An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2012
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| Schlagworte: | |
| Online-Zugang: | Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1071113-1-0 Inhaltsverzeichnis |
| Beschreibung: | 149 Bl. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |c Liang-Seah Tay |
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Datensatz im Suchindex
| _version_ | 1804149189552111616 |
|---|---|
| adam_text | IMAGE 1
CONTENTS
I. INTRODUCTION 1
1.1 PROTEIN TYROSINE KINASES 3
1.1.1 STRUCTURAL CHARACTERISTICS 3
1.1.2 MECHANISM OF ACTIVATION 5
1.2 ANOMALIES IN PTK SIGNALLING AND CANCER 6
1.3 THE ERBB FAMILY OF RECEPTOR PTKS 8
1.3.1 DISCOVERY 8
1.3.2 BASIC CHARACTERISTICS 8
1.3.3 EGFR 10
1.3.3.1 FUNCTIONAL DOMAINS OF EGFR 10
1.3.3.2 SIGNALLING VIA EGFR 12
1.3.3.2.1 EGFR ACTIVATION BY EGF FAMILY OF LIGANDS 12
1.3.3.2.2 TRANS-ACTIVATION OF EGFR 15
1.3.3.2.3 OTHER MEANS OF EGFR ACTIVATION 15
1.3.3.3 GENETIC ANOMALIES LEADING TO A DEREGULATION IN EGFR
SIGNALLING MAY LEAD TO CANCER 16
1.3.3.4 TARGETED CHEMOTHERAPEUTICS AGAINST EGFR 17
1.3.3.4.1 A NEED TO DEVELOP TARGETED CANCER
CHEMOTHERAPEUTICS 17
1.3.3.4.2 SPECIFIC ANTI-EGFR ANTIBODIES 18
1.3.3.4.3 SPECIFIC ANTI-EGFR TKIS 19
1.3.3.5 EGFR-TKI SENSITIZING GENETIC ALTERATIONS 2 1
1.3.3.6 EGFR-TKI RESISTANCE-CONFERRING GENETIC ALTERATIONS 23
1.3.3.6.1 PRIMARY RESISTANCE 23
1.3.3.6.2 SECONDARY RESISTANCE 24
HTTP://D-NB.INFO/1026700078
IMAGE 2
1.4 THE JAK FAMILY OF NON-RECEPTOR PTKS 25
1.4.1 HISTORY AND GENERAL CHARACTERISTICS 25
1.4.2 JAK PROTEINS PRIMARILY SERVE AS CYTOPLASMIC PARTNERS OF CYTOKINE
RECEPTORS 26
1.4.3 FUNCTIONAL DOMAINS OF JAKS 28
1.4.3.1 JH1: KINASE DOMAIN 28
1.4.3.2 JH2: PSEUDOKINASE DOMAIN 28
1.4.3.3 JH3 AND JH4: SH2-LIKE DOMAIN 29
1.4.3.4 JH5 TO JH7: FERM DOMAIN 30
1.4.4 MEDIATORS OF JAK SIGNALLING 3 1
1.4.4.1 THE STAT PROTEINS 3 1
1.4.4.2 THE JAK-STAT SIGNALLING PATHWAY 32
1.4.4.3 STATS ARE EMPLOYED BY OTHER NON-JAK PTKS FOR SIGNALLING 33
1.4.4.4 DIFFERENTIAL ACTIVITIES OF STATS 33
1.4.4.5 INACTIVATION OF JAK-STAT SIGNALLING 34
1.4.5 DISEASES CAUSED BY GENETIC ANOMALIES OF JAK PROTEINS 35
1.4.5.1 DEFICIENCIES IN JAKS 35
1.4.5.1.1 DEFICIENCIES IN JAK1 AND JAK2 CONFER LETHALITY 35
1.4.5.1.2 DEFICIENCY IN JAK3 CAUSES SCID 35
1.4.5.1.3 DEFICIENCY IN TYK2 CAUSES MILD EFFECTS IN MICE
BUT SEVERE DISEASE IN HUMANS 36
1.4.5.2 GENETIC ALTERATIONS IN JAKS IN HUMAN MALIGNANCIES 38
1.4.5.2.1 THE JAK2 V617F MUTATION IS ASSOCIATED WITH
MYELOPROLIFERATIVE DISEASES 38
1.4.5.2.2 OTHER ACTIVATING MUTATIONS O F JAKS ARE FOUND IN
VARIOUS LYMPHOID AND MYELOID LEUKAEMIAS 39
1.4.5.2.3 GENETIC ALTERATIONS OF TYK2 ASSOCIATED WITH
CANCER 40
IMAGE 3
1.4.5.2.4 A POSSIBLE ROLE OF TYK2 IN TUMOUR FORMATION AND
INVASION 4 1
II. SPECIFIC AIMS 42
III. MATERIALS AND METHODS 43
3.1 REAGENTS 43
3.2 MEDIA AND SUPPLEMENTS 44
3.3 ANTIBODIES 45
3.4 GENETIC MATERIAL USED IN CONFIRMATORY MUTATIONAL ANALYSES OF
SELECTED
GENETIC ALTERATIONS 45
3.5 PRIMERS 46
3.6 CELL LINES 50
3.7 PROPAGATION OF MAMMALIAN CELL LINES 51
3.8 EXTRACTION OF TOTAL RNA AND GENOMIC DNA 52
3.9 GENERATION OF CDNA BY REVERSE TRANSCRIPTION 52
3.10 POLYMERASE CHAIN REACTION 53
3.11 AGAROSE GEL ELECTROPHORESIS 54
3.12 DIDEOXY-SEQUENCING AND SEQUENCE ANALYSIS 54
3.13 RESTRICTION DIGESTION OF DNA 55
3.14 LIGATION OF VECTOR AND INSERT 55
3.15 PREPARATION OF COMPETENT E. COLI CELLS 55
3.16 TRANSFORMATION OF PLASMID DNA 56
3.17 LARGE-SCALE PRODUCTION OF PLASMID DNA 56
3.18 SITE-DIRECTED MUTAGENESIS AND CLONING 57
3.19 TRANSFECTION OF PLASMIDS 58
3.20 CELL LYSIS FOR PROTEIN DETECTION 59
IMAGE 4
3.21 WESTERN BLOTTING AND IMMUNO-PRECIPITATION 60
3.22 DETERMINATION OF CELLULAR VIABILITY USING MTT 62
3.23 DETERMINATION OF PROLIFERATION BY CELL COUNTING 63
3.24 SIRNA KNOCK-DOWN OF TYK2 63
3.25 TRANSWELL MIGRATION ASSAY 64
3.26 REAL-TIME PCR 64
3.27 STATISTICAL ANALYSIS 65
IV. RESULTS 66
4.1 ANALYSES OF GENETIC ALTERATIONS OF THE EGFR AND TYK2 GENES 66
4.1.1 GENETIC ALTERATIONS OF THE EGFR GENE 66
4.1.1.1 PREVALENCE OF SOMATIC EGFR MUTATIONS IN PRIMARY TUMOURS 69
4.1.2 GENETIC ALTERATIONS OF THE TYK2 GENE 69
4.1.2.1 PREVALENCE OF TYK2 GENETIC ALTERATIONS IN PRIMARY TUMOURS 70
4.2 BIOCHEMICAL AND FUNCTIONAL ANALYSES OF GENETIC ALTERATIONS OF THE
EGFR GENE.... 73
4.2.1 RESPONSE OF HUMAN CANCER CELL LINES CARRYING SOMATIC EGFR
MUTATIONS
TOWARDS THE EGFR TKI GEFITINIB 73
4.2.2 MUTATIONAL PROFILING OF THE KRAS AND BRAF GENES OF HUMAN CANCER
CELL
LINES CARRYING SOMATIC EGFR MUTATIONS 76
4.2.3 GEFITINIB RESISTANCE OF SW-48 CELLS MAY BE DUE TO A LOW EGFR
PROTEIN
EXPRESSION LEVEL 79
4.2.4 THE DYNAMICS OF PROLIFERATION OF RL95-2 CELLS IN THE PRESENCE OF
GEFITINIB 80
4.2.5 THE P753S SUBSTITUTION INCREASES AUTO-PHOSPHORYLATION OF THE EGFR
PROTEIN 82
4.3 BIOCHEMICAL AND FUNCTIONAL ANALYSES OF GENETIC ALTERATIONS OF THE
TYK2 GENE... 85
4.3.1 BASAL PHOSPHORYLATION LEVELS OF VARIANT TYK2 PROTEINS 85
IMAGE 5
4.3.1.1 BASAL PHOSPHORYLATION LEVELS AT TYROSINE RESIDUES Y1054 AND
Y1055 86
4.3.1.2 BASAL PHOSPHORYLATION LEVELS O F OTHER TYROSINE RESIDUES 88
4.3.2 EFFECTS OF OVER-EXPRESSION OF VARIANT TYK2 PROTEINS ON SELECTED
SIGNALLING PATHWAYS 89
4.3.3 TYK2-R901Q MUTANT PROTEIN IS LIKELY TO BE EXPRESSED IN THE OVARIAN
CANCER CELL LINE IGROV-1 92
4.3.4 EFFECT OF TYK2 KNOCK-DOWN ON PROLIFERATION OF IGROV-1 CELLS 93
4.3.5 TYK2 KNOCK-DOWN DECREASES MIGRATORY CAPABILITY OF IGROV-1 CELLS 94
4.3.6 EFFECTS ON STAT3 PHOSPHORYLATION LEVEL IN IGROV-1 CELLS UPON TYK2
KNOCK-DOWN AND STAT3 INHIBITOR TREATMENT 94
4.3.7 CHANGES IN THE EXPRESSION OF SELECTED DOWNSTREAM TARGET PROTEINS
IN
IGROV-1 CELLS UPON TYK2 SIRNA-MEDIATED ABROGATION OF STAT3
SIGNALLING 97
V. DISCUSSION 99
5.1 PTKS ARE VALID TARGETS FOR ANTI-CANCER THERAPEUTIC INTERVENTION 99
5.2 LARGE-SCALE SCREENING IS A FEASIBLE APPROACH TO IDENTIFY
CANCER-RELATED GENETIC
ALTERATIONS 100
5.3 A CATALOGUE OF GENETIC ALTERATIONS OF PTKS IN HUMAN CANCER CELL
LINES IS
URGENTLY NEEDED 100
5.4 THE ADVANTAGES AND DISADVANTAGES OF SCREENING FOR GENETIC
ALTERATIONS USING
CDNA 102
5.5 CATEGORIZATION OF GENETIC ALTERATIONS DEPENDS ON THE USAGE OF
NON-DISEASE
CONTROL MATERIAL 103
5.6 CHARACTERIZATION OF FUNCTIONALLY IMPORTANT GENETIC ALTERATIONS OF
EGFR 103
5.6.1 EGFR G719S MUTATION IN SW-48 CELLS 106
IMAGE 6
5.6.2 EGFR A289V MUTATION IN RL95-2 CELLS 109
5.6.3 EGFR P753S MUTATION IN SK-MEL-28 CELLS 112
5.7 CHARACTERIZATION OF FUNCTIONALLY IMPORTANT GENETIC ALTERATIONS OF
TYK2 114
5.7.1 TYK2 R901Q MUTATION IN IGROV-1 CELLS 116
5.7.2 OTHER GENETIC ALTERATIONS OF TYK2 120
5.8 SUMMARY AND CONCLUDING REMARKS 121
VI. REFERENCES 124
VII. APPENDIX 149
7.1 DESCRIPTION OF TYK2 DELETIONS AT THE PROTEIN LEVEL 149
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| spelling | Tay, Liang-Seah Verfasser aut An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines Liang-Seah Tay 2012 149 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Techn. Univ., Diss., 2012 Molekulargenetik (DE-588)4039987-4 gnd rswk-swf Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd rswk-swf Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 s Molekulargenetik (DE-588)4039987-4 s DE-604 Protein-Tyrosin-Kinasen (DE-588)4409305-6 s Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:91-diss-20120327-1071113-1-0 http://mediatum.ub.tum.de/node?id=1071113 Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1071113-1-0 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025016320&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Tay, Liang-Seah An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines Molekulargenetik (DE-588)4039987-4 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd |
| subject_GND | (DE-588)4039987-4 (DE-588)4409305-6 (DE-588)4271161-7 (DE-588)4113937-9 |
| title | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |
| title_auth | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |
| title_exact_search | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |
| title_full | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines Liang-Seah Tay |
| title_fullStr | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines Liang-Seah Tay |
| title_full_unstemmed | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines Liang-Seah Tay |
| title_short | An analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |
| title_sort | an analysis of genetic alterations of the epidermal growth factor receptor and the tyrosine kinase 2 in human cancer cell lines |
| topic | Molekulargenetik (DE-588)4039987-4 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd Epidermaler Wachstumsfaktor-Rezeptor (DE-588)4271161-7 gnd |
| topic_facet | Molekulargenetik Protein-Tyrosin-Kinasen Epidermaler Wachstumsfaktor-Rezeptor Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1071113 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20120327-1071113-1-0 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025016320&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT tayliangseah ananalysisofgeneticalterationsoftheepidermalgrowthfactorreceptorandthetyrosinekinase2inhumancancercelllines |