Functional investigation of the class II tumor suppressor gene H-REV107-1 [Elektronische Ressource] / von Irina Nazarenko

Irina Nazarenko

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2003
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Functional investigation of the class II tumor
suppressor gene H-REV107-1
Dissertation
zur Erlangung des akademischen Grades
Doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Irina Nazarenko
geb. am 24. Januar 1975 in Kustanay, Kasachstan
Präsident der Humboldt-Universität zu Berlin:
Prof. Dr. Jürgen Mlynek
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I:
Prof. Dr. Michael Linscheid
Gutachter: 1. Prof. Dr. Thomas Börner
2. Prof. Dr. Reinhold Schäfer
3. Prof. Dr. Dr. Christian Hagemeier
Tag der Einreichung: 11.06.03
Tag der mündlichen Prüfung: 16.09.03Table of Contents
List of Figures....................................................................................................................... IV
List of Tables ........................................................................................................................ VI
List of Abbreviations...........................................................................................................VII
Zusammenfassung............................................................................................................... IX
Abstract.................................................................................................................................. X
1 Introduction .....................................................................................................................1
1.1 Multi-Step Progression of Tumors .................................................................................1
1.1.1 Oncogenes .......................................................................................................................... 1
1.1.2 Tumor Suppressor Genes ................................................................................................... 1
1.1.3 Mechanisms of Gene Silencing........................................................................................... 4
1.2 H-REV107-1 is a Member of the NlpC/P60 Protein Superfamily...................................6
1.2.1 The NlpC/P60 Protein Superfamily ..................................................................................... 6
1.2.2 The LRAT-Like Protein Family .......................................................................................... 11
1.3 Purpose of this Work13
2 Materials and Methods .................................................................................................14
2.1 Materials......................................................................................................................14
2.1.1 Chemicals.......................................................................................................................... 14
2.1.2 Kits..................................................................................................................................... 14
2.1.3 Enzymes......... 15
2.1.4 Antibodies.......................................................................................................................... 15
2.1.5 Fluorophore-Labelled Antibodies ...................................................................................... 16
2.1.6 cDNA Library ..................................................................................................................... 16
2.1.7 Mammalian Cell Lines .......................................................................................................16
2.1.8 E. coli Strains..................................................................................................................... 16
2.1.9 Yeast Strains .................................................................................................................... 17
2.1.10 Plasmids and Expression Constructs................................................................................ 17
2.1.11 Oligonucleotides ................................................................................................................ 19
2.2 Methods.......................................................................................................................21
2.2.1 Yeast Two-Hybrid System................................................................................................. 21
2.2.2 Bacterial Culture....... 34
2.2.3 Enzymatic Manipulation and Analysis of DNA .................................................................. 35
2.2.4 Culturing of Mammalian Cells............................................................................................ 40
2.2.5 Apoptosis Assays .............................................................................................................. 41
2.2.6 Analysis of Proteins ........................................................................................................... 42
I2.2.7 Protein Interaction Analysis...............................................................................................48
2.2.8 Co-Immunoprecipitation .................................................................................................... 50
2.2.9 Immunofluorescence Analysis and Confocal Microscopy ................................................. 51
2.2.10 Phosphatase Assay........................................................................................................... 52
3 Results ...........................................................................................................................53
3.1 Identification of Proteins Interacting with H-REV107-1................................................53
3.1.1 Screening of a Human Kidney cDNA Library to Identify Potential Interacting Partners
of the H-REV107-1 Protein................................................................................................ 53
3.1.2 Sequencing Analysis of Clones Encoding Putative Interaction Partners of the
H-REV107-1 Protein.......................................................................................................... 55
3.1.3 Verification of Specificity of Interactions Using the Mating Test........................................ 56
3.1.4 Examination of Protein Expression in Yeast ..................................................................... 59
3.1.5 Generation of the H-REV107-1V5 and ∆CH-REV107-1HA Expression Vectors. ............. 60
3.2 PC4..............................................................................................................................61
3.2.1 H-REV107-1 Interacts with PC4 in COS-7 Cells............................................................... 61
3.2.2 Examination of the Intracellular Localisation of the Ectopically Expressed H-REV107-1
and PC4 Proteins .............................................................................................................. 62
3.2.3 H-REV107-1 Interacts with Endogenous PC4 in COS-7 Cells.......................................... 63
3.2.4 H-REV107-1, PC4, and STAT1 Form a Protein Complex Related to IFNγ-Signaling....... 65
3.3 PR65............................................................................................................................70
3.3.1 H-REV107-1 Interacts with PR65 in COS-7 Cells............................................................. 70
3.3.2 H-REV107-1 and PR65 are Co-Localised in COS-7 Cells................................................ 72
3.3.3 Hts with PR65 in a Cell-Free System .................................................. 73
3.3.4 Homodimer Formation of H-REV107-1 ............................................................................. 73
3.3.5 Determination of the H-REV107-1 Domains Responsible for Interaction with PR65
and Homodimer Formation................................................................................................ 76
3.4 Investigation of a Role of the H-REV107-1 – PR65 Interaction in Apoptosis ..............79
3.4.1 H-REV107-1 Does not Induces Apoptosis in Rat Fibroblasts FE-8 .................................. 79
3.4.2 The ∆C107-∆N Interaction Deficient Mutant Fails to Induce Apoptosis in
Human Ovarian Carcinoma Cell Lines A27/80 and OVCAR-3 ......................................... 80
3.4.3 Cellular re-distribution of the endogenous PR65 protein correlates with the
H-REV107-1 induced apoptosis in OVCAR-3 cells........................................................... 82
3.4.4 H-REV107-1 inhibits PP2A activity in vitro ........................................................................ 83
3.4.5 Okadaic acid induces apoptosis in OVCAR-3 cells 88
3.4.6 PP2A inhibition in OVCAR-3 cells leads to the activation of procaspase-9 ...................... 89
3.5 Confirmation of interaction between H-REV107-1 and RARG, S100A6, ETF1, and
P14.5 ...........................................................................................................................91
3.5.1 RARG ................................................................................................................................ 91
3.5.2 S100A6, ETF1, and P14.5................................................................................................. 92
II4 Discussion.....................................................................................................................97
4.1 Yeast Two–Hybrid System ..........................................................................................9