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The role of IKKalpha in sporadic and familial colorectal tumorigenesis [Elektronische Ressource] / Serkan İsmail Göktuna

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TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Humanbiologie The role of IKKalpha in sporadic and familial colorectal tumorigenesis Serkan İsmail Göktuna 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. Vorsitzende(r): Univ.-Prof.Dr. J. J. Hauner Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Schemann 2. Priv.-Doz. Dr. F. R. Greten Die Dissertation wurde am 02.11.2009 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 05.02.2010 angenommen. ABSTRACT In this thesis, the role of IKKα in colorectal tumorigenesis was investigated by the use of chemical and genetic mouse models of colorectal tumors. To do this, an inactive IKKα mutant mouse was used in conjunction with these tumor models and the development of colorectal tumors were monitored for morphological and physiological differences. As a result of these experiments, a distinct phenotype of tumor growth retardation and extended survivals of the animals were observed in all tumor models with IKKα inactivation.

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TECHNISCHE UNIVERSITÄT MÜNCHEN


Lehrstuhl für Humanbiologie


The role of IKKalpha in sporadic and familial colorectal tumorigenesis


Serkan İsmail Göktuna

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.

Vorsitzende(r): Univ.-Prof.Dr. J. J. Hauner

Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Schemann
2. Priv.-Doz. Dr. F. R. Greten

Die Dissertation wurde am 02.11.2009 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und
Umwelt am 05.02.2010 angenommen.




ABSTRACT


In this thesis, the role of IKKα in colorectal tumorigenesis was investigated by the use of
chemical and genetic mouse models of colorectal tumors. To do this, an inactive IKKα mutant
mouse was used in conjunction with these tumor models and the development of colorectal
tumors were monitored for morphological and physiological differences. As a result of these
experiments, a distinct phenotype of tumor growth retardation and extended survivals of the
animals were observed in all tumor models with IKKα inactivation.
Molecular analysis of reduced tumor growth in IKKα mutants resulted in a clear IFNγ
upregulation due to myeloid cell recruitment by epithelial secreted factors and autocrine myeloid
cell activation by intrinsic factors like MCP-1 and IL-12. NF-κB activity was found to be crucial
for activation of myeloid cells to release IFNγ, which further caused growth arrest in IKKα
mutant enterocytes.
In conclusion, phospho-activation of IKKα is characterized as an important regulator of
immune responses in colorectal tumorigenesis. Therefore, the primary role of IKKα in colorectal
tumorigenesis is to suppress IFNγ release. However, this role adversely promotes tumor growth in
colorectal cancers. In this respect, these results strongly suggest that IKKα phospho-inactivation
can be a potent therapeutic strategy for the therapy of colorectal tumors.






ZUSAMMENFASSUNG



In der vorliegenden Arbeit die Funktion von IKKα bei der Tumorentstehung wurde durch
chemische und genetische murine kolorektale Karzinom-Modelle untersucht. Inaktivierung von
IKKα führte in allen Modellen durch eine Verzögerung des Tumorwachstums zu einer
signifikanten Verlängerung des Überlebens der Versuchstiere.
Molekularbiologische Untersuchungen zeigten in IKKα defizienten Mäusen eine deutlich
gesteigerte IFNγ Hochregulation aufgrund der Rekrutierung von myeloischen Zellen durch
epithelial sekretierte Faktoren sowie durch autokrine myeloische Zell-Aktivierung durch
intrinsische Faktoren wie MCP-1 und IL-12. Die NF-κB Aktivität ist von entscheidender
Bedeutung, für die Aktivierung der myeloischen Zellen zur Freisetzung von IFNγ, was in Folge
zu konsekutivem Wachstumsstop in IKKα defizienten Enterozyten führt.
Zusammenfassend demonstrieren die Ergebnisse dieser Arbeit, dass die Aktivierung von
IKKα die kolorektale Karzinomentstehung durch Suppression der IFNγ Expression maßgeblich
fördert. Die Möglichkeit einer pharmakologischen Inhibition der IKKα abhängigen IFNγ
Suppression könnte demnach in Zukunft eine neue potentielle Strategie in der Therapie des
kolorektalen Karzinoms darstellen.



ii



ACKNOWLEDGMENTS


First of all, I want to thank Prof. Florian R. Greten for providing this research position in which I have found
plenty of opportunity to develop my technical skills, my theoretical background on various subjects and my scientific
approach to different issues. He is a model scientist for hard-working, commitment to oneself job, critical thinking
and innovative problem solving abilities. He helped me a lot to be able to look subjects from different angles, in
developing skeptic thinking abilities and in finding way out when I was hindered by certain experimentation. It
would not be possible for me to finish this project without his profound guidance. I have every confidence in him
that he will be a leading scientist in his area in the near future. The experience I gained from his advisership will be
one of the most powerful earnings to build up my future academic career. For this reason and many others, it was a
great pleasure and honor for me to have a chance to make research together with him.
I am also thankful to Prof. Roland M. Schmid for being a very good Chief to our department, providing us with
such good facilities and hosting social activities which made each other know.
I am grateful to Prof. Michael Schemann for accepting me as a student in his faculty, being my Doktorvater and
advising me with academic and bureaucratic requirements that helped a lot while preparing this thesis.
I am also thankful to Dr. M. Canan Arkan for her kind collaboration in our research projects and every other
help she gave while I am working in the lab. Moreover, I was benefited from her essential advises both in academic
and personal issues that made my life easier.
Furthermore, I am grateful to Dr. Hana Algül, Dr. Jens Siveke, Dr. Dieter Saur, Dr. Günther Schneider and Dr.
Hassan Nakhai, for their collaboration, advises in research and in personal issues.
I am especially thankful to Dr. Frank Schmidt for invaluable helps in dendritic cell experiments; Dr. Jörg
Magesand Angela Servatius for generating and analyzing Microarray data; Julia Bollrath for excellent and enormous
amount of FACS data that helped a lot in finishing this project; Özge Canlı for exceptional ELISA and macrophage
stimulation data; Tim Nebelsiek for vital kinase assay data and completing expression analysis with double
knockouts; Kristin Retzlaff for brilliant technical assistance for dealing mice genotypes and matings; similarly Birgit
Wittig and Rabea Könitschke for technical assistance and the rest of the lab for providing assistance whenever I was
in the need.
My personal thanks go to my comrades Jamil Khasawneh, Dr. Alexander A. Fingerle, (soon to be Dr.) Moritz
Bennecke, Tim Nebelsiek, Dr. Arun K. Mankan and Özge Canlı for their friendship and assistance in technical and
iii practical problems. I am grateful to them not only being the best colleagues one can have but also being such good
friends. Especially their kind advises and limitless humor helped me a lot in coping with the difficulties of the
research life and persuading me to go on no matter how hard the difficulties I faced.
My other lab mates Julia Bollrath, Sarah Schwitalla, Vivian Daer, Simone Zach, Manon Schulz and Çiğdem
Atay, I am also thankful to you for countless helps and the good times we had in the lab and in extracurricular
ndactivities. Thank you so much all other people from 2 Internal Medicine who helped me in any single way or just
shared their friendship.
I am thankful to Prof. Michael Karin (Univ. of Calif. San Diego, USA), Prof. Yinglin Hu (Univ. of Texas MD
Anderson Cancer Centre, USA), Roland M. Schmid, Prof. Falk Weih (Leibniz Institut für Alterforschung,
Germany), Prof. Makato M. Taketo (Kyoto University, Japan), and Prof. Sylvie Robin (Institut Curie, France) for
kindly providing the mouse models we used in this study. Moreover, I thank Prof. Davis Artis for providing IL-12b
neutralizing antibody.
I am thankful to my friends Dr. Özgür Şahin (DKFZ, Heidelberg), Özlem Sener Şahin (Univ. Heidelberg),
Ufuk Bayburt (METU, Ankara), Dr. Çetin Baloğlu (Cambridge Univ., UK), Dr. Günseli Bayram Akçapınar
(Sabancı Univ., İstanbul), Dr. Çağhan Kızıl (MPI Dresden), Dr. Güneş Özhan Kızıl (MPI Dresden) for personal
assistance they provided to complete my documents, being there whenever I needed somebody to talk and infinite
support and trust they provided.
Lastly, I cannot find words to describe my gratefulness for my parents Nesrin and Mete and my sister Canan
Sezin for providing support in every possible way one can imagine. I am also thankful to my uncle Fethi Göktuna for
his limitless supports and trust that made this thesis possible. And the rest of my family and cousins I could not
mention specifically here, thank you very much.
I am dedicating this thesis to my family and to my beloved cousins Güzide and Levent who were like elder sister
and elder brother to me and have departed from this world to leave me in solitude. I hope you are in peace wherever
you went.



Serkan İsmail Göktuna

Munich, October 2009





iv





Güzide Ablama,

Levent Ağabeyime,

Öncü Dayıma,

ve hiçbir zaman desteklerini

eksik etmeyen aileme







Imagination will often carry us to worlds that never were.
But without it we go nowhere.

Carl Sagan



Indexes



INDEX

ABSTRACT.............................................................................................................................................................I
ZUSAMMENFASSUNG....... II
ACKNOWLEDGMENTS....III
INDEX..................................VII
INDEX OF FIGURES............X
INDEX OF TABLES............ XI
ABBREVIATIONS..............XII
1. INTRODUCTION ............................................................................................................................................1
1.1. BACKGROUND...........4
1.1.1. Intestinal Cell Development and Differentiation........................................................................................4
1.2. COLORECTAL CANCER ...........................................................................................................................9
1.2.1. Epidemiology of CRC ..............................................................................................................................9
1.2.2. Etiology of CRC.....10
1.2.3. Genetics and Pathology of CRC ..............................................................................................................11
1.2.3.1. Apc and Wnt Signaling...................13
1.2.4. Inflammation and Colorectal Cancer......................................................................................................16
1.2.4.1. Tumor Immunosurveillance, Immunotolerance and Escape .........................................................................17
1.2.4.1.1. Innate Immunity in Tumor Surveillance................................................................................................18 .2. Adaptive Immunity in Tumor Surveillance ............................................................................................20
1.2.4.1.3. Immunotolerance and Immune Escape ..................................................................................................21
1.2.4.2. NF-κB Signaling...........................................................................................................................................23
1.2.4.2.1. IKK Complex ........................................................................................................................................28 .2. IKKβSpecific Roles.30
1.2.4.2.3. IKKαSpecific Roles32
2. PURPOSE OF THE STUDY ..........................................................................................................................36
3. MATERIALS AND METHODS.....................................................................................................................37
3.1. MATERIALS ..............................................................................................................................................37
3.1.1. Chemicals..............37
3.1.2. Mouse Models........38
min/+Apc.......................................38
T2Vil-Cre-ER...............................38
+/lox(ex3)Ctnnb1.............................38
AA/AAIkka......................................38
fl/flIkka.........................................38
fl/flIkkb ...............................................................................................................................
-/-Nfkb2........................................38
fl/flRelb
-/-Ifnar1.......................................38
-/-Ifng...........................................38
3.1.3. Cell Lines...............39
3.1.5. Commercial Kits....39
vii
Indexes
3.1.6. Bacterial Strains....................................................................................................................................40
3.1.7. Buffers and Solutions .............................................................................................................................41
3.1.7.1. Culture Media ...............................................................................................................................................41
3.1.7.1.1. LB broth.................41 .2. LB agar...................41
3.1.7.2. Buffers ..........................................................................................................................................................42
3.1.7.2.1 DNA Assay Buffers..42 .1.1 TE Buffer.............42
3.1.7.2.1.2 Tail Lysis Buffer...................................................................................................................................42
3.1.7.2.1.3 Agarose Gel Buffer: TAE (50x) ............................................................................................................42 .2.2. Western Blot Buffers and Gels ............................................................................................................43
3.1.7.3. Histological Staining Buffers and Solutions .................................................................................................48
3.1.7.4. Solutions for Animal Experiments................................................................................................................49
3.2. METHODS.................50
3.2.1. Animal Treatments50
3.2.1.1. Animal Handling ..........................................................................................................................................50
3.2.1.2. Genotyping of Mice......................................................................................................................................51
3.2.1.3. Proliferation Index via Bromouridine Injections...........................................................................................51
3.2.1.4. Inducing Distal Colon Tumors via Azoxymethane (AOM) ..........................................................................52
3.2.1.5. Induction of Cre Recombinase via Tamoxifen..............................................................................................52
3.2.1.6. Blood Serum and Culture Media Supernatant Isolation.............52
3.2.1.7. Cell Isolation and Primary Cell Culturing ....................................................................................................53
3.2.2. Histological Protocols.............................................................................................................................58
3.2.2.1. General Histological Preparations ................................................................................................................58
3.2.2.2. Immunohistochemistry .................................................................................................................................60
3.2.2.3. Immunohistofluorescence.............................................................................................................................61
3.2.2.4. TUNEL Assay ..............................................................................................................................................61
3.2.3. Cell Based Assays ...................................................................................................................................62
3.2.3.1. Mammalian Cell and Tissue Culture Based Assays......................................................................................62
3.2.3.2. Bacterial Cell Based Methods.......................................................................................................................63
3.2.4. Nucleic Acid Based Assays and Preparations ............................................................................................64
3.2.4.1. DNA Isolation Mouse Tails and Tissues ......................................................................................................64
3.2.4.2. Mini and Maxiprep of Plasmid DNAs ..........................................................................................................65
3.2.4.3. RNA Isolation...............................................................................................................................................65
3.2.4.4. cDNA Synthesis............................................................................................................................................65
3.2.4.5. Real Time PCR (RT-PCR)............................................................................................................................66
3.2.4.6. Restriction Enzyme Digestion of Plasmids...................................................................................................67
3.2.4.7. Agarose Gel Electrophoresis.........................................................................................................................67
3.2.4.8. DNA Chip Assay and Evaluation of Microarray Data..................................................................................68
3.2.5. Protein Based Assay and Preparations .....................................................................................................68
3.2.5.1. Protein Isolation.......................................68
3.2.5.2. Protein Content Assay ..................................................................................................................................69
3.2.5.3. SDS-PAGE ...................................................................................................................................................69
3.2.5.4. Immunoblotting ............................................................................................................................................70
3.2.5.5. EMSA...........................................................................................................................................................70
3.2.5.6. Coomassie Staining ......................................................................................................................................72
3.2.5.7. Quick Coomassie Staining............................................................................................................................72
3.2.5.8. Co-Immunoprecipitation...............................................................................................................................72
3.2.5.9. Kinase Assay ................................................................................................................................................73
4. RESULTS..........................................................................................................................................................75
AA/AA4.1. CHARACTERIZATION OF IKKα MICE ...........................................................................................76
4.2. BASIC OBSERVATIONS WITH SPORADIC AND FAMILIAL CRC MODELS.....................................77
4.2.1. Azoxymethane Induced Sporadic CRC77
4.2.1.1. Tumor Number, Size and Proliferation in AOM Induced CRC ....................................................................77
4.2.1.2. Tumor Morphology in AOM Induced CRC..................................................................................................79
4.2.2. APC Loss Induced Genetic Intestinal Adenocarcinoma .............................................................................80
min/+4.2.2.1. Survival in Apc Mice..............................................................................................................................80
min/+4.2.2.3. Tumor Number, Size and Distribution in 4 Months Old Apc Mice ........................................................81
min/+ 4.2.2.4. Anemia in 4 Months Old Apc Mice ........................................................................................................82
min/+ 4.2.2.5. Proliferation in Apc Mice Tumors at Month 4 ........................................................................................82
viii Indexes
4.2.3. Constitutive β-catenin Activation Induced Genetic Intestinal Adenoma ....................................................84
T2 +/lox(ex3) min/+4.2.3.1. Vil-Cre-ER Ctnnb1 as a Good Representation of Apc Model.....................................................84
CA AA/AA4.2.3.2. Survival of β-cat Ikkα Mice ................................................................................................................86
CA AA/AA4.2.3.3. Morphology of β-cat Ikkα Mice..........................................................................................................87
CA AA/AA4.2.3.4. General Morphology of β-cat Ikkα Mice at Day 15............................................................................87
4.3. IDENTIFICATION OF MOLECULAR MECHANISM BEHIND IKKα INACTIVATION IN CRC.....90
4.3.1. Involvement of Alternative NF-κB Activation in IKKα Inactivation ........................................................90
CA4.3.2. General Expression Analysis in β-cat Mice............................................................................................92
CA4.3.2.1. Hightroughput Expression Analysis in β-cat Mice via Microarray...........................................................92
4.3.2.1.1. Hierarchical Clustering of Expression Data ............................................................................................92 .2. Functional Classification of Expression Data and Identifying Molecular Signatures................................95
CA4.3.2.2. Expression Analysis in β-cat Mice via RT-PCR .......................................................................................99
CA4.3.2.3. Expression Analysis in β-cat Mice via Protein Immunoblotting.............................................................101
CA AA/AA4.2.3.4. Cell Cycle Arrest as a Result of IFNγ Upregulation in β-cat Ikkα Mice...........................................103
4.3.3. Understanding Regulation of Downstream Targets of IKKα Inactivation .................................................104
CA AA/AA4.3.3.1. Reversion of Extended Survival in β-cat Ikkα Mice via Ifng Deletion .............................................104
4.3.3.2. Contribution of Epithelial and Hematopoietic Cell Compartments ............................................................105
4.3.3.3. Analysis of Hematopoietic Cells to Identify Molecular Machinery Behind IFNγ Activation.....................107
4.3.3.3.1. Lack of Th1 Activation Regardless of Increase in IL-12 ........................................................................107 .2. NK Cell Involvement and Lack of Tumor Specific Cytotoxicity...........................................................111
CA AA/AA4.3.3.3.3. Myeloid Cell Activation and Expansion in β-cat Ikkα Mice114
4.3.3.4. Requirement of Active NF-κB Signaling in IKKα Inactivation .................................................................122
5. DISCUSSION.................................................................................................................................................125
5.1. CHARACTERIZATION OF PHENOTYPE ASSOCIATED WITH IKKα INACTIVATION IN CRC.125
AA/AA5.1.1. AOM Induced Ikkα Mice Have Tumors with Reduced Size and Number.........................................126
min/+ AA/AA5.1.2. Apc Ikkα Mice Have Retarded Tumor Initiation and Development.............................................127
CA AA/AA5.1.3. Extended Survival and Reduced Proliferation of β-cat Ikkα Mice...................................................129
5.2. IDENTIFICATION OF THE ROLE OF IKKα IN CRC .........................................................................130
AA/AA5.2.1. Survival in Ikkα Mice is Independent of Alternative NF-κB Signaling130
AA/AA5.2.2. Decreased Proliferation in Ikkα Mice is not due to Direct Control over Wnt Signaling ......................131
CA AA/AA 5.2.3. General Expression Analysis Revealed Interferon Related Gene Upregulation in β-cat Ikkα Mice ......133
CA AA/AA5.2.4. IFNγ Induced Cell Cycle Arrest is the Key Mechanism in Reduced Proliferation in β-cat Ikkα Mice..135
AA/AA5.2.5. Both Epithelial and Hematopoietic Cells Contribute to the Ikkα Phenotype.......................................137
CA AA/AA5.2.6. Hematopoietic Origin of IFNγ Secretion in β-cat Ikkα Mice..........................................................139
CA AA/AA5.2.6.1. Adaptive Immunity is not Involved in the Suppression of Proliferation in β-cat Ikkα Mice.............139
CA AA/AA5.2.6.2. Myeloid Cell Activation as a Source of IFNγ in β-cat Ikkα Mice.....................................................141
AA/AA5.2.7. NF-κB Activation is Required for Reduced Proliferation in Ikkα Mice .............................................143
5.2.8. Identifying Kinase Specific and Structural Roles of IKKα in Immune Suppression ....................................145
6. CONCLUSION..............................................................................................................................................148
7. REFERENCES.................150
8. APPENDICES169
8.1. SUPPLEMENTARY MICROARRAY DATA ...........................................................................................169
8.2. PRIMERS..................176
8.3 PLASMIDS.................180
8.4. ANTIBODIES...........182
8.5 LIST OF INSTRUMENTS........................................................................................................................185
8.6. STATISTICS ............................................................................................................................................187
CURRICULUM VITAE......188
LEBENSLAUF.....................189

ix Indexes



INDEX OF FIGURES

FIGURE 1.1: HUMAN GASTROINTESTINAL SYSTEM....................................................................................................5
F1.2: ARCHITECTURE OF SMALL INTESTINE AND COLON...............................................................................6
FIGURE 1.3: INTESTINAL EPITHELIAL CELL DIFFERENTIATION..................................................................................8
F1.4: GENETICS OF CRC. .............................................................................................................................12
FIGURE 1.5: WNT SIGNALING.................................................................................................................................15
F1.6: REL FAMILY OF PROTEINS....................................................................................................................24
FIGURE 1.7: NF-κB ACTIVATION PATHWAYS. .........................................................................................................26
F1.8: IKK SUBUNITS......28
FIGURE 1.9: MODEL FOR IKK DIMERIZATION AND PHOSPHO-ACTIVATION ............................................................29

AA/AAFIGURE 4.1 BASIC CHARACTERIZATION OF IKKα MOUSE INTESTINE.. ...............................................................76
F4.2: AOM INDUCED MICE WEIGHT DIFFERENCE CURVE ..........................................................................77
FIGURE 4.3: BASIC TUMOR CHARACTERIZATION IN AOM INDUCED MICE. ...........................................................78
F4.4:TUMOR MORPHOLOGY IN AOM INDUCED MICE.................................................................................79
MIN/+ AA/AAFIGURE 4.5: SURVIVAL OF APC IKKα MICE....................................................................................................80
MIN/+ AA/AAFIGURE 4.6: BASIC TUMOR CHARACTERIZATION IN 4 MONTH APC IKKα MICE.. .........................................81
MIN/+ AA/AAFIGURE 4.7: ANEMIC STATUS OF APC IKKα MICE AT MONTH 4....................................................................82
MIN/+ AA/AAFIGURE 4.8: PROLIFERATION INAPC IKKα MICE INTESTINES AT MONTH 4...................................................83
MIN/+ AA/AAFIGURE 4.9: GENERAL MORPHOLOGY OF APC IKKα TUMORS AT 4 MONTHS.................................................84
CA FL/FL FIGURE 4.10: GSEA ENRICHMENT PLOT FOR COMPARING β-CAT MODEL WITH APC UPREGULATED GENES..85
CA AA/AAFIGURE 4.11: SURVIVAL OF β-CAT IKKα MICE..................................................................................................86
CA AA/AAFIGURE 4.12: MORPHOLOGY OF DUODENUM AT THE TIME OF SACRIFICE IN β-CAT IKKα MICE.. ....................87
CA AA/AAFIGURE 4.13: H&E STAINING OF β-CAT IKKα MICE AT DAY 15......................................................................89
CA AA/AAFIGURE 4.14: BRDU AND C-MYC IHC OF β-CAT IKKα MICE AT DAY 15. ........................................................89
CA AA/AAFIGURE 4.15: CYCLIN D1 AND CYCLIN D2 IHC OF β-CAT IKKα MICE AT DAY 15. .........................................90
ΔCA -/- CA - IEC FIGURE 4.16: SURVIVAL OF β-CAT P52 AND β-CAT RELB MICE.91
F4.17: HIERARCHICAL CLUSTERING OF 732 SIGNIFICANTLY REGULATED GENES IN HEATMAP AND
DENDOGRAM..........................................................................................................................................................93
FIGURE 4.18: K-MEANS CLUSTERING OF SIGNIFICANTLY REGULATED 732 GENES IN MICROARRAY ANALYSIS. ......94
FIGURE 4.19: CLOSER LOOK AT THE CLUSTER WITH 79 TRANSCRIPTS THAT ARE UPREGULATED IN 15 DAY β-
CA AA/AA
CAT IKKα MICE.................95
CA AA/AAFIGURE 4.20: FUNCTIONAL CLASSIFICATION OF THE GENES IN β-CAT IKKα MICE UPREGULATED CLUSTER..96
F4.21: GSEA OF MICROARRAY DATA WITH INTERFERON INDUCTION SIGNATURE GENES. .........................98
CA FL/FL FIGURE 4.22: GSEA ENRICHMENT PLOT FOR COMPARING β-CAT MODEL WITH APC UPREGULATED GENES..99
CA AA/AAFIGURE 4.24: IFNγ PRODUCTION IS INCREASED IN β-CAT IKKα MICE............................................................100
CA AA/AAFIGURE 4.25: WB ANALYSIS IN β-CAT IKKα MICE AT DAY 15.........................................................................102
MIN/+F4.26: WB ANALYSIS IN APC TUMORS AT MONTH 4...............................................................................103
CA AA/AAFIGURE 4.27: CELL CYCLE ARREST IN β-CAT IKKα MICE AT DAY 15. .............................................................104
-/- -/- CA AA/AAFIGURE 4.28: IFNγ BUT NOT IFNAR1 REVERTS β-CAT IKKα PHENOTYPE.. ..................................................105
ΔCA IECFIGURE 4.29: SURVIVAL GRAPHS FOR INTESTINAL SPECIFIC IKKα DELETION MODEL β-CAT IKKα MICE AND
AA/AAIKKα BONE MARROW TRANSPLANTED MICE.....................................................................................................106
CA AA/AAFIGURE 4.30: INVESTIGATION OF T-CELL ACTIVATION IN β-CAT IKKα MICE.................................................109
x