TARGET IDENTIFICATION AND PROBE DEVELOPMENT FOR PANCREATIC NEUROENDOCRINE TUMORS Identifizierung von Zielstrukturen und Sondenentwicklung für pankreatische neuroendokrine Tumore Von der Fakultät Energie‐, Verfahrens‐ und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte Abhandlung vorgelegt von Jan Lennart Körner aus Warstein Hauptberichter: Prof. Dr. Roland Kontermann Nebenberichter: Prof. Dr. Christina Wege Tag der mündlichen Prüfung: 19.02.2015 Institut für Zellbiologie und Immunologie Universität Stuttgart 2015 1 INDEX II Table of Contents 1. Glossary .............................................................................................................................. V 2. Abstract ............................................................................................................................ VII 2.1. Zusammenfassung ................................................................................................................ VIII 3. Introduction ........................................................................................................................ 1 3.1. Rationale .................................................................................................................................. 1 3.2. G Protein‐Coupled Receptors .................................................................................................. 1 3.3. GPCR Signal Transduction ........................................................................................................ 3 3.4. GPCRs as Drug Target Structures ............................................................................................. 4 3.5. Peptides and their G Protein‐Coupled Receptors .................................................................... 5 3.5.1. GIP and its Receptor ...................................................................................................... 6 3.5.2. MC1R – Melanocortin Receptor 1 ................................................................................. 7 3.6. Neuroendocrine Tumors .......................................................................................................... 9 3.6.1. NETs of the Pancreas ..................................................................................................... 9 3.6.2. The Role of Somatostatin in NETs ............................................................................... 10 3.7. Alternative Splicing ................................................................................................................ 12 3.7.1. Alternative Splicing in Cancer ...................................................................................... 13 3.8. DNA Microarrays .................................................................................................................... 14 3.8.1. Detection of Alternative Splicing ................................................................................. 15 3.9. Aims of this Work ................................................................................................................... 16 4. Materials ........................................................................................................................... 18 4.1. Instruments ............................................................................................................................ 18 4.2. Chemicals and Peptides ......................................................................................................... 19 4.2.1. Cell Culture Reagents ................................................................................................... 19 4.2.2. Kits ............................................................................................................................... 20 4.2.3. Buffers and Solutions ................................................................................................... 20 4.2.4. Peptides and Small Molecules ..................................................................................... 22 4.3. Enzymes ................................................................................................................................. 22 4.4. Antibodies .............................................................................................................................. 22 4.5. Plasmids ................................................................................................................................. 23 4.6. Organisms .............................................................................................................................. 23 4.6.1. Eukaryotic Cell Lines .................................................................................................... 23 4.6.2. Stably Transfected Cell Lines ....................................................................................... 23 4.6.3. Prokaryotes .................................................................................................................. 24 4.7. Software ................................................................................................................................. 24 5. Methods ........................................................................................................................... 25 5.1. Cell Culture ............................................................................................................................. 25 1 INDEX III 5.1.1. Cultivation of Cell Lines ............................................................................................... 25 5.1.2. Transient Transfection ................................................................................................. 25 5.1.3. Establishing recombinant cell lines ............................................................................. 25 5.1.4. Cryoconservation of Cell Lines ..................................................................................... 26 5.2. Cellular Assays ........................................................................................................................ 26 5.2.1. Measurement of Intracellular cAMP Levels ................................................................ 26 5.2.2. Ca2+‐Mobilization ......................................................................................................... 27 5.3. Radioactive Peptide‐Binding Studies ..................................................................................... 28 5.3.1. Iodination of Peptides ‐ Chloramine T Method ........................................................... 28 5.3.2. Cell Membrane Isolation ............................................................................................. 28 5.3.3. Radioactive Binding Measurements ............................................................................ 29 5.4. Molecular biology .................................................................................................................. 29 5.4.1. Polymerase Chain Reaction – PCR ............................................................................... 29 5.4.2. Restriction and Ligation of DNA .................................................................................. 30 5.4.3. Electrophoretic Separation of DNA ............................................................................. 30 5.4.4. Transformation of Escherichia Coli .............................................................................. 30 5.4.5. Total RNA Preparation ................................................................................................. 31 5.4.6. RT‐PCR ......................................................................................................................... 31 5.4.7. Quantitative Real‐time PCR ......................................................................................... 31 5.4.8. Sequencing .................................................................................................................. 32 5.4.9. Immunofluorescence off Cultured Cells ...................................................................... 32 5.4.10. Immunohistochemistry on Human Tissue ................................................................... 33 5.4.11. Cell Lysis – Protein Preparation ................................................................................ 33 5.4.12. Bicinchoninic Acid Assay – BCA Assay ..................................................................... 34 5.4.13. SDS‐PAGE ..................................................................................................................... 34 5.4.14. Western‐Blotting ......................................................................................................... 34 5.5. DNA‐Microarray Technology ................................................................................................. 35 5.5.1. RNA‐Preparation for Hybridization .............................................................................. 35 5.5.2. Data Analysis ................................................................................................................ 35 5.5.3. Splicing Index ............................................................................................................... 35 5.6. Curve Fitting and Calculation of IC/EC50 Values ..................................................................... 36 6. Results .............................................................................................................................