The role of the mitotic spindle checkpoint in chemotherapy-induced apoptosis [Elektronische Ressource] / vorgelegt von Celia Vogel

Andrej Shishkin

Sujets

• Gesundheit

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Aus dem Institut für Molekularbiologie und Tumorforschung
der Philipps-Universität Marburg
Geschäftsführender Direktor: Prof. Dr. Rolf Müller







The Role of the Mitotic Spindle Checkpoint in
Chemotherapy-Induced Apoptosis




Inaugural-Dissertation

zur Erlangung des Doktorgrades
der Humanbiologie
(Dr. rer. physiol.)

dem Fachbereich Medizin der







vorgelegt von
Celia Vogel
aus Oldenburg

Marburg 2008











































Angenommen vom Fachbereich Medizin
der Philipps-Universität Marburg am 05.08.2008.

Gedruckt mit Genehmigung des Fachbereichs.

Dekan: Prof. Dr. Matthias Rothmund
Referent: PD Dr. Holger Bastians
Korreferent: Prof. Dr. Czubayko
















































The only way of discovering the limits of the possible is to venture a little way past them
into the impossible.
Arthur C. Clarke
Table of Contents
Table of Contents
Table of Contents ..............................................................................................................................I
Summary........................................................................................................................................... 1
Zusammenfassung............................................................................................................................ 3
Introduction...................................................................................................................................... 5
CANCER AS MEDICAL AND SCIENTIFIC PROBLEM ................................................................ 5
TUMORIGENESIS IS INDUCED BY MULTIPLE MECHANISMS .................................................. 6
THE EUKARYOTIC CELL CYCLE .......................................................................................... 7
The G2/M transition ...................................................................................................... 9
Mitosis is comprised of distinct phases and is regulated by phosphorylation............. 10
Regulation of mitosis by regulated protein proteolysis............................................... 13
CELL CYCLE CHECKPOINTS................................................................................................ 14
The DNA damage checkpoints.................................................................................... 14
The intra-S phase checkpoints................................................................................. 14
The G1/S DNA damage checkpoint ........................................................................ 15
The G2/M DNA damage checkpoint....................................................................... 16
p53 protects genomic integrity ................................................................................ 17
The spindle checkpoint ensures genomic integrity by controlling chromatid
Cdc20segregation mediated by the APC/C ..................................................................... 19
Mechanism of spindle checkpoint signaling ........................................................... 21
Mad2 activation ....................................................................................................... 24
Altered expression of spindle checkpoint genes can lead to aneuploidy, cancer
and premature aging in mice and men..................................................................... 24
The postmitotic G1 checkpoint prevents propagation of tetraploid cells generated
by a failed mitosis.................................................................................................... 26
APOPTOSIS AND OTHER FORMS OF CELL DEATH................................................................ 27
Apoptosis..................................................................................................................... 27
Other forms of cell death............................................................................................. 31
CHEMOTHERAPY................................................................................................................. 32
Introduction ................................................................................................................. 32
DNA damaging agents ................................................................................................ 33
Topoisomerase inhibitors ........................................................................................ 33
Alkylating agents..................................................................................................... 35
Antimetabolites........................................................................................................ 35
Spindle damaging agents............................................................................................. 36
Vinca alkaloids ........................................................................................................ 37
Taxanes.................................................................................................................... 37
Epothilones .............................................................................................................. 38
New mitotic targets...................................................................................................... 38
Cyclin dependent kinase inhibitors.......................................................................... 38
Aurora inhibitors ..................................................................................................... 39
Plk inhibitors ........................................................................................................... 40
Eg5/KSP inhibitors.................................................................................................. 41
G2 CP abrogation .................................................................................................... 41
AIMS OF THE STUDY ............................................................................................................ 43
Materials and Methods.................................................................................................................. 44
MATERIALS ......................................................................................................................... 44
Chemicals, antibodies and probes ............................................................................... 44
Lab ware ...................................................................................................................... 50
Equipment.................................................................................................................... 51
Table of Contents
Plasmids....................................................................................................................... 53
Bacteria........................................................................................................................ 54
Cell lines...................................................................................................................... 54
Data bases, software .................................................................................................... 56
METHODS ............................................................................................................................ 57
Molecular Biology....................................................................................................... 57
Generation of transformation competent bacteria ................................................... 57
Transformation of bacteria ...................................................................................... 57
Plasmid DNA preparation from bacteria................................................................. 57
Restriction digest ..................................................................................................... 58
PCR.......................................................................................................................... 58
PCR purification...................................................................................................... 58
Cloning .................................................................................................................... 59
Agarose gel electrophoresis..................................................................................... 59
Gel extraction .......................................................................................................... 59
DNA Sequencing..................................................................................................... 60
Chromatin