The fibroblast growth factor-binding protein (FGF-BP) and the human epidermal growth factor receptor-2 (HER-2) [Elektronische Ressource] : functional studies on two gene products relevant in ovarian cancer / vorgelegt von Shaker Abuharbeid

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• Gesundheit

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• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2005
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Aus dem Institut für Pharmakologie und Toxikologie
Geschäftsführender Direktor: Prof. Dr. med. Thomas Gudermann
des Fachbereichs Medizin der Philipps-Universität Marburg
und des Universitätsklinikums Gießen und Marburg, Standort Marburg




The Fibroblast Growth Factor-binding Protein (FGF-BP) and the Human
Epidermal Growth Factor Receptor-2 (HER-2): Functional Studies on Two
Gene Products Relevant in Ovarian Cancer




INAUGURAL–DISSERTATION
zur Erlangen des Doktorgrades der Humanbiologie
(Dr. rer. physiol.)

dem Fachbereich Medizin der Philipps-Universität Marburg


vorgelegt von

Shaker Abuharbeid
aus Gaza, Palästina


Marburg, 2005






































Angenommen vom Fachbereich Humanmedizin der Philipps-Universität Marburg
am: 01.11.2005

Gedruckt mit Genehmigung des Fachbereichs


Dekan: Prof. Dr. B. Maisch
Referent: Prof. Dr. F. Czubayko
Korreferent: Prof. Dr. G. Aumüller






















To my dear parents and daughters
Nour, Anwar and Rana Table of contents I
TABLE OF CONTENTS

1 INTRODUCTION.........................................................................................1
1.1 Basic tumor biology and biological role of oncogenes in cancer................... 1
1.2 Angiogenesis in tumor growth........................................................................ 2
1.3 Fibroblast growth factors and the fibroblast growth factor-binding protein... 3
1.3.1 Fibroblast growth factors (FGFs).................................................................... 3
1.3.2 The fibroblast growth factor-binding protein (FGF-BP)................................ 6
1.3.3 FGF-BP expression in neoplastic tissues and its regulation in skin and
colon carcinogenesis....................................................................................... 7
1.3.4 FGF-BP expression in normal tissues and its regulation during embryonic
development and tissue repair......................................................................... 9
1.3.5 Regulation of FGF-BP expression by TPA, EGF, fetal bovine serum and
retinoids.......................................................................................................... 11
1.3.6 Structural characterization of FGF-BP............................................................ 13
1.3.7 The mechanism of FGF-BP action................................................................. 14
1.4 The HER-2 Receptor....................................................................................... 16
1.4.1 Structure of HER receptors............................................................................. 17
1.4.2 Ligands of HER receptors............................................................................... 19
1.4.3 HER-2-induced signaling pathways............................................................... 21
1.4.4 The relevance of the HER-2 network in cancer.............................................. 22
1.4.5 Effects of HER-2 overexpression on chemotherapeutic drug sensitivity in
tumor cells....................................................................................................... 23
1.4.6 HER-2-targeting strategies.............................................................................. 25
1.5 Antineoplastic agents...................................................................................... 28
1.5.1 Taxol............................................................................................................... 28
1.5.2 rViscumin........................................................................................................ 29
1.6 Biology of ovarian cancer............................................................................... 31

2 OBJECTIVES AND STRUCTUR OF THIS THESIS.............................. 32

3 MATERIALS AND METHODS................................................................. 33
3.1 Materials......................................................................................................... 33
3.1.1 Reagents......................................................................................................... 33
3.1.2 Chemotherapeutic agents and phosphotyrosine kinase inhibitors.................. 34
3.1.3 Kits and enzymes............................................................................................ 34
3.1.4 Antibodies....................................................................................................... 34
3.1.5 Oligonucleotides and primers......................................................................... 35
3.1.6 Bacterial cells and vectors.............................................................................. 36
3.1.7 Tissue culture media and reagents.................................................................. 36
3.1.8 Cell lines......................................................................................................... 36
3.1.9 Equipment, devices and working materials.................................................... 37
3.1.10 Standard solutions, buffers and bacterial growth media................................. 37

Table of contents II
3.2 Methods...................................................................................................... 42
3.2.1 Cell culture methods................................................................................... 42
3.2.1.1 Handling of COS-7, SW-13, HepG2, SKOV-3 and SF-9 cells.................. 42
3.2.1.2 Thawing of cultured cell lines.................................................................... 42
3.2.1.3 Maintenance of cells in culture.................................................................. 42
3.2.1.4 Preparation of freeze-stocks of cultured cell lines..................................... 43
3.2.1.5 Transient and stable transfection of COS-7 and SW-13 cells.................... 43
3.2.1.6 Growth assays............................................................................................ 43
3.2.1.6.1 WST-1 proliferation assay......................................................................... 43
3.2.1.6.2 Soft agar assay............................................................................................ 44
3.2.2 Biochemical and immunochemical methods.............................................. 45
3.2.2.1 Immunohistochemistry............................................................................... 45
3.2.2.2 Immunofluorescence.................................................................................. 45
3.2.2.3 Purification of recombinant FGF-BP......................................................... 46
3.2.2.4 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blotting........................................................................................................ 47
3.2.2.5 Dot blotting................................................................................................ 49
3.2.2.6 Protein staining with Coomassie brillant blue........................................... 49
1253.2.2.7 [ I]-labeling of FGF-BP and rViscumin.................................................. 49
1253.2.2.8 Analysis of cellular uptake of [ I]-FGF-BP in COS-7 cells through
subcellular fractionation............................................................................. 49
3.2.2.9 Analysis of cellular rViscumin binding/uptake.......................................... 50
3.2.3 Molecular biological methods.................................................................... 51
3.2.3.1 Polymerase chain reaction (PCR)............................................................... 51
3.2.3.2 Electrophoretic separation of DNA fragments in agarose gels.................. 52
3.2.3.3 Phenol-chloroform extraction of DNA...................................................... 53
3.2.3.4 Linearization of plasmid DNA and restriction digest of PCR products..... 53
3.2.3.5 Dephosphorylation of digested DNA......................................................... 54
3.2.3.6 DNA isolation from agarose gel................................................................. 55
3.2.3.7 Ligation...................................................................................................... 55
3.2.3.8 Preparation of chemically competent Escherichia coli cells...................... 55
3.2.3.9 Transformation of chemically competent Escherichia coli cells................ 56
3.2.3.10 Bacterial culture.........................