Cystine-import and regulation of apoptosis in B-lymphocytes [Elektronische Ressource] / Ana Banjac

Ana Banjac

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

A thesis submitted of the requirements for the degree of Doctorate (Dr. rer. nat) from
the Faculty of Biology at the Ludwig-Maximilians-University, Munich, Germany:








Cystine-Import and Regulation of
Apoptosis in B-Lymphocytes







Ana Banjac





GSF Research Centre for Environment and Health GmbH
Institute for Clinical Molecular Biology and Tumor Genetics, Munich, Germany


Munich, November 2005









































First Examiner: Prof. Dr. Dirk Eick

Second Examiner: PD Dr. Angelika Böttger



Date of the oral examination: Munich 08.11.2005

Contents

Contents:


LIST OF ABBREVIATIONS..........................................................................................1
INTRODUCTION...........................................................................................................3
1.1 Oxygen Radicals and Antioxidative Defense System .................................................. 3
1.2 Burkitt Lymphoma Cells and Oxidative Stress........................................................... 6
1.3 The Heterodimeric Amino Acid Transporters ............................................................ 9
1.3.1 The Heavy Chains rbAT and 4F2hc....................................................................... 10
1.3.2 The Light Chains .................................................................................................... 11
1.4 The Cystine Glutamate Antiporter............................................................................. 12
1.4.1 Physiological Role of the Cystine Glutamate Antiporter in the Cellular
Antioxidative Defense System ............................................................................... 13
1.5 Glutathione (GSH)........................................................................................................ 15
1.5.1 Role of Intracellular Glutathione............................................................................ 15
1.5.2 Compartmentation of Glutathione.......................................................................... 17
1.6 The Multiple Roles of Cysteine in Cells...................................................................... 19
2 THE AIM OF THE WORK....................................................................................22
3 MATERIALS ........................................................................................................23
3.1 Chemical reagents 23
3.2 Enzymes......................................................................................................................... 24
3.3 Antibodies...................................................................................................................... 24
3.4 Radioactive Isotopes..................................................................................................... 25
3.5 Disposables and Kits 25
3.6 Bacteria.......................................................................................................................... 26
3.7 Cell line 26
3.8 Stably transfected cell lines.......................................................................................... 26
3.9 Parental cell line ........................................................................................................... 26 Contents
3.10 Materials for cloning .................................................................................................... 27
3.10.1 Oligonucleotides..................................................................................................... 27
3.10.2 Linkers for cloning ................................................................................................. 27
3.10.3 Vectors used for cloning......................................................................................... 28
3.10.4 Maps of vectors used for the expression of mouse and human xCT and
mouse 4F2 hc.......................................................................................................... 29
4 METHODS ...........................................................................................................31
4.1 Culture of Bacteria ....................................................................................................... 31
4.1.1 Preparation of competent bacteria .......................................................................... 31
4.1.2 Transformation of bacteria ..................................................................................... 32
4.1.3 Miniprep of plasmid DNA...................................................................................... 32
4.1.4 Maxi-prep of plasmi.................................................................................... 33
4.2 Molecular biology techniques 34
4.2.1 Cloning of plasmid DNA........................................................................................ 34
4.2.2 Dephosporylation of 5’ ends .................................................................................. 35
4.2.3 Filling in DNA overhangs 35
4.2.4 Ligation of DNA ends /or with DNA linkers ......................................................... 35
4.2.5 Hybridisation of complementary oligonucleotides................................................. 35
4.2.6 Polymerase chain reaction (PCR)........................................................................... 36
4.2.7 Isolation of DNA for cloning and analysis............................................................. 36
4.2.8 Generation of eucaryotic expression vectors for human and murine xCT and
murine 4F2hc.......................................................................................................... 37
4.3 Eucaryotic cell culture ................................................................................................. 39
4.3.1 Cultivation of Burkitt’s lymphoma cells ................................................................ 39
4.3.2 Assessments of cell number ................................................................................... 39
4.3.3 Cryo-conservation and thawing of eucaryotic cell(s) lines .................................... 39
4.3.4 Stable transfection of BL cells................................................................................ 40
4.3.5 Dilution experiments .............................................................................................. 40
4.3.6 BSO treatment ........................................................................................................ 41
4.3.7 Co-culture assay ..................................................................................................... 41
4.4 Methods for the analysis of DNA, RNA and proteins ............................................... 41
4.4.1 Radioactive labelling of DNA fragments ............................................................... 41
4.4.2 Isolation of total RNA ............................................................................................ 42
4.4.3 Northern blot analysis............................................................................................. 42
4.4.4 SDS-PAGE Electrophoresis ................................................................................... 43
4.4.5 Protein extraction and immunobloting ................................................................... 44
4.5 Biochemical methods.................................................................................................... 45
4.5.1 Measurement of L-cystine uptake activity ............................................................. 45
4.5.2 Determination of intracellular glutathione 46
4.5.3 ination of total thiol containing compounds............................................... 47
4.5.3.1 Determination of total extracellular thiols.......................................................... 47
4.5.3.2 ination of acid soluble thiols 47
4.5.4 HPLC determination of extra- and intracellular cysteine....................................... 48
TM4.6 FACS -Analysis.......................................................................................................... 48 Contents
4.7 Apoptotic assays............................................................................................................ 49
4.7.1 Measurement of cell death by Annexin-V-FITC and propidium iodide staining .. 49
4.7.2 Assessment of genomic DNA fragmentation by flow cytometric measurement
of nuclear DNA content.......................................................................................... 49
4.7.3 Detection of active caspases................................................................................... 49
4.7.4 Determination of the mitochondrial membrane potential....................................... 50
4.7.5 ination of intracellular reactive oxygen species (ROS) levels.................... 50
5 RESULTS ............................................................................................................51
5.1