Regulationsmechanismen Des Transkriptionsfaktors HIF
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Regulation of the Hypoxia-Inducible Factor-1 by Prolonged and Severe Low Oxygen Tension Dem Fachbereich Biologie der Technischen Universität Kaiserslautern zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) eingereichte Dissertation vorgelegt von Tobias Schmid Betreuer: Prof. Dr. Bernhard Brüne Kaiserslautern, im August 2004 Vorsitzender der Prüfungskommision: Prof. Dr. Dr. Heinrich Zankl 1. Gutachter: Prof. Dr. Bernhard Brüne 2. Gutachter: Prof. Dr. Thomas Kietzmann “Love is like oxygen. You get too much you get too high, not enough and you're gonna die.” (The Sweet, 1978) Acknowledgements After an enormously interesting, difficult, enjoyable, exhausting, exciting, and also sometimes frustrating 3 years, there are a lot of people whom I want to thank for being with me and supporting me in many different ways during that time. Before I begin, I want to apologize to all the people I have unintentionally forgotten to mention in the following. After weeks of writing, my brain seems to be a little drained. So please forgive me and feel included. First of all, I want to thank my supervisor Prof. Bernhard Brüne for offering me the opportunity of working on my Ph.D. thesis in his labs, first in Erlangen and then in Kaiserslautern. His discussions and support greatly facilitated my work, as, of course, did his financial support. Major thanks go to Dr. Jie Zhou, who always supported and mentored me and my work. His scientific as well as personal advice strongly enhanced my work and stimulated my scientific work and thinking. He has been a great influence and has grown to be a good friend during the past few years. I also want to thank Prof. Thomas Kietzmann for agreeing to evaluate my thesis, and Prof. Heinrich Zankl for chairing the final exam. A special thanks goes to Andrea Trinkaus for her excellent technical assistance, as well as for always being ready to help when there was a need, be it work-related or private. She also greatly increased my working efficiency by keeping me and the rest of the lab in a certain state of order. Furthermore, I want to thank all members of the “hypoxia”-lab: Melvin Callapina for interesting discussions about HIF-1 and other parts of the world; Sandra Christmann for helping me in all respects and for being great fun to work with; Steffen Schnitzer for discussing difficult hypoxic (and other) issues with me, and for being there for the occasional coffee. As every scientist will agree, this work would not have been possible without the help, discussions, and invaluable comments of the entire lab during the seminars, coffee breaks, barbecues and elsewhere. Specifically, I want to thank Dr. Andreas von Knethen (for cloning and FACS would not have been possible without your help), Dr. Roman Köhl (for in vitro binding has never been easier than with your help), Axel Johann (for lending me another point of view on hypoxic and other problems), Heike Sternike (for enabling me to avoid most of the administrative stuff and ordering business), Silvia Kühnert (for working is easier with clean tools), and Frau Watt (for being the soul of the biology department). Thanks to Merck & Co., West Point (USA) for providing me with the opportunity to present my data at the 40th Annual meeting of the SOT in Nashville in 2002 (thank you Werner for providing shelter). Additional thanks to the IUBMB for selecting me for participation at the young scientists program at the Joint Meeting of the HUPO and the IUBMB in 2003 in Montreal. Thank you Caro, Sabine, Steffi, Gerhard, and Jörg, i.e., TCS & Co. With our 5th semester, my life in science began. You have always been and still are a great bunch of people to be around, to talk to, to drink with, and to discuss problems with (scientific or not). Moreover, I want to thank my parents for always supporting me, my (scientific) career and all other aspects of my life. Without you, none of the following would have been possible. The same applies to my brothers and sisters, who helped me become the person I am now. Last, but definitely not least, I want to thank the two most important people in my life, who, at the same time, had to endure the most difficult part of my thesis – namely me! Without both of you, I wouldn’t have been able to get through this part of my life. Thank you very much, Steffi, for always being there and giving me so much love during the past 7.5 years. The invaluable corrections of my English and all the distractions from and discussions about my work gave me the energy to pull through. And thank you, Amelie, first, for setting the deadline for my thesis, and then, after arriving earlier than expected, for allowing me to finish all the same! Index I Index 1 Summary ...................................................................................................1 2 Zusammenfassung ....................................................................................3 3 Introduction ..............................................................................................5 3.1 Introductory remarks............................................................................................5 3.2 Normoxia/Hypoxia/Anoxia ...................................................................................5 3.3 Hypoxia and the hypoxia-inducible factor 1 (HIF-1).........................................6 3.3.1 Structure of HIF-1 .............................................................................................6 3.3.2 Classical regulation of HIF-1 ............................................................................8 3.3.3 Alternative regulation of HIF-1.......................................................................11 3.3.3.1 Alternative regulators.............................................................................11 3.3.3.2 Regulation via phosphorylation..............................................................14 3.3.4 Adaptive responses to oxygen depletion .........................................................15 3.3.5 HIF-1 and apoptosis.........................................................................................17 3.3.6 HIF-1 and diseases...........................................................................................18 3.4 Hypoxia and p53 ..................................................................................................19 3.5 Hypoxia and transcriptional regulation ............................................................22 3.6 Aims of this study ................................................................................................24 4 Materials and Methods ..........................................................................25 4.1 Materials...............................................................................................................25 4.1.1 Cells.................................................................................................................25 Index II 4.1.2 Bacteria............................................................................................................25 4.1.3 Chemicals and reagents ...................................................................................25 4.1.4 Antibodies........................................................................................................27 4.1.5 Plasmids...........................................................................................................28 4.1.6 Primers.............................................................................................................30 4.1.7 Instruments and Software ................................................................................30 4.2 Methods ................................................................................................................32 4.2.1 Cell biology .....................................................................................................32 4.2.1.1 Cell culture .............................................................................................32 4.2.1.2 Hypoxia and anoxia................................................................................32 4.2.1.3 Transient transfection.............................................................................32 4.2.2 Biochemistry....................................................................................................33 4.2.2.1 Protein determination (Lowry method) ..................................................33 4.2.2.2 SDS-PAGE/Western blot analysis ..........................................................34 4.2.2.3 Coomassie Blue staining ........................................................................34 4.2.2.4 Expression and preparation of (GST-fusion) proteins in bacteria.........35 4.2.2.5 In vitro transcription/translation............................................................35 4.2.2.6 Peptide array assay ................................................................................35 4.2.2.7 Competition binding assay .....................................................................36 4.2.3 Molecular Biology...........................................................................................36 4.2.3.1 Reporter gene analysis ...........................................................................36 4.2.3.2 Caspase activity assay............................................................................37 4.2.3.3 RNA isolation..........................................................................................37 4.2.3.4