Analysis of retinoblastoma protein function in the regulation of apoptosis [Elektronische Ressource] = Analyse der Funktion des Retinoblastoma-Proteins in der Apoptose-Regulation / von Anja Masselli

Andreas Karkavitsas

Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Analysis of retinoblastoma protein function
in the regulation of apoptosis

Analyse der Funktion des Retinoblastoma-Proteins in der
Apoptose-Regulation




Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)



vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena

von Diplom-Biochemikerin
Anja Masselli

geboren am 25.07.1976 in Göttingen





Jena, 2005

Table of contents

Table of contents
1 Introduction............................................................................................................1
1.1 Apoptosis as cell fate and cellular stress response............................................1
1.2 Central mediators of the apoptotic program......................................................2
1.3 The intrinsic apoptosis pathway........................................................................3
1.4 Death receptor-induced apoptosis .....................................................................6
1.5 Regulation of apoptosis pathways9
1.6 The retinoblastoma protein as a regulator of proliferation and apoptosis.......13
1.7 Objectives........................................................................................................17
2 Results ...................................................................................................................18
2.1 Effect of constitutively active RB variants on cell death response to different
stimuli ............................................................................................................18
2.1.1 Doxorubicin-induced activation of caspases is attenuated in Rat-16 cells
arrested by PSM-RB ................................................................................19
2.1.2 Staurosporine-induced apoptosis of Rat-16 cells is not prevented by
PSM-RB...................................................................................................26
2.1.3 Inducible expression of RB variants sensitizes Rat-16 cells to TNF-
induced apoptosis.....................................................................................28
MI/MI2.2 Analysis of altered TNF response in Rb fibroblasts.................................34
MI/MI2.2.1 Response of wild-type and Rb cells to selective activation of TNF
receptors34
MI/MI
2.2.2 Gene expression analysis of TNF response in Rb cells ....................36
2.2.3 Analysis of mitochondria-mediated apoptosis in TNF-treated wild-type
MI/MI
and Rb cells .......................................................................................45
2.2.4 In vitro analysis of cytochrome c release.................................................47
MI/MI2.2.5 TNF dosage effects in Rb cells .........................................................51
MI/MI2.2.6 Effect of caspase inhibition on TNF response in wild-type and Rb
cells ..........................................................................................................54

Table of contents
3 Discussion..............................................................................................................58
MI/MI3.1 Rb-MI-dependent suppression of apoptosis in Rb fibroblasts .................60
3.2 Post-transcriptional suppression of mitochondrial apoptosis by Rb-MI.........61
3.2.1 Role of nucleo-cytoplasmic signaling during TNF-induced apoptosis....64
MI/MI3.2.2 Caspase-independent cell death in TNF treated Rb fibroblasts ........66
3.3 Constitutively active RB variants have contrasting effects on the apoptotic
response to different stimuli.............................................................................67
3.3.1 Effect of PSM-RB induced growth arrest on apoptosis...........................68
3.3.2 Cell-cycle independent effects on apoptosis by overexpression of RB
variants .....................................................................................................70
4 Summary...............................................................................................................72
5 Materials and Methods........................................................................................75
5.1 Abbreviations and Symbols ............................................................................75
5.1.1 Abbreviations...........................................................................................75
5.1.2 Amino acid symbols.................................................................................77
5.1.3 Prefixes for measurement units................................................................77
5.2 Materials..........................................................................................................78
5.2.1 Bacteria strains.........................................................................................78
5.2.2 Bacterial culture media and solutions ......................................................78
5.2.3 Plasmids and Plasmid constructs .............................................................78
5.2.4 Cell culture media and solutions..............................................................78
5.2.5 Buffers and solutions ...............................................................................79
5.2.6 Antibodies ................................................................................................84
5.2.7 Caspase substrates and inhibitors84
5.2.8 Enzymes...................................................................................................85
5.2.9 Miscellaneous reagents and materials......................................................85
5.3 Methods...........................................................................................................86
5.3.1 Transformation of E. coli.........................................................................86
5.3.2 Preparation of plasmid DNA from E. coli ...............................................86
5.3.3 Spectrophotometric quantification of nucleic acids.................................87
5.3.4 Enzymatic manipulation of DNA ............................................................87
5.3.5 Isolation, purification and characterization of nucleic acids....................88
Table of contents
5.3.6 Construction of Rat-16 cell lines .............................................................89
5.3.7 Cell culture...............................................................................................90
5.3.8 Clonogenic survival assay........................................................................90
5.3.9 Flow cytometry ........................................................................................90
5.3.10 Immunofluorescence microscopy ............................................................91
5.3.11 DNA microarray analysis of gene expression..........................................92
5.3.12 Preparation of cell lysates95
5.3.13 Isolation of fractionated cell extracts.......................................................95
5.3.14 Isolation of mitochondria/heavy membrane fraction from mouse liver ..95
5.3.15 Determination of protein concentration ...................................................96
5.3.16 Caspase activity assay..............................................................................96
5.3.17 Immunoblotting........................................................................................96
5.3.18 Immunoprecipitation................................................................................97
5.3.19 Analysis of in vivo cytochrome c release by cell fractionation................97
5.3.20 In vitro cytochrome c release assay .........................................................97
6 References .............................................................................................................98
7 Acknowledgements.............................................................................................115
8 Lebenslauf...........................................................................................................116
9 Selbstständigkeitserklärung..............................................................................117

Introduction
1 Introduction
1.1 Apoptosis as cell fate and cellular stress response
Programmed cell death is a process that is indispensable for the normal development
of multicellular organisms and is of vital importance throughout their life. During
embryonic development, elimination of surplus cells is required for the proper shaping
of organs and body parts and the creation of complex multicellular tissues. In the
developing vertebrate nervous system, for instance, most types of neurons are initially
produced in excess. Surplus neurons will eventually be eliminated after reaching their
target tissue, based on competition for survival factors that are released by the target
cells in limited amounts. This allows the number of neurons to be exactly matched to
the number of target cells. Similar mechanisms are thought to operate both during
development and adulthood of metazoans to balance the numbers of different cell