Constitutive activation of Jak2-STAT5-signalling pathway in MedB-1 cells due to deficient SOCS-1 [Elektronische Ressource] / submitted by Alexandra Juliana Bucur

Bucur

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Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2005
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Institute for Pathology and Forensic Medicine
Department of Pathology
Director: Prof. Dr. med. P. Möller






Constitutive Activation of Jak2/STAT5-
Signalling Pathway
in MedB-1 cells Due to Deficient SOCS-1

Dissertation
for obtaining the Doctor Degree of Human Biology
at the Faculty of Medicine, University of Ulm


submitted by:
ALEXANDRA JULIANA BUCUR
from
Cluj-Napoca, Romania





ULM, 2005
Amtierender Dekan:
Prof. Dr. med. K.-M. Debatin


Erstgutachter:
Prof. Dr. med. P. Möller


Zweitgutachter:
Prof. Dr. med. T. Seufferlein





Tag der Promotion:
28.10.2005































TABLE OF CONTENTS 3
TABLE OF CONTENTS
TABLE OF CONTENTS .................................................................................................. 3
ABBREVIATIONS ........................................................................................................... 6
1. INTRODUCTION ......................................................................................................... 8
1.1 Supressors of Cytokine Signaling (SOCS)................................................................. 8
1.1.1 Molecular Mechanisms of SOCS action................................................................ 10
1.1.2 Physiological Role of SOCS-1 .............................................................................. 11
1.1.3 Cytokine Induced Expression of SOCS Introduction............................................. 12
1.1.4 STATs Mediate Induction of SOCS Transcription................................................. 12
1.2 The Role of STATs in Transcriptional Control .................................................... 14
1.2.1 Structure of STAT Proteins ................................................................................... 14
1.2.1.1 N-terminal Domain ............................................................................................. 14
1.2.1.2 Coiled-Coil Domain ............................................................................................ 14
1.2.1.4 Linker Domain.................................................................................................... 15
1.2.1.5 SH2 Domain....................................................................................................... 15
1.2.1.6 Transcriptional Activation Domain...................................................................... 15
1.2.2 Cytokines and Growth Factors Implicated in STAT Activation .............................. 15
1.2.3 Function of STATs in Oncogenesis....................................................................... 16
1.2.4 STAT-regulated Genes ......................................................................................... 17
1.2.5 STAT-interacting proteins that Modulate STAT signaling ..................................... 17
1.2.6 Targeting STAT Proteins for Therapeutic Intervention.......................................... 18
1.3 Janus Kinases........................................................................................................ 19
1.3.1 Structure and Organisation ................................................................................... 19
1.3.2 Janus Kinases and Cytokine Signal Transduction ................................................ 20
1.3.3 Models of Jak2 Activattion .................................................................................... 22
1.3.4 Attenuation of Jak/STAT signaling ........................................................................ 23
1.3.4.1 NonReceptor Type Protein Tyrosine Phosphatases .......................................... 23
1.3.4.2 Receptor Type Protein Tyrosine Phosphatases................................................. 23
1.3.4.3 Ubiquitination and ISGylation............................................................................. 24
1.3.4.4 PIAS Family Members ....................................................................................... 24
1.3.4.5 SOCS Regulation and Protein Interaction.......................................................... 24
1.4 MedB-1 a Tumor Cell Line Derived from a PMBL................................................ 25 TABLE OF CONTENTS 4
1.4.1 Immunological Characteristics of MBL.................................................................. 26
1.4.2 Molecular Abnormalities of MBL ........................................................................... 26
1.4.3 Cytogenetics of MBL............................................................................................. 27
1.4.4 Molecular Diagnosis of MBL ................................................................................. 28
1.5 Karpas 1106P a Mediastinal Lymphoblastic B-cell non-Hodgkin
Lymphoma.............. 30
2. AIMS OF THE PROJECT.......................................................................................... 31
3. MATERIALS AND METHODS .................................................................................. 32
3.1 Cell Culture and Transfection............................................................................... 32
3.1.1 Cell Counting ........................................................................................................ 33
3.1.2 Cell Freezing......................................................................................................... 33
3.1.3 Cell Thawing 33
3.2. Cell Lyses and Western blotting.......................................................................... 34
3.3 Antibodies .............................................................................................................. 37
3.4 RNA-isolation, RT-PCR and Real-Time PCR........................................................ 37
3.4.1 RT-PCR ................................................................................................................ 38
3.5 Analysis of Cell Proliferation ................................................................................ 39
3.6 Electrophoretic Mobility Shift Assay (EMSA)...................................................... 40
3.6.1 Determination of Protein Concentration ................................................................ 40
323.6.2 P-labelling of STAT5-DNA Binding Probe .......................................................... 41
3.7 Analysis of SOCS-1 cDNA Sequence................................................................... 42
3.8 Analysis of SOCS-1 Mutations in PMBL .............................................................. 46
3.9 Wt-SOCS-1 Expression Plasmid Preparation...................................................... 46
3.9.1 Quantification of DNA ........................................................................................... 47
3.10 Pulse-Chase Experiments................................................................................... 47
3.10.1 Immunoprecipitation and Determination of Jak-2 Degradation ........................... 47
3.10.2 Table 2................................................................................................................ 49
3.11 Chemicals, Reagents and Devices ..................................................................... 49
3.11.1 Enzymes ............................................................................................................. 49
3.11.2 Standards............................................................................................................ 49
3.11.3 Ready-to-use Kits ............................................................................................... 50
3.11.4 Used Materials.................................................................................................... 50
3.11.5 Membranes and Filters ....................................................................................... 51
3.11.6 Devices ............................................................................................................. 51 TABLE OF CONTENTS 5
3.11.7 Chemicals ........................................................................................................... 53
4. RESULTS .................................................................................................................. 55
4.1 Jak2 DNA Level and its Expression in MedB-1 ................................................... 55
4.1.1 Determination of Jak2 DNA Level ......................................................................... 56
4.1.2 Determination of Jak2 mRNA Level ...................................................................... 56
4.1.3 Jak2 Protein Expression ....................................................................................... 57
4.2 STAT5 E