NF-_k63B-pathway [IKK2-NF-kappaB-pathway] in c-MYC-induced lymphomagenesis [Elektronische Ressource] / vorgelegt von Kay Oliver Klapproth

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• Publié par: universitat_ulm
• Publié le: 01 janvier 2009
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 6 Mo

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FAKULTÄT FÜR NATURWISSENSCHAFTEN
UNIVERSITÄT ULM
Investigation of the function
of the IKK2/NF-κB-pathway in
c-MYC-induced lymphomagenesis
DISSERTATION
zur Erlangung des Doktorgrades (Dr. rer. nat.)
an der Fakultät für Naturwissenschaften
der Universität Ulm
vorgelegt von
Kay Oliver Klapproth
aus Marburg
2009Dekan: Prof. Dr. Axel Groß
Erstgutachter: Prof. Dr. Thomas Wirth
Institut für Physiologische Chemie
Universität Ulm
Zweitgutachter: Prof. Dr. Klaus-Dieter Spindler
Allgemeine Zoologie und Endokrinologie
Universität Ulm
Drittgutachter: Prof. Dr. Dirk Eick
Institut für Klinische Molekularbiologie und Tumorgenetik
GSF-Forschungszentrum für Umwelt und Gesundheit, München
Tag der Promotionskolloquiums: 25. März 2010
Die Arbeiten im Rahmen der vorliegenden Dissertation wurden im Institut für
Physiologische Chemie der Universität Ulm durchgeführt und von Herrn Prof. Dr.
Thomas Wirth betreut.
2TABLE OF CONTENTS
TABLE OF CONTENTS
SUMMARY .............................................................................................................6
ZUSAMMENFASSUNG..........................................................................................8
1 INTRODUCTION ...........................................................................................10
1.1 The c-MYC transcription factor...............................................................10
1.1.1 Structure and function .....................................................................11
1.1.2 MYC regulated genes......................................................................14
1.2 MYC in cancer........................................................................................16
1.2.1 MYC-induced malignant transformation ..........................................16
1.2.2 MYC-induced apoptosis ..................................................................17
1.3 The NF-κB transcription factor................................................................19
1.3.1 Structure and function .....................................................................19
1.3.2 NF-κB regulated genes ...................................................................23
1.4 NF-κB in lymphomas ..............................................................................24
1.5 Burkitt´s lymphoma (BL).........................................................................26
2 MATERIAL AND METHODS ........................................................................30
2.1 Cell lines and cell culture........................................................................30
2.1.1 Cell lines used in this study .............................................................30
2.1.2 Culture conditions............................................................................31
2.1.3 Doxycycline treatment and NF-κB activation...................................31
2.1.4 Stimulation with anti-CD40 ..............................................................31
2.2 Retroviral transductions..........................................................................31
2.2.1 Retroviral vectors and producer cell lines........................................31
2.2.2 Transient transfection of producer cell lines ....................................32
2.2.3 Retroviral transduction of lymphoma cells.......................................33
2.3 Tumor transplantation and in vivo competition assay.............................33
2.4 Conditional expression of NF-κB modulators in human cell lines...........34
2.4.1 Vectors ............................................................................................34
2.4.2 Transfections...................................................................................35
2.5 Viable cell counts and apoptosis detection.............................................36
3TABLE OF CONTENTS
2.6 Fas surface expression ..........................................................................36
2.7 Stimulation with anti-Fas antibodies .......................................................36
2.8 Knockdown of caspase-8 .......................................................................37
2.9 Exogenous expression of CFLAR...........................................................37
2.10 Immunoblot analysis and electrophoretic mobility shift assay.............38
2.10.1 Protein extracts ...............................................................................38
2.10.2 Immunoblotting................................................................................38
2.10.3 Electrophoretic mobility shift assay .................................................39
2.11 Gene expression profiling ...................................................................40
2.11.1 Sample preparation.........................................................................40
2.11.2 Affymetrix Gene Chip ......................................................................40
2.11.3 Data analysis...................................................................................40
2.12 RNA-isolation and RT-PCR ................................................................41
2.12.1 RNA preparation..............................................................................41
2.12.2 RT-PCR...........................................................................................41
2.12.3 Quantitative PCR.............................................................................41
2.13 Specific materials................................................................................42
2.13.1 Chemicals........................................................................................42
2.13.2 Reagents and materials ..................................................................43
2.13.3 Cell culture ......................................................................................44
2.13.4 Buffers.............................................................................................45
3 RESULTS......................................................................................................48
3.1 NF-κB activity in MYC-dependent murine lymphoma cells.....................48
3.1.1 A transgenic mouse model for conditional MYC expression ...........48
3.1.2 Basal and induced NF-κB activity in MYC-driven lymphoma cells ..50
3.1.3 Influence of MYC expression on NF-κB activity ..............................52
3.2 Modulation of NF-κB activity in murine lymphoma cells .........................54
3.2.1 Retroviral modulation of NF-κB activity ...........................................54
3.2.2 Effects of NF-κB modulation on growth and viability .......................57
3.3 CD40-mediated NF-κB activation in murine lymphoma cells..................61
3.4 Inhibition of NF-κB in an in vivo competition assay ................................64
4TABLE OF CONTENTS
3.5 Modulation of NF-κB activity in BL cells .................................................66
3.5.1 A conditional expression system for NF-κB modulators ..................66
3.5.2 Effects of NF-κB modulation on growth and viability .......................69
3.6 CA-IKK2-induced apoptosis in BL cells ..................................................73
3.6.1 Inhibition of caspase activity............................................................73
3.6.2 Role of CFLAR in CA-IKK2-induced apoptosis................................74
3.6.3 Role of caspase-8 in CA-IKK2-induced apoptosis...........................75
3.7 Specificity of CA-IKK2-induced apoptosis ..............................................77
3.8 NF-κB dependency of CA-IKK2-induced effects in BL cells ...................78
3.8.1 Inhibition of NF-κB activity in the presence of CA-IKK2 ..................78
3.8.2 NF-κB inhibition in CA-IKK2 expressing Ramos..............................80
3.9 CD40-mediated NF-κB activation in BL cells..........................................83
3.10 Gene Expression profiling of CA-IKK2 expressing Ramos .................85
3.10.1 Regulation of antigen presentation and cell adhesion genes ..........87
3.10.2 Regulated genes involved in cell cycle progression ........................89
3.10.3 Regulated genes involved in apoptosis...........................................90
3.11 Fas-induced apoptosis in BL cells.......................................................91
3.11.1 Fas surface expression following CA-IKK2 expression ...................91
3.11.2 Fas sensitivity of BL cells following CA-IKK2 expression ................93
3.11.3 NF-κB dependency of Fas-induced apoptosis.................................94
3.11.4 Effects of neutralizing anti-FasL on CA-IKK2-induced apoptosis ....95
4 DISCUSSION ................................................................................................97
5 ABBREVIATIONS....................................................................................... 110
6 REFERENCES ............................................................................................ 113
PUBLICATIONS AND POSTERS ...................................................................... 127
ERKLÄRUNG..................................................................................................... 129
ACKNOWLEDGEMENTS .................................................................................. 130
CURRICULUM V