The role of glutathionperoxidase 4 (GPX4) in hematopoiesis and leukemia von Kira Célénie Stahnke Inaugral- Dissertation zur Erlangung der Doktorwürde der TierärztliChen Fakultät der Ludwig-Maximilians-Universität MünChen The role of glutathionperoxidase 4 (GPX4) in hematopoiesis and leukemia von Kira Célénie Stahnke aus Dortmund München 2016 Aus dem VeterinärwissensChaftliChen Department der TierärztliChen Fakultät der Ludwig-Maximilians-Universität München Lehrstuhl für Molekulare Tierzucht und Biotechnologie Arbeit angefertigt unter der Leitung von Univ.-Prof. Dr. Eckhard Wolf Angefertigt am Institut für Experimentelle TumorforsChung Universitätsklinikum Ulm Mentor: Prof. Dr. Christian Buske GedruCkt mit Genehmigung der TierärztliChen Fakultät der Ludwig-Maximilians-Universität München Dekan: Univ.-Prof. Dr. JoaChim Braun Berichterstatter: Univ.-Prof. Dr. Eckard Wolf Korreferent/en: Priv.-Doz. Dr. Bianka Schulz Tag der Promotion: 06.02.2016 Table of contents List of Abbreviations..............................................................................2 1 Introduction ........................................................................................6 1.1 ROS and oxidative stress in physiology and pathology..................................................... 6 1.1.1 SourCes of Reactive Oxygen SpeCies..................................................................................................6 1.1.2 PhysiologiCal role of ROS........................................................................................................................8 1.1.3 Oxidative stress, prinCiple pathology of ROS.................................................................................9 1.1.3.1 Oxidative stress in diseases........................................................................................................................... 9 1.1.3.2 Oxidative stress in CanCer .............................................................................................................................. 9 1.2 Antioxidative mechanisms .......................................................................................................11 1.2.1 Non-enzymatiC antioxidant mechanisms.....................................................................................12 1.2.2 EnzymatiC antioxidant meChanisms...............................................................................................14 1.3 Antioxidant mechanisms in tumorigenesis and as target for anti cancer drugs....19 1.4 Normal and malignant hematopoiesis..................................................................................20 1.4.1 Normal hematopoiesis .........................................................................................................................21 Postpartal hematopoiesis...................................................................................................................................21 1.5 Malignant hematopoiesis- leukemia .....................................................................................23 1.6 Aims of the present study..........................................................................................................31 2 Materials and Methods ................................................................. 32 2.1 Materials .........................................................................................................................................32 2.1.1 Reagents .....................................................................................................................................................32 2.1.2 Cell Culture.................................................................................................................................................33 2.1.3 Disposables ...............................................................................................................................................34 2.1.4 Kits ................................................................................................................................................................35 2.1.5 Equipment .................................................................................................................................................35 2.1.6 Cell lines and Culture Conditions......................................................................................................36 2.1.6.1 Human leukemia Cell lines...........................................................................................................................36 2.1.6.2 Mouse Cell lines.................................................................................................................................................37 2.1.6.3 Mouse Cell lines established from leukemiC miCe..............................................................................38 2.1.6.4 PaCkaging Cell lines .........................................................................................................................................38 2.1.7 Antibodies for FACS-staining ............................................................................................................39 2.1.8 Primer for Standard PCR.....................................................................................................................40 2.1.9 shRNA hairpin SequenCes...................................................................................................................40 2.1.10 Plasmids...................................................................................................................................................41 2.1.11 Assays for qRT-PCR.............................................................................................................................42 2.1.12 Antibodies for detection of protein..............................................................................................43 2.1.13 Enzymes...................................................................................................................................................43 2.1.14 Patient samples.....................................................................................................................................43 2.1.15 Mouse strains.........................................................................................................................................43 2.1.16 Software and statistiCal analysis...................................................................................................44 2.2 Methods...........................................................................................................................................45 2.2.1 Isolation of DNA ......................................................................................................................................45 2.2.2 Isolation of RNA ......................................................................................................................................45 2.2.3 cDNA synthesis........................................................................................................................................45 2.2.4 QuantifiCation of RNA and DNA........................................................................................................46 2.2.5 Standard PCR (polymerase Chain reaCtion)................................................................................47 2.2.6 Quantitative RT-PCR .............................................................................................................................48 2.2.7 Agarose gel eleCtrophoresis...............................................................................................................48 2.2.8 ExtraCtion of protein from whole Cells..........................................................................................49 2.2.9 ExtraCtion of nuclear and CytoplasmatiC protein .....................................................................49 2.2.10 DeteCtion of protein............................................................................................................................50 2.2.11 Immunoblot............................................................................................................................................51 2.2.12 Heat shoCk transformation of DH5α Competent E. Coli Cells.............................................53 2.2.13 Isolation of plasmid DNA (Miniprep, Maxiprep)....................................................................53 2.2.14 RestriCtion analysis.............................................................................................................................54 2.2.15 SequenCing..............................................................................................................................................54 2.2.16 General Culture Conditions...............................................................................................................54 2.2.17 Freezing and thawing of mammalian Cells ...............................................................................54 2.2.18 Cell Counting and determination of Cell viability ...................................................................55 2.2.19 Measuring ROS with DCFDA ...........................................................................................................55 2.2.20 Annexin V staining...............................................................................................................................56