Anti-angiogenic effects of Cyclin dependent kinase inhibitors [Elektronische Ressource] : novel function of Cyclin dependent kinase-5 in endothelial cell migration / Johanna Liebl

Johanna Liebl

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 27
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

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





Anti-angiogenic effects of Cyclin dependent kinase inhibitors -
Novel function of Cyclin dependent kinase 5 in endothelial cell migration





Johanna Liebl
aus Pfarrkirchen
2008



Erklärung:
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn PD Dr. Stefan Zahler betreut am Lehrstuhl für Pharmazeutische
Biologie von Frau Prof. Dr. Angelika M. Vollmar.





Ehrenwörtliche Versicherung:
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.





München, 13. Oktober 2008




Johanna Liebl






Dissertation eingereicht am: 13. Oktober 2008
1. Gutachter: PD Dr. Stefan Zahler
2. Gutachter: Prof. Dr. Martin Biel
Mündliche Prüfung am: 21.November 2008







Für Mama und Papa




CONTENTS I
1 CONTENTS
II CONTENTS
1 CONTENTS...................................................................................I
2 INTRODUCTION........................................................................ 1
2.1 Background and aim of the study ........................................................2
2.2 Cyclin dependent kinases......................................................................4
2.2.1 Functions................................................................................................................ 4
2.2.2 Regulation .............................................................................................................. 5
2.3 Cyclin dependent kinase 5 (Cdk5) .......................................................6
2.3.1 Regulation of Cdk5 ................................................................................................ 6
2.3.2 Function of Cdk5 in the nervous system................................................................ 8
2.4 Cyclin dependent kinase inhibitors....................................................10
2.4.1 Roscovitine (Seliciclib, CYC202)........................................................................ 10
2.4.2 Derivatives of roscovitine: LGR561, LGR848, and LGR849 ............................. 11
2.5 Angiogenesis .........................................................................................12
2.5.1 Angiogenic cascade.............................................................................................. 12
2.6 Endothelial cell migration...................................................................14
2.6.1 Cell types.............................................................................................................. 14
2.6.2 Regulation of endothelial cell migration.............................................................. 14
2.6.2.1 Cell polarization........................................................................................... 15
2.6.2.2 Extension of membrane protrusions ............................................................ 16
2.6.2.3 Adhesion assembly and disassembly ........................................................... 17
2.6.2.4 Cell contraction............................................................................................ 18
2.6.3 Rho GTPases........................................................................................................ 19
2.6.3.1 Regulation .................................................................................................... 19
2.6.3.2 Rac1 ............................................................................................................. 20
2.6.3.3 Cdc42 ........................................................................................................... 21
2.6.3.4 RhoA ............................................................................................................ 22
CONTENTS III
2.6.4 Focal adhesion kinase (FAK) ...............................................................................23
3 MATERIALS AND METHODS .............................................. 25
3.1 Materials............................................................................................... 26
3.1.1 Roscovitine and its derivatives LGR561, LGR848, and LGR849 .......................26
3.1.2 Biochemicals and inhibitors .................................................................................26
3.2 Cell Culture.......................................................................................... 27
3.2.1 Solutions and reagents27
3.2.2 Endothelial cells ...................................................................................................28
3.2.3 Passaging ..............................................................................................................29
3.2.4 Freezing and thawing............................................................................................29
3.3 Angiogenesis assays ............................................................................. 30
3.3.1 Proliferation assay (crystal violet staining assay) ................................................30
®3.3.2 CellTiter-Blue Cell Viability Assay ...................................................................30
3.3.3 Scratch assay ........................................................................................................31
3.3.4 Chemotaxis assay .................................................................................................32
3.3.5 Tube formation assay ...........................................................................................33
3.3.6 Mouse aortic ring assay ........................................................................................34
3.3.7 Chick chorioallantoic membrane (CAM) assay ...................................................35
3.3.8 Mouse cornea micropocket angiogenesis assay36
3.4 Western Blot analysis.......................................................................... 38
3.4.1 Preparation of samples .........................................................................................38
3.4.2 SDS-PAGE electrophoresis..................................................................................39
3.4.3 Electroblotting ......................................................................................................40
3.4.4 Protein detection...................................................................................................41
3.4.4.1 Specific protein staining with antibodies......................................................41
3.4.4.2 Unspecific protein staining of gels and membranes.....................................44
3.4.5 Membrane stripping and reprobing ......................................................................44
IV CONTENTS
3.5 Protein quantification..........................................................................45
3.5.1 Bicinchoninic (BCA) Protein Assay.................................................................... 45
3.5.2 Bradford Assay .................................................................................................... 45
3.6 Transfection of cells.............................................................................46
3.7 Confocal laser scanning microscopy..................................................47
3.7.1 Microscopy with fixed cells................................................................................. 48
3.7.2 Life cell imaging .................................................................................................. 49
3.7.3 Histological staining ............................................................................................ 50
3.8 Flow Cytometry (FACS) .....................................................................51
3.9 Tubulin fractionation ..........................................................................53
3.10 Pull-down assay....................................................................................54
3.11 Adhesion assay .....................................................................................55
3.12 Statistical analysis................................................................................55
4 RESULTS ................................................................................... 57
4.1 Roscovitine, LGR561, LGR848, and LGR849 exert anti-angiogenic
effects in vitro and in vivo....................................................................58
4.1.1 Roscovitine and the LGRs inhibit endothelial cell proliferation ......................... 58
4.1.2 Effects of roscovitine and the LGRs on cell cycle and apoptosis........................ 59
4.1.3 LGRs on endothelial cell migration..................... 61
4.1.4 Effects of roscovitine on endothelial cell chemotaxis ......................................... 62
4.1.5 Roscovitine and the LGRs inhibit tube formation ............................................... 63
4.1.6 e and the LGRs reduce vessel sprouting out of mouse aortic rings.... 64
4.1.7 Roscovi