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SPON2 and Its Implication in Epithelial-Mesenchymal Transition

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SPON2 and Its Implication in Epithelial-Mesenchymal Transition SPON2 and its implication in epithelial-mesenchymal transition Dissertation zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr.rer.nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von Artur Muradyan aus Yerevan, Armenien September 2009 Diese Dissertation wurde in der Zeit vom Januar 2005 bis Mai 2009 am Max-Planck- Institut für molekulare Genetik in Berlin, in der Abteilung von Herrn Prof. Dr. Hans-Hilger Ropers, in der Arbeitsgruppe von Herr Dr. Reinhard Ullmann angefertigt. The work presented in this thesis has been conducted from January 2005 till May 2009 at Max Planck Institute for Molecular Genetics (Berlin), in the department of Prof. Dr. Hans- Hilger Ropers under supervision of Dr. Reinhard Ullmann. 1. Gutachter: Prof. Dr. Hans-Hilger Ropers Max-Planck-Institut für molekulare Genetik 2. Gutachter: Prof. Dr. Constance Scharff Freie Universität Berlin Tag der Disputation: 04.02.2010 Day of the Disputation: 04.02.2010 Hiermit erkläre ich, die vorliegende Arbeit selbständig und ohne unerlaubte Hilfe angefertigt zu haben und alle verwendeten Hilfsmittel und Inhalte aus anderen Quellen als solche kenntlich gemacht zu haben. Des Weiteren versichere ich, dass die vorliegende Arbeit nie in dieser oder anderer Form Gegenstand eines früheren Promotionsverfahrens war. I hereby declare that the work presented in this thesis has been conducted independently and without any inappropriate support and that all sources of information, be it experimental or intellectual, are aptly referenced. I hereby declare that this thesis has not been submitted, either in the same or a different form, to this or any other university for a degree. Artur Muradyan Berlin, September 2009 All information is up-to-date as of September 2009. However, electronic resources can change rapidly, which may lead to some outdated information at the time this work is published. Nothing from this thesis, including text, photographs, pictures and diagrams should be reproduced or published in any manner without prior written permission. To my parents, brother and in loving memory of my sister Irina │ i ACKNOWLEDGMENT I would like to thank Prof. Dr. Hans.-H. Ropers for establishing a scientific framework, in which I pursued my PhD, and for giving me an opportunity to carry out my thesis in his department. I am indebted to Prof. Dr. Constance Scharff for reviewing and evaluating my thesis. I want to highlight with sincere gratitude my group leader Dr. Reinhard Ullmann, who was persistently supporting and guiding over the years I spent in his group. I highly appreciate him for shaping my self-motivation and independence in science. I acknowledge Max Planck Society for financial support. A special chapter of compliments deserved a brilliant and kind person Dr. Sybille Krauss for her generous and immense help in many experiments and fruitful discussions. I express my gratitude to Dr. Chandan Goswami for valuable discussions and technical tips. I am thankful to Prof. H.Popper, Prof. R.Pfragner, Dr. I.Halbwedl and Dr. H.Kothmeier for samples and SCLC cell line. Special thanks goes to my dear Ines Mueller and Hannelore Madle, who assisted in some experiments and made my life in laboratory easier, to Fikret, Gennadiy, Murat, Masoud, Corinna and Lars for being always kind and helpful. I thank also my group members Ralph, Marei, Jan, Katrin, Stephanie, Grit, Anne, Vivien and Alischo. Ein herzliches Dankeschön meinem besten Freund und Kollegen Georg Wieczorek, mit dem ich viele gute und unvergessliche Zeiten in Deutschland verbracht habe, und seiner Frau Christiana, die immer so nett und gastfreundlich war. Georg ist derjenige, der mir die Schönheiten Deutschlands gezeigt hat. During my entire project I benefited from the expertise of the whole department, therefore I express my sincere gratitude and sympathy. Particularly, I want to point out the healthy atmosphere, being in tune with harmony and productive collaboration. I am thankful to secretariat of our department, namely Gabriele Eder and Hannelore Markert for their continuous support in all kinds of paperwork and legality. │ ii I am thankful to my friend and colleague Dr. Stephan Heymann not only for scientific cooperation, but also for kind attitude and hospitality. I owe my appreciation to his wife Margarita as well. I am grateful to Dr. Peter Stosiek and Arpik Nshdejan for their constant motivation and care. I am thankful to my friends Meline, Bedros, Karine, Andranik, Hendrik, Vigen, Borik, Mariam, Nazeli, Masis and many others from Armenian Community in Berlin. I do love them all ! Looking back at the years I spent in Germany, I want to thank Berlin, one of my lovely cities in the world that gave me so much hospitality, positive impressions and memories. I owe a special gratitude to my lovely and beautiful girlfriend Katerina Kraft for her love, kindness, support, care and charm. Finally, I owe my deepest gratitude and appreciation to my parents Karine Gasparyan and Grant Muradyan, to my brother Armen and his wife Lusine for their permanent encouragement, everlasting support, attention, care and love. I thank my delightful nephew Gor and sweet niece Natalie, who with their endearing words were passing me supreme emotions and were making my life full of joy and love. I remember with gratitude my grandmother Eugenia Sahakyan, who was a big part of my education. And the last but not least, a very special merit belongs to my lovely, unforgettable and missed sister Irina Muradyan, who had an immense impact on my education and life. │ iii TABLE OF CONTENTS ACKNOWLEDGMENT .............................................................................................................i TABLE OF CONTENTS ..........................................................................................................iii ABBREVIATIONS...................................................................................................................v SUMMARY .............................................................................................................................1 ZUSAMMENFASSUNG............................................................................................................2 INTRODUCTION ................................................................................................................. 4 I-1 CANCER BIOLOGY .........................................................................................................5 I-2 CANCER EVOLUTION ......................................................................................................6 I-2.1 KNUDSON’S TWO-HIT HYPOTHESIS AND FIELD CANCERISATION...........................................6 I-2.2 STEM CELL THEORY ...............................................................................................8 I-3 EPITHELIAL TISSUE ORGANIZATION AND CARCINOMAS ............................................................10 I-4 CELL ADHESION.......................................................................................................... 11 I-5 TUMOUR MICROENVIRONMENT AND TUMOUR-STROMA INTERACTION .......................................... 14 I-6 INFLAMMATION AND CANCER .......................................................................................... 15 I-7 ROLE OF FIBROBLASTS IN CANCER ................................................................................... 16 I-7.1 SEED AND SOIL THEORY AND METASTASIS .................................................................. 17 I-8 CANCER CELL AND MOTILITY .......................................................................................... 18 I-9 EPITHELIAL-MESENCHYMAL (EMT) AND MESENCHYMAL-EPITHELIAL TRANSITION (MET) ................. 20 MATERIALS AND METHODS............................................................................................... 26 II-1 MATERIALS ............................................................................................................... 27 II-1.1 CHEMICALS AND CHEMICAL COMPOUNDS .................................................................... 27 II-1.2 BIOLOGICAL REAGENTS......................................................................................... 29 II-1.3 CELL LINES ....................................................................................................... 32 II-1.4 KITS AND MICROARRAYS....................................................................................... 33 II-1.5 MATERIALS, PLASTIC-/GLASSWARE AND DISPOSABLES.................................................... 34 II-1.6 EQUIPMENT....................................................................................................... 34 II-1.7 BIOINFORMATICS TOOLS AND SOFTWARE ................................................................... 35 II-1.8 ONLINE RESOURCES AND DATABASES........................................................................ 36 II-2 METHODS................................................................................................................. 37 II-2.1 GENERAL METHODS ............................................................................................. 37 II-2.2 SAMPLE PREPARATION FOR MICROARRAY HYBRIDIZATION ............................................... 40 II-2.3 MICROARRAY PLATFORMS...................................................................................... 43 II-2.4 MICROARRAY INTERPRETATION............................................................................... 50 II-2.5 MICROARRAY VALIDATION....................................................................................
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