PKC isozymes in the control of fate of prostate cancer cells [Elektronische Ressource] / Vvivian Annaluise von Burstin (geb. Därr)

Vivian Von Burstin

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 33
• 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
PKC isozymes in the control of fate of prostate cancer cells
Vivian Annaluise von Burstin (geb. Därr)
aus
Houston, U.S.A.
2010Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 (in der Fassung der vierten Änderungssatzung vom 26.
November 2004) von Herrn Prof. Dr. Marcelo G. Kazanietz betreut und von Frau Prof.
Dr. Angelika M. Vollmar von der Fakultät für Chemie und Pharmazie vertreten.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.
München, den 02.12.2010
Vivian A. von Burstin
Dissertation eingereicht am: 02.12.2010
1. Gutachter: Prof. Dr. Angelika M. Vollmar
2. Gutachter: Prof. Dr. Marcelo G. Kazanietz
Mündliche Prüfung am: 08.02.2011TABLE OF CONTENTS
I INTRODUCTION..................................................................................................... 5
I.1. Prostate Cancer .............................................................................................. 5
I.2 Apoptosis ......................................................................................................... 9
I.2.1 General features of apoptosis.................................................................... 9
I.2.2 Caspases .................................................................................................. 9
I.2.3 Pathways of caspase activation............................................................... 11
I.2.4 Apoptosis versus necrosis ....................................................................... 14
I.2.5 B-cell lymphoma-2 (BCL-2) family ........................................................... 14
I.2.6 The apoptosome...................................................................................... 16
I.2.7 Induction of apoptosis by DNA double strand breaks............................... 19
I.3 PKC isozymes................................................................................................ 21
I.3.1 Structure of PKC isozymes ...................................................................... 21
I.3.2 Mechanisms of PKC activation and inactivation....................................... 22
I.4 Regulation of apoptotic pathways by PKC isozymes ...................................... 25
I.5 Naturally occurring DAG analogs: Phorbol esters and Bryostatins.................. 29
I.6 Aims of the study............................................................................................ 32
II MATERIALS AND METHODS............................................................................. 34
II.1 Compounds for cell culture treatment ............................................................ 34
II.2 Cell Culture ................................................................................................... 34
II.2.1 Materials................................................................................................. 35
II.2.2 Cultivation of cells................................................................................... 34
II.2.3 Freezing and thawing of LNCaP cells ..................................................... 36
II.2.4 Inactivation of fetal bovine serum (FBS) ................................................. 37
II.3 Apoptosis assay ............................................................................................ 38
II.3.1 Materials................................................................................................. 38
II.3.2 DAPI staining.......................................................................................... 39II.3.3 Generation of Conditioned medium (CM)................................................ 40
II.3.4 Treatment of LNCaP cells with etoposide ............................................... 41
II.3.5 Treatment of LNCaP cells with cycloheximide ........................................ 41
II.4 Transfection .................................................................................................. 41
II.4.1 Transfection by electroporation............................................................... 42
II.5 Immunofluorescence and confocal microscopy ............................................. 43
II.6 Measurement of cytokines in the CM............................................................. 44
II.6.1 ELISA for humhan TNFα (Sandwich ELISA)........................................... 45
II.6.2 RayBio® Human Cytokine Antibody Array .............................................. 47
II.7 Western Blot Analysis.................................................................................... 48
II.7.1 Isolation of proteins................................................................................. 48
II.7.2 Protein Quantification ............................................................................. 49
II.7.3 SDS-PAGE............................................................................................. 49
II.7.4 Western Blotting and detection ............................................................... 51
II.8. Generation of myristoylated PKCδ constructs............................................... 54
II.8.1 Cloning of myristoylated PKCδ constructs............................................... 54
II.8.2 Transformation ....................................................................................... 59
II.9 RNA interference (RNAi) ............................................................................... 60
II.9.1 siRNAs for experimental gene silencing.................................................. 61
II.10 RNA isolation............................................................................................... 62
II.10.1 RNA isolation with the Qiagen RNeasy Mini Kit .................................... 62
II.10.2 RNA isolation for microarray ................................................................. 62
II.11 cDNA synthesis........................................................................................... 63
II.12 Reverse transcriptase real-time PCR .......................................................... 64
II.12.1 Experimental conditions........................................................................ 64
-ΔΔCtII.12.2 Quantification with the 2 method (comparative C method).............. 66t
II.12.3 Materials and primers for RT-PCR using TaqMan®.............................. 66II.13 DNA Microarray and Analysis of Array Data ................................................ 67
II.14 Validation of data by quantitative RT-PCR................................................... 70
II.15. Statistical analysis ...................................................................................... 70
III RESULTS ........................................................................................................... 71
III.1 Phorbol ester and bryostatin have opposing effects in LNCaP cells ............. 71
III.1.1 PMA induces apoptosis in the prostate cancer cell line LNCaP ............. 71
III.1.2 The C1 domain ligand bryostatin 1 fails to induce cell death and inhibits
PMA-induced apoptosis in LNCaP cells........................................................... 72
III.1.3 Bryostatin 1 does not affect the expression of survival factors or the
phosphorylation status of Akt or p38................................................................ 73
III.1.4 Conditioned medium (CM) from bryostatin 1-treated LNCaP cells inhibits
PMA- ................................................. 75
III.1.5 Analysis of the secreted factors to the CM using a cytokine array did not
reveal significant changes upon treatment with bryostatin 1 ............................ 78
III.1.6 Bryostatin 1 prevents PMA-stimulated release of TNFα from
LNCaP cells..................................................................................................... 81
III.1.7 PKCδ is a key regulator of PMA-induced TNFα release from
LNCaP cells..................................................................................................... 83
III.1.8 Bryostatin 1 selectively inhibits the translocation of PKC
membrane in LNCaP cells ............................................................................... 85
III.1.9 Translocation of PKCδ to the plasma membrane is essential for the
induction of apoptosis in LNCaP cells.............................................................. 86
III.2. Genome-wide expression analysis reveals PKCδ as master regulator of
transcription in LNCaP cells ................................................................................ 89
III.2.1 Inhibition of protein synthesis by cycloheximide significantly decreased
PMA-induced apoptosis................................................................................... 89
III.2.2 PMA rapidly induces changes in gene expression levels ....................... 90
QYLQDROV1LSVPXFHHDGDDOSVRDSGWVLOFW3R&/WHK'HQIII.2.3 Selection of PMA-regulated genes according to their Gene Ontologies . 91
III.2.4 Isozyme-specific depletion of PKCs in LNCaP cells............................... 92
III.2.5 Reproducibilty between two independent microarray experiments......... 93
III.2.6 Principal component analysis................................................................. 95