Diverse functions of Stat3 in intestinal epithelial cells during inflammation-associated and sporadic carcinogenesis [Elektronische Ressource] / Julia Bollrath

Julia Bollrath

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Humanbiologie




Diverse functions of Stat3 in intestinal epithelial cells
during inflammation-associated and sporadic
carcinogenesis


Julia Bollrath






Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum
Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen
Universität München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.




Vorsitzender: Univ.-Prof. Dr. D. Haller
Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Schemann
2. Priv.-Doz. Dr. F. R. Greten
3. Univ.-Prof. Dr. B. Küster




Die Dissertation wurde am 28.04.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 03.10.2010 angenommen.


TABLE OF CONTENTS

1. Introduction..................................................................................... 1

1.1 Role of the tumor microenvironment in cancer development......... 2

1.2 Inflammatory bowel disease as a predisposing factor for colorectal
cancer development....................................................................................7

1.3 Colon cancer........................................................................................ 9
1.3.1 Molecular events in the development of colon cancer............ 10
1.3.2 Wnt signaling: a commonly hyperactivated pathway in
colon cancer.................................................................................... 11

1.4 The Stat3 signaling pathway............................................................... 12
1.4.1 Components and its functions................................................. 12
1.4.2 The Stat3 pathway in cancer development..............................14
1.4.3 The Stat3 pathway in colon cancer......................................... 15

1.5 Mouse models of colon cancer............................................................ 16

2. Objective of this study.................................................................... 18

3. Materials and Methods............................................ 19

3.1 Mice....................................................................................................... 19
3.1.1 Mouse models.........................................................................
3.1.2 LPS measurement................................................................... 20
3.1.3 Bone marrow transplantation.................................................. 21
3.1.4 Genotyping of mice................................................................. 21
3.1.5 Mouse treatment...................................................................... 24
3.1.6 Sacrifice of mice..................................................................... 24
3.1.7 T-cell stimulation and intracellular staining for
Fluorescence Activated Cell Sorting (FACS).................................. 26

3.2 Histology............................................................................................... 27
3.2.1 Haematoxylin & Eosin staining (H&E).................................. 27
3.2.2 Alcian Blue staining................................................................ 27
3.2.3 Azure Eosin staining............................................................... 27
3.2.4 Immunohistochemical staining (IHC)..................................... 28
3.2.5 TUNEL staining (TdT-mediated dUTP-biotin nick end
labeling)........................................................................................... 29

3.3 RNA Analysis....................................................................................... 29
3.3.1 RNA-isolation from epithelial cells or tissue..........................
3.3.2 cDNA synthesis....................................................................... 30
3.3.3 Realtime (RT) – PCR..............................................................
3.3.4 RNA microarray analysis........................................................ 33




3.4 Protein analysis.................................................................................... 33
3.4.1 Epithelial cell lysis for protein lysates.................................... 33
3.4.2 Immunoblot analysis............................................................... 34
3.4.3 Kinase assay............................................................................ 36

3.5 Mutation analysis for Ctnnb mutations............................................. 37

3.6 Statistics................................................................................................ 39

4. Results.............................................................................................. 40

4.1 Intestinal epithelial cell (IEC)-specific ablation of Stat3 does not
induce any overt phenotype in unchallenged animals............................ 40

4.2 Lack of Stat3 in IEC protects from tumor formation in a mouse
model of CAC............................................................................................. 41

4.3 Stat3 is an essential factor for the protection of enterocytes from
apoptosis during early tumor promotion................................................. 45

∆IEC 4.4 Stat3 mice develop more severe acute colitis in response
to DSS treatment and show an impaired wound healing response....... 46

4.5 Stat3 controls cell cycle progression by regulating key factors
at G1/S and G2/M phase transition.......................................................... 49

Y757F4.6 The hyperproliferative effect in gp130 mice is dependent
on non-haematopoietic cells and is induced by IL-6, as well as by
IL-11............................................................................................................ 52

4.7 Lack of Stat3 prolongs survival in a model of sporadic
intestinal carcinogenesis............................................................................ 55

4.8 Survival advantage is not dependent on cell autonomous changes
in proliferation or induction of apoptosis in IEC, but on activation of
haematopoietic cells................................................................................... 58

c.a ∆IEC4.9 What is causing the immune activation in β-cat Stat3
animals?...................................................................................................... 63

4.10 What are the factors responsible for hyperactivation of the
c.a. Stat3 signaling pathway in β-cat animals?.......................................... 65










5. Discussion......................................................................................... 67

5.1 Stat3 in signaling in CAC.................................................................... 68
5.1.1 Stat3 regulates apoptosis and cell-cycle progression of IEC
during CAC...................................................................................... 68
5.1.2 Stat3 signaling in IEC as a central factor in mucosal
wound-healing.................................................................................. 69
5.1.3 Stat3 is induced by IL-6 family cytokines in the CAC
model................................................................................................ 71

5.2 Stat3 in sporadic intestinal carcinogenesis........................................ 73
5.2.1 In a model of sporadic intestinal carcinogenesis, Stat3 sig-
naling does not control proliferation, but induction of apoptosis.... 73
c.a ∆IEC5.2.2 Survival advantages in β-cat Stat3 depend on activated
immune cells.................................................................................... 74
∆IEC5.2.3 Why does immune cell activation occur in Stat3 mice?... 75
5.2.4 What drives Stat3 signaling in this model?............................. 77

5.3 Stat3 signaling in IEC is a key factor in intestinal carcinogenesis.. 78

6. Summary.......................................................................................... 80

7. References........................ 82

8. Abbreviations................................................................................... 96
Introduction
______________________________________________________________________
1. Introduction

As the median survival of the population increases, the lifetime risk of developing
cancer does as well. This development is further accelerated by changes in
environmental and life-style derived factors which contribute to tumor development.
Well-known predisposing factors include smoking as a risk-factor for the development
of lung cancer, excessive UV-exposure as a risk-factor for the development of
melanoma or an unbalanced nutrition leading