Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors:
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Item Description: | Zsfassung in dt. u. engl. Sprache |
| Physical Description: | 143 Bl. Ill., graph. Darst. |
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Record in the Search Index
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| adam_text | IMAGE 1
TABLE OF CONTENTS PAGE
1 SUMMARY 7
2 ZUSAMMENFASSUNG 8
3 LIST OF ABBREVIATIONS 9
4 INTRODUCTION 11
4.1 BCR-ABL AND CHRONIC MYELOID LEUKEMIA 11
4.1.1 HEMATOPOIESIS AND LEUKEMIA 11
4.1.2 THE ONCOGENIC TYROSINE KINASE BCR-ABL IS THE PRODUCT OF THE
PHILADELPHIA CHROMOSOME AND THE DRIVER OF CHRONIC MYELOID LEUKEMIA 12
4.1.3 BCR-ABL CONTAINS MULTIPLE DOMAINS CONTRIBUTING TO TRANSFORMATION
14 4.1.4 SPECIFIC INHIBITORS OF THE TYROSINE KINASE ACTIVITY OF BCR-ABL
AS MOLECULAR
TARGETED THERAPY FOR CML 16
4.1.5 RESISTANCE TO BCR-ABL INHIBITORS CAN BE CAUSED BY VARIOUS
MECHANISMS... 18 4.1.6 THE BCR-ABL/GRB2/GAB2 AXIS 20
4.2 THE GAB2 DOCKING PROTEIN 22
4.2.1 THE GAB FAMILY OF DOCKING PROTEINS INTEGRATES AND AMPLIFIES
SIGNALS FROM MULTIPLE RECEPTORS 22
4.2.2 FUNCTIONS AND REGULATION OF GAB2 26
4.2.3 GAB2 IS INVOLVED IN THE PATHOGENESIS OF HUMAN CANCER 29
4.3 AIM OF THIS STUDY 31
5 RESULTS 32
5.1 EXPRESSION LEVELS OF THE GAB2 DOCKING PROTEIN MODULATE THE
SENSITIVITY OF CML CELLS TO BCR-ABL INHIBITORS 32
5.1.1 LOWERING GAB2 EXPRESSION LEVELS INCREASES THE SENSITIVITY OF K562
CELLS TO IMATINIB-INDUCED CELL DEATH 32
5.1.2 INDUCIBLE OVEREXPRESSION OF GAB2 CONFERS RESISTANCE TO MULTIPLE
BCR-ABL INHIBITORS 36
5.2 SIGNALING-IMPAIRED MUTANTS OF GAB2 FAIL TO CONFER RESISTANCE TO
BCR-ABL INHIBITORS 43
5.2.1 THE ACTIVITY OF THE BCR-ABL/GRB2/GAB2 AXIS IS MODULATED BY THE
14-3-3 BINDING POTENTIAL OF GAB2 43
5.2.2 GAB2 MODULATES IMATINIB-SENSITIVITY VIA THE PI3-KINASE/AKT AND THE
SHP2/ERK PATHWAYS 49
5.3 IDENTIFICATION OF DRUG COMBINATIONS TO OVERCOME GAB2-MEDIATED TKI-
RESISTANCE 51
5.3.1 BCR-ABL INHIBITORS FAIL TO ABOLISH GAB2 PHOSPHORYLATION AND
SIGNALING OUTPUT COMPLETELY 51
5.3.2 DASATINIB COMBINED WITH JAK2 INHIBITION IS NOT SUFFICIENT TO
OVERCOME GAB2-MEDIATED TKI-RESISTANCE 57
5.3.3 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS CONTRIBUTE TO
TYROSINE PHOSPHORYLATION OF GAB2 IN CML CELLS 59
5.3.4 DASATINIB COMBINED WITH INHIBITORS OF THE PI3-KINASE/AKT/MTOR
PATHWAY OVERCOMES GAB2-MEDIATED TKI-RESISTANCE 64
HTTP://D-NB.INFO/1031457798
IMAGE 2
5.4 HIGHER GAB2 EXPRESSION LEVELS CORRELATE WITH TKI-RESISTANCE 69
5.5 REDUCTION OF GAB2 EXPRESSION SENSITIZES PRIMARY CELLS TO TKIS 76
5.5.1 KNOCKDOWN OF GAB2 RE-SENSITIZES PRIMARY HUMAN IMATINIB-RESISTANT
CML CELLS 76
5.5.2 LOWERING GAB2 EXPRESSION LEVELS SENSITIZES BCR-ABL-TRANSFORMED
PRIMARY MURINE BONE MARROW CELLS TO TKIS 78
6 DISCUSSION 80
6.1 GAB2 - A NOVEL MODULATOR OF TKI-SENSITIVITY? 80
6.2 THE MOLECULAR MECHANISM OF GAB2-MEDIATED TKI-RESISTANCE 82 6.2.1
SIGNALING EVENTS DOWNSTREAM OF GAB2 MEDIATING TKI-RESISTANCE 82 6.2.2
REGULATION OF BCR-ABL-DRIVEN GAB2 SIGNALING BY 14-3-3 PROTEINS 85 6.2.3
KINASES DRIVING GAB2 TYROSINE PHOSPHORYLATION IN THE ABSENCE OF BCR-ABL
ACTIVITY 87
6.2.4 THE GAB2 PHOSPHORYLATION MAP IN CML CELLS 91
6.3 DOES GAB2 OVEREXPRESSION OCCUR IN CML? 93
6.3.1 GAB2 EXPRESSION LEVELS IN CML ARE HETEROGENEOUS AND INCREASE
DURING DISEASE PROGRESSION 93
6.3.2 MECHANISMS REGULATING GAB2 GENE EXPRESSION 95
6.4 THE BCR-ABL/GRB2/GAB2 AXIS AS A TARGET FOR CML THERAPY 96
6.4.1 TARGETING THE GRB2/GAB2 INTERFACE 97
6.4.2 STABILIZING THE GAB2/14-3-3 INTERACTION 98
7 CONCLUSION AND OUTLOOK 99
8 MATERIAL AND METHODS 100
8.1 CELL BIOLOGICAL METHODS 100
8.1.1 CULTIVATION OF ESTABLISHED CELL LINES 100
8.1.2 ISOLATION AND CULTIVATION OF HUMAN PRIMARY BLOOD LEUKOCYTES 101
8.1.3 ISOLATION AND CULTIVATION OF MURINE PRIMARY CELLS 102
8.1.4 CRYO-CONSERVATION OF CELLS 102
8.1.5 TRANSFECTION 103
8.1.5.1 TRANSFECTION WITH POLYETHYLENIMINE AND PRODUCTION OF
RETROVIRUSES 103 8.1.5.2 TRANSFECTION WITH ARREST-IN* REAGENT AND
PRODUCTION OF LENTIVIRUSES 103 8.1.5.3 ELECTROPORATION OF K562 CELLS AND
ESTABLISHMENT OF STABLE CELL POOLS 104 8.1.6 RETROVIRAL AND LENTIVIRAL
TRANSDUCTION AND ESTABLISHMENT OF STABLY INFECTED
CELL POOLS 104
8.1.7 INDUCTION OF INDUCIBLE EXPRESSION AND KNOCKDOWN SYSTEMS 105 8.1.8
TREATMENT WITH KINASE INHIBITORS 105
8.1.9 FLUORESCENCE ACTIVATED CELL SORTING 106
8.1.9.1 FLOW CYTOMETRIC DETECTION OF GFP OR RFP 106
8.1.9.2 FLOW CYTOMETRIC DETECTION OF THE MEGAKARYOCYTE SURFACE MARKER
CD41 106 8.1.9.3 FLOW CYTOMETRIC DETECTION OF APOPTOSIS USING ANNEXIN-V
STAINING 107 8.1.9.4 FLOW CYTOMETRIC DETECTION OF CELL DEATH USING 7-AAD
STAINING 107 8.2 PROTEIN BIOCHEMISTRY 108
8.2.1 PREPARATION OF TOTAL CELL LYSATES 108
8.2.2 SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS 108
8.2.3 WESTERN BLOT AND IMMUNODETECTION 109
8.2.4 PURIFICATION OF GAB2 BY IMMUNOPRECIPITATION 110
8.2.5 FLUOROMETRIC CASPASE-3/7 ACTIVITY ASSAY 111
8.3 MOLECULAR BIOLOGY 112
8.3.1 GENERATION AND CULTIVATION OF CHEMICALLY COMPETENT ESCHERICHIA
COLI BACTERIA 112
8.3.2 CRYO-CONSERVATION OF E. COLI BACTERIA 112
IMAGE 3
8.3.3 TRANSFORMATION OF COMPETENT E. COLI BACTERIA 113
8.3.4 PREPARATION OF PLASMID DNA IN SMALL AND LARGE SCALE 113
8.3.5 PREPARATION OF GENOMIC DNA FROM MOUSE TAIL BIOPSIES 113
8.3.6 RESTRICTION ENDONUCLEASE DIGEST OF DNA 114
8.3.7 ELECTROPHORETIC SEPARATION OF DNA 114
8.3.8 PURIFICATION OF DNA FRAGMENTS FROM AGAROSE GELS 114
8.3.9 DEPHOSPHORYLATION OF DNA FRAGMENTS 114
8.3.10 LIGATION OF DNA FRAGMENTS 114
8.3.11 POLYMERASE CHAIN REACTION 115
8.3.11.1 GENOTYPING OF GAB2-DEFICIENT MICE 115
8.3.12 CLONING OF PCR PRODUCTS 115
8.3.13 SITE-SPECIFIC MUTAGENESIS 116
8.3.14 SEQUENCING OF PLASMID DNA AND PCR FRAGMENTS 116
8.3.15 GENERATION OF PTET FROM PWHE636 117
8.3.16 GENERATION OF PTET/GAB2 CONSTRUCTS 117
8.3.17 GENERATION OF PLEGOHU6-GAB2SH-G 117
8.4 LIST OF ANTIBODIES 118
8.4.1 PRIMARY ANTIBODIES FOR WESTERN BLOT 118
8.4.2 HORSERADISH PEROXIDASE (HRP) COUPLED SECONDARY ANTIBODIES FOR
WESTERN BLOT 119
8.4.3 ANTIBODIES AND REAGENTS FOR FLOW CYTOMETRY 119
8.5 LIST OF OLIGONUCLEOTIDES 120
8.6 LIST OF DNA VECTORS 121
8.7 CHEMICALS 122
9 REFERENCES 123
10 DANKSAGUNG 143
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| spelling | Wöhrle, Franziska Ursula Verfasser aut Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors vorgelegt von Franziska Ursula Wöhrle 2012 143 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. u. engl. Sprache Freiburg i. Br., Univ., Diss., 2012 Enzyminhibitor (DE-588)4152479-2 gnd rswk-swf Signalkette (DE-588)4409935-6 gnd rswk-swf Chronisch-myeloische Leukämie (DE-588)4199205-2 gnd rswk-swf Resistenz (DE-588)4136408-9 gnd rswk-swf Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Chronisch-myeloische Leukämie (DE-588)4199205-2 s Signalkette (DE-588)4409935-6 s Protein-Tyrosin-Kinasen (DE-588)4409305-6 s Enzyminhibitor (DE-588)4152479-2 s Resistenz (DE-588)4136408-9 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025682242&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Wöhrle, Franziska Ursula Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors Enzyminhibitor (DE-588)4152479-2 gnd Signalkette (DE-588)4409935-6 gnd Chronisch-myeloische Leukämie (DE-588)4199205-2 gnd Resistenz (DE-588)4136408-9 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd |
| subject_GND | (DE-588)4152479-2 (DE-588)4409935-6 (DE-588)4199205-2 (DE-588)4136408-9 (DE-588)4409305-6 (DE-588)4113937-9 |
| title | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors |
| title_auth | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors |
| title_exact_search | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors |
| title_full | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors vorgelegt von Franziska Ursula Wöhrle |
| title_fullStr | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors vorgelegt von Franziska Ursula Wöhrle |
| title_full_unstemmed | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors vorgelegt von Franziska Ursula Wöhrle |
| title_short | Gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple Bcr-Abl inhibitors |
| title_sort | gab2 signaling in chronic myeloid leukemia cells confers resistance to multiple bcr abl inhibitors |
| topic | Enzyminhibitor (DE-588)4152479-2 gnd Signalkette (DE-588)4409935-6 gnd Chronisch-myeloische Leukämie (DE-588)4199205-2 gnd Resistenz (DE-588)4136408-9 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd |
| topic_facet | Enzyminhibitor Signalkette Chronisch-myeloische Leukämie Resistenz Protein-Tyrosin-Kinasen Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025682242&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT wohrlefranziskaursula gab2signalinginchronicmyeloidleukemiacellsconfersresistancetomultiplebcrablinhibitors |