DISSERTATION Titel der Dissertation Expression of G protein-coupled receptor 19 in human lung cancer cells is triggered by entry into S phase and supports G2/M cell cycle progression Verfasser Stefan Kastner angestrebter akademischer Grad Doktor der Naturwissenschaften (Dr.rer.nat.) Wien, 2012 Studienkennzahl lt. Studienblatt: A 091 490 Dissertationsgebiet lt. Studienblatt: Dr.-Studium der Naturwissenschaften Molekulare Biologie Betreuerin / Betreuer: Univ.-Doz. Dr. Wolfgang Sommergruber Acknowledgement Acknowledgement I would like to thank Boehringer Ingelheim RCV – particularly my supervisor Dr. habil. Wolfgang Sommergruber – for the kind offer to conduct the research for my doctoral thesis in their laboratories. Wolfgang, your door was always open for great discussions about all the things a PhD student needs to ask during the course of a thesis and beyond! Prof. Michael Freissmuth from the Institute of Pharmacology at the Medical University of Vienna deserves my deepest gratitude for his willingness to support my doctoral thesis. Mischa, your help and advice – ranging from the design of experiments over your offer to partly conduct them in your laboratories to assistance with manuscript preparation – were of greatest value. Despite being a ‘hamster in a running wheel’ you always offered me a time slot for discussions on my project. Thank you so much for your help! I would further like to thank Dr. Christina Glöckel and Simon Keuerleber for their help with the adenylyl cyclase assay and the ‘exchange’ of expression constructs. Special thanks go to Prof. Walter Berger and Dr. habil. Renate Voit for their willingness to be the appraisers of my doctoral thesis. I’m also very thankful for excellent technical assistance by Teresa Gottschamel, Nathalie Harrer, Herwig Machat, and Diane Thompson, who assisted me in performing various molecular and cellular biology techniques – it was a pleasure to learn from your experience! Besides, thank you very much, Teresa, for taking care of my cells during my days off. Dr. Anja Ebert from the Institute of Molecular Pathology in Vienna kindly offered her well-established chromatin immunoprecipitation protocol. Dr. Tilman Voss helped me a lot with explanations regarding the high-throughput screening and the vast possibilities this technique has to offer – especially with regards to data analysis. Dr. Andreas Wernitznig was always happy and patient to discuss bioinformatic issues regarding our inhouse TANGO database. And last but not least: Special thanks to all the people from the F&E at Boehringer that helped my right from the beginning of my doctoral thesis. It has been a great time getting to know you and working together with you! Table of contents Table of contents Abbreviations ......................................................................................................................... 9 I. Abstract .......................................................................................................................... 13 II. Zusammenfassung ........................................................................................................ 14 III. Introduction ................................................................................................................... 15 1. Cancer – a highly diverse disease ............................................................................................ 15 1.1. Fundamental characteristics of cancer ........................................................................... 16 2. Lung cancer ................................................................................................................................. 19 2.1. Small cell lung cancer (SCLC) ............................................................................................ 20 2.2. Non-small cell lung cancer (NSCLC) ................................................................................. 21 2.3. The neuroendocrine phenotype of lung cancer ............................................................ 21 2.4. Molecular characterization of lung cancer ..................................................................... 22 3. Cell cycle ...................................................................................................................................... 23 3.1. Cyclins and cyclin-dependent kinases (CDKs) ................................................................ 24 3.2. Cell cycle checkpoints ........................................................................................................ 25 4. G protein-coupled receptors (GPCRs) .................................................................................... 27 4.1. Classification of GPCRs ........................................................................................................ 28 4.2. Classical GPCR signaling and GPCR-interacting proteins (GIPs) ................................. 29 4.3. GPCR oligomerization .......................................................................................................... 30 4.4. Endocytosis of GPCRs from the plasma membrane ....................................................... 32 4.5. GPCRs are involved in cell cycle regulation and cancer ............................................. 33 5. G protein-coupled receptor 19 (GPR19) ................................................................................. 34 5.1. Gene and protein information ........................................................................................... 34 5.2. Expression of Gpr19 .............................................................................................................. 36 5.3. Signal transduction of GPR19 ............................................................................................. 36 5.4. Functional implications for GPR19 ..................................................................................... 37 6. Project definition .......................................................................................................................... 37 IV. Materials ......................................................................................................................... 39 1. Devices and accessory .............................................................................................................. 39 2. Software ........................................................................................................................................ 41 3. Chemicals ..................................................................................................................................... 41 4. Kits, enzymes, and reagents ...................................................................................................... 41 5. Buffers and solutions .................................................................................................................... 44 6. Plasmid constructs ....................................................................................................................... 46 7. DNA oligodeoxynucleotides ...................................................................................................... 49 8. Short interfering ribonucleic acids (siRNAs) ............................................................................. 50 9. TaqMan® assays .......................................................................................................................... 50 10. Primary antibodies ....................................................................................................................... 51 11. Cell lines ........................................................................................................................................ 52 12. Animals .......................................................................................................................................... 52 5 Table of contents V. Methods ......................................................................................................................... 53 1. Molecular biology and biochemistry (in vitro analyses) ........................................................ 53 1.1. Nucleic acid reconstitution ................................................................................................. 53 1.2. Nucleic acid concentration measurement – NanoDrop™ ........................................... 53 1.3. Isolation of total RNA from cultured cells .......................................................................... 53 1.4. Complementary-deoxyribonucleic acid (cDNA) synthesis ........................................... 54 1.5. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) gene expression and data analysis ................................................................................... 54 1.6. Preparation of bacterial culture plates (selective medium) ......................................... 55 1.7. Bacterial transformation ...................................................................................................... 55 1.8. Bacteria culture .................................................................................................................... 56 1.9. Bacteria glycerol stock ........................................................................................................ 56 1.10. Plasmid preparation (Maxi Prep) ....................................................................................... 56 1.11. Plasmid