Analysis of the role of the E-(epithelial) cadherin in murine lung tumorigenesis [Elektronische Ressource] / Fatih Ceteci

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

ANALYSIS OF THE ROLE OF THE E-(Epithelial)
CADHERIN IN MURINE LUNG
TUMORIGENESIS



Dissertation

For completion of the Doctorate degree in Natural Sciences at the
Bayerische Julius-Maximilians-Universität Würzburg








Oleg Tyrsin



Fatih Ceteci

from Solingen, Germany
Würzburg 2008 The hereby submitted thesis was completed from Jun 2002 until Jun 2008 at the Institute for
Medical Radiation and Cell Research (MSZ), Bayerische Julius-Maximilians University,
Würzburg under the supervision of Prof. Dr. Ulf R. Rapp (Faculty of Medicine) and PD Dr. R.
Götz (Faculty of Medicine).
























Submitted on:
Members of the thesis committee:
Chairman: Prof. Dr. Ulf R. Rapp
Examiner: PD Dr. R. Götz
Examiner: Prof. Dr. R. Benavente
Date of oral exam:
Certificate issued on:


ACKNOWLEDGMENTS


It is a pleasure to thank the many people who made this thesis possible.

I would like to express my deep and sincere gratitude to Prof. Dr. Ulf R. Rapp who allowed me to
undertake my Ph.D. research in the MSZ Institute. It is difficult to overstate my gratitude to him
not only for inviting me to the MSZ but also for his strong scientific guidance as well as for
providing enthusiasm, inspiration, and strong motivation during my studies. His wide knowledge
and his logical way of thinking have been of great value for me. His encouraging and personal
guidance have provided a good basis for the present thesis.

I am deeply grateful to my supervisor, Dr. Rudolf Götz, for his continuous support, detailed
constructive comments, and fruitful discussions and for his important support throughout this
work.

I would like to thank Prof. Dr. Ricardo Benavente for his contribution to my research seminars
and for his critical thesis evaluation.

I am indebted to thank all the members of mouse club for their important contribution and
stimulating discussions. I would like to thank them for the many social and scientific events we
have shared and for providing a stimulating and fun environment. I would not forget to thank our
ex-members for the past in common. I am grateful to all technicians of our group for their perfect
expertise in every aspect of mice.

I am grateful to many other co-workers in MSZ-Institute.

During this work I have collaborated with many colleagues for whom I have great regard, and I
wish to extend my warmest thanks to all those who have helped me with my work.

I would like to thank all the members and organizers of the GK-639 for their excellent
supervision, support and guidance for this work.

A penultimate thank-you goes to my wonderful parents. For always being there when I needed
them most, and never once complaining about how infrequently I visit, they deserve far more
credit than I can ever give them.

My final, and most heartfelt, acknowledgment must go to my wife and also my excellent-talented
colleague Semra. Her support, encouragement, and companionship have turned my scientific
journey into a pleasure. It is difficult to express in a few sentences the gratitude for my wife. The
greatest acknowledgement I reserve for her to whom I dedicate this dissertation.

Table of Contents

TABLE OF CONTENTS


ZUSAMMENFASSUNG ............................................................................................1
SUMMARY .....................................................................................................................2
1. INTRODUCTION
1.1. Lung Cancer ............................................................................................................ 3
1.1.1. Definition and histopathology of lung cancer .................................................3
1.1.2. Molecular biology of lung cancer ...................................................................4
1.1.3. Factors play role in progression of NSCLC to metastasis ..............................4
1.1.4. SP-C C-RAF BXB as a transgenic mouse NSCLC model .............................6
1.2. Cell adhesion molecules and cancer ........................................................................7
1.2.1. Cadherin superfamily ......................................................................................9
1.2.2. E-cadherin as an adhesion molecule ..............................................................12
1.2.3. E-cadherin as a tumor suppressor ..................................................................17
1.2.4. Cross talk of E-cadherin with Wnt/ß-catenin signalling ................................18
1.3. Experimental design and aim of the project ...........................................................20
1.3.1. DOX inducible dn E-cadherin strategy …… ……………………………….21
1.3.2. DOX inducible Cre recombinase-mediated Cdh1 gene inactivation ............22
2. RESULTS
2.1. Construction of PBi5 dn E-cadherin plasmid ........................................................23
2.2. In vitro analysis of dn E-cadherin expression ........................................................24
2.3. Generation and characterisation of dn E-cadherin expressing transgenic mice .....25
2.4. Loss of E-cadherin function in alveolar type II epithelial cells promotes
hyperplasia, but is insufficient for tumorigenesis ...................................................29
2.5. dn E-cadherin expression in SP-C C-RAF BXB adenomas ...................................33
2.6. Tumor progression in SP-C C-RAF BXB lung tumors after E-cadherin
disruption ................................................................................................................34
2.7. Expression of dn E-cadherin induces tumor vasculature .......................................38
2.8. Restoration of E-cadherin expression reverses the angiogenic switch ..................39
2.9. Oncogenic C-RAF and E-cadherin ablation cooperate in progression to micro-
metastasis ..............................................................................................................42
2.10. Lack of evidence for EMT in metastatic lung adenocarcinoma ..........................44
i Table of Contents
2.11. Consequences of E-cadherin ablation in normal and oncogenic type II
pneumocytes of mouse lung ................................................................................46
2.12. Analysis of dn E-cadherin-mediated angiogenesis ..............................................53
2.13. Chronic expression of dn E-cadherin in SP-C RAF BXB expressing lung
epithelial cells up-regulates -catenin-responsive genes that include
endodermal and other lineage markers ................................................................60
3. DISCUSSION
3.1. Is interference with E-cadherin function oncogenic? ...........................................62
3.2. Induction of angiogenic switch by disruption of E-cadherin ................................62
3.3. E-cadherin disruption promotes tumor progression to micrometastasis ...............63
3.4. Disruption of E-cadherin activates -catenin signalling .......................64
3.5. Up-regulation of -catenin target genes after E-cadherin ablation ......................66
4. MATERIALS AND METHODS
4.1. Materials ................................................................................................................68
4.1.1. Instruments ………………………………………………………………68
4.1.2. Chemical reagents and general materials .....................69
4.1.3. Cell culture materials .................................................................................71
4.1.4. Antibodies ..................................................................................................72
4.1.5. Enzymes .....................................................................................................73
4.1.6. Kits .............................................................................................................73
4.1.7. Plasmid DNA ………………………………………………………… ….74
4.1.8. Oligonucleotides 74
4.1.9. Cell lines, mouse lines and bacterial strains ..............................................77
4.2. Solutions and buffers .............................................................................................77
4.2.1. Bacterial medium and DNA isolation buffers ............................................77
4.2.2. DNA buffers ...............................................................................................78
4.2.3. Protein analysis buffers ..............................................................................79
4.2.4. Histological buffers ....................................................................................80
4.3. Methods ………………………………………………………………… ………80
4.3.1. Bacterial manipulation .......................................................................