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Reelin/Disabled-1-Signaling in Glioma Cell Migration and Invasion

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Reelin/Disabled-1-Signaling in Glioma Cell Migration and Invasion AUS DER ABTEILUNG FÜR NEUROPATHOLOGIE Prof. Dr. med. Markus J. Riemenschneider DER FAKULTÄT FÜR MEDIZIN DER UNIVERSITÄT REGENSBURG REELIN/DISABLED-1-SIGNALING IN GLIOMA CELL MIGRATION AND INVASION Inaugural Dissertation zur Erlangung des Doktorgrades der Medizin der Fakultät für Medizin der Universität Regensburg vorgelegt von Stefan Bernd Swoboda 2018 AUS DER ABTEILUNG FÜR NEUROPATHOLOGIE Prof. Dr. med. Markus J. Riemenschneider DER FAKULTÄT FÜR MEDIZIN DER UNIVERSITÄT REGENSBURG REELIN/DISABLED-1-SIGNALING IN GLIOMA CELL MIGRATION AND INVASION Inaugural Dissertation zur Erlangung des Doktorgrades der Medizin der Fakultät für Medizin der Universität Regensburg vorgelegt von Stefan Bernd Swoboda 2018 Dekan: Prof. Dr. Dr. Torsten E. Reichert 1. Berichterstatter: Prof. Dr. med. Markus J. Riemenschneider 2. Berichterstatter: Prof. Dr. rer. nat. Eugen Kerkhoff Table of Contents Table of Contents Abbreviations .................................................................... - 7 - 1 Introduction ............................................................... - 9 - 1.1 Reelin/Disabled-1 signaling pathway ........................................................................ - 9 - 1.1.1 Reelin ................................................................................................................ - 9 - 1.1.2 Disabled-1 ....................................................................................................... - 15 - 1.1.3 Overview of known downstream targets ......................................................... - 17 - 1.1.4 Reelin/Disabled-1 signaling in other cancers .................................................. - 20 - 1.2 General information on glioblastoma ...................................................................... - 21 - 1.2.1 Overview and epidemiology ........................................................................... - 22 - 1.2.2 Etiology ........................................................................................................... - 24 - 1.2.3 Symptoms, diagnosis and treatment ................................................................ - 25 - 1.2.4 Molecular diagnosis and pathology of gliomas ............................................... - 26 - 1.3 Reelin/DAB1 signaling in glioblastoma .................................................................. - 28 - 1.3.1 RELN promoter hypermethylation .................................................................. - 29 - 1.3.2 DAB1 induced proliferation block .................................................................. - 29 - 1.4 Aims of this study .................................................................................................... - 30 - 2 Materials .................................................................. - 32 - 2.1 Chemicals ................................................................................................................ - 32 - 2.2 Cell culture reagents ................................................................................................ - 33 - 2.3 Cell culture materials ............................................................................................... - 34 - 2.4 Primers ..................................................................................................................... - 34 - 2.5 Antibodies for western blotting ............................................................................... - 34 - 2.5.1 Primary antibodies ........................................................................................... - 34 - 2.5.2 Secondary antibodies ....................................................................................... - 35 - - 3 - Table of Contents 2.6 Antibodies and dyes for immunocytochemistry ...................................................... - 35 - 2.7 Cell lines .................................................................................................................. - 36 - 2.7.1 HEK 293T cells ............................................................................................... - 36 - 2.7.2 Human glioblastoma cells ............................................................................... - 36 - 2.8 Devices .................................................................................................................... - 39 - 2.9 Software ................................................................................................................... - 40 - 2.10 Kits .......................................................................................................................... - 40 - 2.11 Other materials ........................................................................................................ - 41 - 2.12 Solutions, buffers and media ................................................................................... - 41 - 2.12.1 Cell culture .................................................................................................. - 41 - 2.12.2 Western blotting .......................................................................................... - 42 - 2.12.3 2D migration assay ...................................................................................... - 44 - 2.12.4 Transwell invasion assay ............................................................................. - 44 - 2.12.5 Immunocytochemistry ................................................................................. - 44 - 3 Methods .................................................................... - 46 - 3.1 Cell biological methods ........................................................................................... - 46 - 3.1.1 Cell lines and cell culture conditions .............................................................. - 46 - 3.1.2 Plate coating .................................................................................................... - 47 - 3.1.3 Preparation of Reelin/GFP conditioned medium ............................................ - 47 - 3.2 Molecular biological methods ................................................................................. - 48 - 3.2.1 RNA isolation .................................................................................................. - 48 - 3.2.2 cDNA synthesis ............................................................................................... - 48 - 3.2.3 Real-time quantitative PCR ............................................................................. - 49 - 3.3 Protein biochemistry ................................................................................................ - 51 - 3.3.1 Western blotting .............................................................................................. - 51 - 3.3.2 Immunocytochemistry and F-actin staining .................................................... - 55 - 3.4 2D migration assay .................................................................................................. - 58 - - 4 - Table of Contents 3.4.1 Cell seeding ..................................................................................................... - 58 - 3.4.2 Cell tracking .................................................................................................... - 58 - 3.5 Transwell invasion assay ......................................................................................... - 59 - 3.5.1 Cell seeding ..................................................................................................... - 59 - 3.5.2 Cell staining and computer-based analysis ..................................................... - 60 - 3.6 Data analysis ............................................................................................................ - 60 - 4 Results ...................................................................... - 61 - 4.1 Reelin induced DAB1 phosphorylation in U87 and U251 glioblastoma cell lines . - 61 - 4.1.1 Reelin produced by HEK 293T ....................................................................... - 61 - 4.1.2 DAB1 phosphorylation in U87/U251 cells ..................................................... - 62 - 4.2 Analysis of migration under the influence of Reelin/DAB1 signaling ................... - 64 - 4.2.1 Fibronectin ...................................................................................................... - 65 - 4.2.2 Laminin ........................................................................................................... - 69 - 4.3 Influences of the Reelin/DAB1 pathway on cell invasion ...................................... - 73 - 4.4 Analysis of the cytoskeleton under the influence of Reelin/DAB1 signaling ......... - 76 - 4.4.1 Analysis of the actin cytoskeleton ................................................................... - 77 - 4.4.2 Analysis of the P-Cofilin to Cofilin ratio ........................................................ - 81 - 4.5 Analysis of downstream targets in Reelin/DAB1 signaling .................................... - 83 - 5 Discussion ................................................................ - 87 - 5.1 Reelin stimulation induces DAB1 phosphorylation in glioblastoma cells .............. - 88 - 5.1.1 DAB1 levels in glioblastoma cell lines U87 and U251 .................................. - 88 - 5.1.2 Reelin induced DAB1 activation levels in U87 and U251 .............................. - 89 - 5.2 Reelin/DAB1 preferentially reduces cell migration in glioblastoma cells .............. - 90 - 5.2.1 Cell migration .................................................................................................
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