Complex redundancy between the mammalian thioredoxin and glutathione systems in cell proliferation and tumorigenesis [Elektronische Ressource] / by Pankaj Kumar Mandal

Pankaj Kumar Mandal

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

From the
Department of Veterinary Sciences
Faculty of Veterinary Medicine
Ludwig-Maximilians-Universität München
Chair for Molecular Animal Breeding and Biotechnology
Univ.-Prof. Dr. Eckhard Wolf


This work was conducted at
Helmholtz Zentrum München
Institute of Clinical Molecular Biology and Tumor Genetics
Head: Prof. Dr. Georg W. Bornkamm
Under the supervision of Dr. Marcus Conrad




Complex Redundancy between the Mammalian Thioredoxin and
Glutathione Systems in Cell Proliferation and Tumorigenesis



Thesis for the attainment of the title Doctor in Veterinary Medicine
from the Faculty of Veterinary Medicine of the
Ludwig-Maximilians-Universität München

by
Pankaj Kumar Mandal
from
Jamalpur, India
Munich, April 2009

Aus dem
Department für Veterinärwissenschaften
Tierärztliche Fakultät
Ludwig-Maximilians-Universität München
Lehrstuhl für Molekulare Tierzucht und Biotechnologie
Univ.-Prof. Dr. Eckhard Wolf


Die Untersuchungen wurden durchgeführt am
Helmholtz Zentrum München
Institut für Klinische Molekularbiologie und Tumorgenetik
Direktor: Prof. Dr. Georg W. Bornkamm
Unter der Leitung von Dr. Marcus Conrad



Komplexe Redundanz zwischen dem Thioredoxin- und
Glutathion-abhängigen System in der Zellproliferation
und Tumorenstehung


Inaugural-Dissertation
zur Erlangung der tiermedizinischen Doktorwürde
der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München

von
Pankaj Kumar Mandal
aus
Jamalpur, Indien
München, April 2009




Gedruckt mit Genehmigung der Tierärztlichen Fakultät
der Ludwig-Maximilians-Universität München






Dekan: Univ.-Prof. Dr. Braun
Berichterstatter: Univ.-Prof. Dr. Wolf
Korreferent/en: Univ.-Prof. Dr. Hirschberger
Summa cum laude
Priv.-Doz. Dr. André Univ.-Prof. Dr. Wanke
Univ.-Prof. Dr. Gabius







thTag der Promotion: 17 Juli 2009









Dedicated to

Dear Father and Uncle Table of contents

LIST OF ABBREVIATIONS.........................................................................................I
1. INTRODUCTION.....................................................................................................1
2. REVIEW OF LITERATURE.....................................................................................2
2.1 REACTIVE OXYGEN SPECIES IN PHYSIOLOGY AND DISEASE DEVELOPMENT ............2
2.1.1 Reactive oxygen species- sources and effects ..........................................2
2.1.2 ROS as signaling molecules.......................................................................3
2.1.3 Thiol-based regulatory switches and principles of redox regulation ...........4
2.1.4 Oxidative stress: The dark side of ROS .....................................................5
ANTIOXIDANT DEFENSE SYSTEMS.........................................................................6 2.2
2.2.1 Non-enzymatic antioxidants6
2.2.2 Enzymatic antioxidants...............................................................................7
-2.3 SYSTEM X AND THE CYSTINE/CYSTEINE CYCLE...................................................7 C
2.4 THE GLUTATHIONE-DEPENDENT SYSTEM ..............................................................8
2.4.1 Glutathione .................................................................................................8
2.4.2 Glutaredoxin and glutathione reductase10
2.4.3 Glutathione peroxidase (GPx) ..................................................................11
2.5 THE THIOREDOXIN/THIOREDOXIN REDUCTASE (TRX-TRXR) SYSTEM ....................11
2.5.1 The mammalian thioredoxin system.........................................................13
2.5.2 Thioredoxins .............................................................................................13
2.5.3 The mammalian thioredoxin reductases...................................................14
2.5.3.1 The mitochondrial thioredoxin reductase (TrxR2)..............................15
2.5.3.2 The cytosolic thioredoxin reductase (TrxR1) .....................................16
2.5.3.2.1 Genomic organization of mouse and human cytosolic thioredoxin
reductase...................................................................................................16
2.5.3.2.2 Structural organization and catalytic mechanism of TrxR1.........17
2.5.3.2.3 Physiological function of TrxR1 ..................................................18
2.5.4 Peroxiredoxins..........................................................................................20
2.6 REDUNDANCIES BETWEEN THE GSH-DEPENDENT AND THE THIOREDOXIN-
DEPENDENT ANTIOXIDANT SYSTEMS.........................................................................21


2.7 INTERPLAY OF ROS AND ANTIOXIDANTS IN TUMOR DEVELOPMENT......................23
2.7.1 Components of the antioxidant system as drug targets for cancer
chemotherapy....................................................................................................25
2.7.2 The thioredoxin system in cancer.............................................................26
2.8 AIMS OF THE PRESENT STUDY............................................................................28
3. MATERIALS AND METHODS..............................................................................29
3.1 MATERIALS .......................................................................................................29
3.2 METHODS .........................................................................................................36
3.2.1 Cloning techniques ...................................................................................36
3.2.2 Methods of gene delivery into target cells ................................................41
3.2.3 Immunoblotting and immunocytochemistry ..............................................46
3.2.4 Cell culture related techniques .................................................................48
3.2.5 Biochemical techniques............................................................................52
3.2.6 Tumor transplantation protocol55
4. RESULTS..............................................................................................................56
4.1 ANALYSIS OF THIOREDOXIN REDUCTASE 1 FUNCTION EX VIVO.............................56
4.1.1 Establishment of mouse embryonic fibroblast cell lines from conditional
Txnrd1 knockout mice .......................................................................................56
4.1.2 Txnrd1 knockout cells are highly susceptible to GSH depletion...............57
4.1.3 Reconstitution of Txnrd1 expression restored resistance against BSO....58
4.1.4 Txnrd1 knockout cells were not rescued by antioxidants .........................61
4.1.5 Txnrdcells underwent necrosis or oxidative stress-induced cell
death upon BSO treatment................................................................................61
4.1.6 xCT over-expression failed to rescue Txnrd1 knockout cells from GSH
depletion............................................................................................................63
4.1.7 xCT over-expressing Txnrd1 knockout cells underwent rapid cell death as
compared to mock-transfected control cells upon GSH depletion.....................65
4.1.8 xCT over-expressing Txnrd1 knockout cells suffered from severe oxidative
stress upon GSH depletion................................................................................66
4.1.8.1 ROS accumulation in xCT over-expressing Txnrd1 knockout cells was
comparable to that in mock-transfected cells ................................................67
4.1.8.2 Activation of the SAPK/JNK pathways led to execution of rapid cell
death in xCT-over-expressing Txnrd1 knockout cells....................................67

4.1.9 Co-culture of Txnrd1 knockout cells with xCT over-expressing cells failed
to prevent BSO-induced cell death....................................................................69
-/-4.1.10 Reconstitution of Txnrd1 expression in xCT over-expressing Txnrd1
cells provided resistance against BSO ..............................................................70
4.2 ANALYSIS OF TXNRD1 FUNCTION IN ONCOGENE-TRANSFORMED CELL LINES .......73
V124.2.1 In vitro transformation of MEFs using the c-myc and Ha-ras oncogenes
...........................................................................................................................73
4.2.2 Induction of Txnrd1 knockout in transformed cells using Tat-Cre protein.73
4.2.2.1 Single cell cloning and the generation of knockout cell lines.............73
4.2.2.2 Confirmation of the Txnrd1 knockout in transformed single cell clones
.......................................................................................................................74
4.2.3 Effect of Txnrd1 knockout on the proliferation of transforme