Mitogen-inducible gene-6 is a negative regulator of the HER-family of receptor tyrosine kinases [Elektronische Ressource] / Markus Oliver Reschke

Reschke

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Genetik



Mitogen-Inducible Gene-6 is a Negative Regulator of the
HER-Family of Receptor Tyrosine Kinases


Markus Oliver Reschke



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. R. Hückelhoven

Prüfer der Dissertation: 1. Univ.-Prof. Dr. A. Gierl

2. Hon.-Prof. Dr. A. Ullrich
(Eberhard-Karls-Universität Tübingen)

3. Univ.-Prof. Dr. W. Wurst




Die Dissertation wurde am 14.07.2008 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 05.11.2008 angenommen.
















To my family

























Parts of this work presented in this thesis have been published in Clinical Cancer Research.

HER3 is a Determinant for Poor Prognosis in Melanoma
Markus Reschke, Daniela Mihic-Probst, Edward Htun van der Horst, Pjotr Knyazev, Peter J.
Wild, Markus Hutterer, Stefanie Meyer, Reinhard Dummer, Holger Moch and Axel Ullrich
Clinical Cancer Research, in press
















1. INTRODUCTION........................................................................................... 1
1.1 The EGF receptor family ....................................................................................... 1
1.1.1 The EGF receptor (EGFR, HER1, ErbB1).................................................................... 2
1.1.2 HER2 (ErbB2/neu)........................................................................................................ 5
1.1.3 HER3 (ErbB3)............................................................................................................... 5
1.1.4 HER4 (ErbB4).. 7
1.2 Targeting human EGF receptors for cancer therapy ......................................... 8
1.3 Negative regulators of receptor tyrosine kinase signalling............................... 10
1.3.1 Mitogen-inducible gene-6 (Mig-6).............................................................................. 11
1.4 Melanoma .............................................................................................................. 15
1.5 Liver regeneration and human liver cancer....................................................... 17
2. SPECIFIC AIMS........................................................................................... 20
3. MATERIAL AND METHODS.................................................................... 21
3.1 Materials ................................................................................................................ 21
3.1.1 Laboratory chemicals .................................................................................................. 21
3.1.2 Enzymes ...................................................................................................................... 22
3.1.3 “Kits“ and other materials 22
3.1.4 Growth factors and ligands ......................................................................................... 22
3.2 Media...................................................................................................................... 23
3.2.1 Bacterial media............................................................................................................ 23
3.2.2 Cell culture media ....................................................................................................... 23
3.3 Stock solutions and commonly used buffers ...................................................... 23
3.4 Cells.. 25
3.4.1 Eukaryotic cell lines .................................................................................................... 25
3.4.2 E. Coli strains.... 26
3.5 Antibodies .............................................................................................................. 26
3.5.1 Primary antibodies....................................................................................................... 26
3.5.2 Secondary antibodies................................................................................................... 28
3.6 Plasmids and oligonucleotides ............................................................................. 28
3.6.1 Primary vectors ........................................................................................................... 28
3.6.2 Constructs.................................................................................................................... 28
3.6.3 Oligonucleotides.......................................................................................................... 29
3.7 Methods of molecular cloning.............................................................................. 30
3.7.1 Plasmid preparation..................................................................................................... 30
3.7.2 Enzymatic manipulation of DNA................................................................................ 30
3.7.3 Introduction of plasmid DNA into E.coli.................................................................... 31
3.7.4 Enzymatic amplification of DNA by polymerase chain reaction (PCR) .................... 31
7.5 DNA sequencing ............................................................................................................ 31
3.8 Methods of mammalian cell culture 32
3.8.1 Calcium-Phosphate transfection.................................................................................. 32
3.8.2 Transfection of plasmid DNA using lipofectamine® ................................................. 32
3.8.3 Transfection of siRNAs using oligofectamine®......................................................... 32
3.8.4 Stimulation of cells...................................................................................................... 32
3.9 Methods of Biochemistry and Cell Biology ........................................................ 32
3.9.1 Lysis of cells with Triton X-100 lysis buffer .............................................................. 32
3.9.2 Determination of total protein concentration in lysates .............................................. 32
3.9.3 Immunoprecipitation ................................................................................................... 33
3.9.4 SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE) 33
3.9.5 Transfer of proteins on nitrocellulose membranes...................................................... 33
3.9.6 Immunoblot detection ................................................................................................. 33
3.9.7 RNA isolation and RT-PCR analysis.......................................................................... 33
3.9.8 Northern and Southern blot analysis ........................................................................... 34
3.9.9 Proliferation assay....................................................................................................... 34
3.9.10 Migration assay ......................................................................................................... 34
3.9.11 Cell branching and Invasion assay ............................................................................ 34
3.9.12 HER3 blocking antibody – Proliferation, Migration and Invasion assay ................. 34
3.9.13 Apoptosis assay and cell cycle analysis by propidium iodide staining..................... 35
3.9.14 Chromatin Immunoprecipitation (ChIP) ................................................................... 35
3.9.15 5-dC-Azacytidine treatment of melanoma cells........................................................ 36
3.9.16 Indirect flow cytometry............................................................................................. 36
3.9.17 Stable isotope labelling by amino acids in cell culture (SILAC) and mass
spectrometry ......................................................................................................................... 36
3.10 Methods of mouse genetics................................................................................. 36
3.10.1 Mice and gene targeting ............................................................................................ 36
3.10.2 2-stage skin carcinogenesis ....................................................................................... 36
3.10.3 Isolation of mouse embryonic fibroblasts (MEFs).................................................... 36
3.10.4 Partial hepatectomy ................................................................................................... 36
3.10.5 KI-67 Immunohistochemistry on mouse liver sections......................