Transactivation of the EGFR signal in human cancer cells [Elektronische Ressource] / Beatrix Schäfer

Schaefer Beatrix

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München







Transactivation of the EGFR Signal in Human Cancer Cells




Beatrix Schäfer

aus

Wissen










2004








Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw 4 der
Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Horst Domdey
betreut

Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, den 09.03.2004



Beatrix Schäfer




Dissertation eingereicht am 09.03.2004
1. Gutachter Prof. Dr. Axel Ullrich
2. Gutachter . Dr. Horst Domdey
Mündliche Prüfung am 15.06.2004

Contents


Contents ......................................................................................................................................3
1 Introduction ........................................................................................................................7
1.1 Protein tyrosine kinases ............................................................................................7
1.1.1 Receptor tyrosine kinases (RTK) ........................................................................8
1.1.2 EGF-like ligands..................................................................................................9
1.1.3 Ligand-induced activation of receptor tyrosine kinases......................................
1.1.4 Cytoplasmic tyrosine kinases............................................................................10
1.1.5 Recruitment of downstream signalling molecules ............................................11
1.2 Mitogen-activated-protein-kinase (MAPK) pathways.........................................13
1.3 Protein kinase B/Akt ...............................................................................................14
1.4 G protein-coupled receptors...................................................................................14
1.5 EGFR signal transactivation ..................................................................................16
1.6 Metalloproteases ......................................................................................................21
1.6.1 ADAMs.............................................................................................................22
1.6.2 The Matrix Metalloproteinases (MMPs) ...........................................................23
1.7 Molecular oncology and aberrant signalling in cancer........................................24
1.8 Cancer cell characteristics ......................................................................................25
1.9 Aim of the study.......................................................................................................26
2 Materials and Methods.....................................................................................................28
2.1 Materials...................................................................................................................28
2.1.1 Laboratory chemicals and biochemicals............................................................28
2.1.2 Enzymes............................................................................................................29
2.1.3 Radiochemicals..................................................................................................2
2.1.4 "Kits" and other materials..................................................................................29
2.1.5 Growth factors and ligands................................................................................30
2.1.6 Media and buffers..............................................................................................30
2.1.6.1 Media for E. coli bacteria ..............................................................................3
2.1.6.2 Cell culture media..........................................................................................30
2.1.7 Stock solutions and buffers ...............................................................................30
2.1.8 Bacteria strains (E. coli) ....................................................................................32
2.1.9 Cell lines............................................................................................................32
2.1.10 Antibodies..........................................................................................................3
2.1.11 Plasmids and oligonucleotides ..........................................................................33
2.1.11.1 Primary vectors..........................................................................................33
2.1.11.2 Constructs..................................................................................................34
2.2 Methods in molecular biology ................................................................................35
2.2.1 Plasmid preparation for analytical purpose .......................................................35
2.2.2 id on in preparative scale...........................................................35
2.2.3 Enzymatic manipulation of DNA......................................................................35
2.2.3.1 Digestion of DNA samples with restriction endonucleases ..........................35
2.2.3.2 Dephosphorylation of 5’-termini with calf intestine alkaline phosphatase
(CIAP) 35
2.2.3.3 DNA insert ligation into vector DNA ...........................................................35
2.2.4 Agarose gel electrophoresis...............................................................................36
2.2.5 Isolation of DNA fragments using low melting temperature agarose gels........36
2.2.6 Introduction of plasmid DNA into E.coli cells..................................................36
2.2.6.1 Preparation of competent E. coli bacteria......................................................36
2.2.6.2 Transformation of competent E. coli bacteria ...............................................36
2.2.7 Enzymatic amplification of DNA by polymerase chain reaction (PCR)...........36
2.2.8 DNA sequencing...............................................................................................37
2.3 Methods in mammalian cell culture ......................................................................37
2.3.1 General cell culture techniques..........................................................................37
2.3.2 Transfection of cultured cell lines .....................................................................37
2.3.2.1 Transfection of cells with calcium phosphate ...............................................37
2.3.2.2 RNA interference...........................................................................................38
2.3.3 Retroviral gene transfer in cell lines..................................................................38
2.4 Protein analytical methods .....................................................................................38
2.4.1 Lysis of eucaryotic cells with Triton X100 .......................................................38
2.4.2 Determination of protein concentration in cell lysates......................................38
2.4.3 Immunprecipitation and in vitro association with fusion proteins ....................39
2.4.4 SDS-polyacrylamide-gelelectrophoresis...........................................................39
2.4.5 Transfer of proteins on nitrocellulose membranes ............................................3
2.4.6 Immunoblot detection........................................................................................39
2.5 Biochemical and cell biological assays...................................................................39
2.5.1 Stimulation of cells............................................................................................39
2.5.2 ERK1/2 and AKT/PKB phosphorylation ..........................................................40
2.5.3 ERK/MAPK activity.........................................................................................40
2.5.4 Flow cytometric analysis of cell surface proteins .............................................4
32.5.5 Incorporation of H-thymidine into DNA .........................................................40
2.5.6 Distribution of cell cycle phases........................................................................40
2.5.7 In vitro wound closure.......................................................................................40
2.5.8 Migration and invasion......................................................................................41
2.6 Statistical analysis....................................................................................................41
3 Results ...............................................................................................................................42
3.1 A variety of GPCR agonists stimulate EGFR tyrosine phosphorylation in
k