p53 MUTATIONS AS A SOURCE OF ABERRANT β-CATENIN ACCUMULATION IN CANCER ...

176 Pages·2002·3.78 MB·English

by  Tolga Çağatay

p53 MUTATIONS AS A SOURCE OF ABERRANT b -CATENIN

ACCUMULATION IN CANCER CELLS

A THESIS SUBMITTED TO
THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS
AND
THE INSTITUTE OF ENGINEERING AND SCIENCE OF
BÝLKENT UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

By
TOLGA ÇAGATAY
September, 2002
TToo MMyy FFaammiillyy;;
ÇÇAAGATAYs
I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

________________________________
Prof. Dr. Mehmet Öztürk

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

________________________________
Prof. Dr. Wayne Criss

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

________________________________
Prof. Dr. Ahmet Koman

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

________________________________
Assist. Prof. Ugur Yavuzer

I certify that I have read this thesis and that in my opinion it is fully adequate, in
scope and in quality, as a thesis for the degree of Doctor of Philosophy.

________________________________
Assist. Prof. Isik. G. Yulug

Approved for the Institute of Engineering and Science

_______________________________
Prof. Dr. Mehmet Baray
Director of Institute of Engineering and Science

ii
ABSTRACT

p53 MUTATIONS AS A SOURCE OF ABERRANT b -CATENIN

ACCUMULATION IN CANCER CELLS

TOLGA ÇAGATAY
Ph.D. in Molecular Biology and Genetics
Supervisor: Prof. Dr. Mehmet Öztürk
September 2002, 157 Pages

b -catenin is involved in both cell-cell interactions and wnt pathway-dependent cell

fate determination through its interactions with E-cadherin and TCF/LEF
transcription factors, respectively. Cytoplasmic/nuclear levels of b -catenin are

important in regulated transcriptional activation of TCF/LEF target genes. Normally,
these levels are kept low by proteosomal degradation of â-catenin through Axin1-
and APC-dependent phosphorylation by CKI and GSK-3b . Deregulation of b -catenin

degradation results in its aberrant accumulation, often leading to cancer.
Accordingly, aberrant accumulation of b-catenin is onberved at high frequency in
many cancers. This accumulation correlates with either mutational activation of
CTNNB1 (b -catenin) or mutational inactivation of APC and Axin1 genes in some

tumors. However, there are many tumors that display b -catenin accumulation in the

absence of a mutation in these genes. Thus, there must be additional sources for
aberrant b -catenin accumulation in cancer cells. Here, we provide experimental

evidence that wild-type b -catenin accumulates in hepatocellular carcinoma (HCC)

cells in association with mutational inactivation of p53 gene. We also show that
worldwide p53 and b -catenin mutation rates are inversely correlated in HCC. These

data suggest that inactivation of p53 is an important cause of aberrant accumulation
of b-catenin in cancer cells.

iii
ÖZET

KANSER HÜCRELERiNDEK SIRADISI b -KATENiN BiRiKiMiNiNE

p53 MUTASYOUNUNUN KATKISI

TOLGA ÇAGATAY
Doktora Tezi, Moleküler Biyoloji ve Genetik Bölümü
Tez Yöneticisi: Prof. Dr. Mehmet Öztürk
Eylül 2002, 157 sayfa

b -katenin, E-kaderin ve TCF/LEF ifade faktörleri ile etkileserek hücre-hücre

etkilesim ve wnt-bagimli hücre kaderi belirlenmesinde rol alir. b -kateninin

sitoplazmik/çekirdek seviyeleri TCF/LEF hedefi olan genlerin kontrollu ifadesi için
önemlidir. Nomalde bu seviyeler b -kateninin Aksin-1’e ve APC’ye bagli olarak CK-

I ve GSK-3b tarafindan fosforlanmasini takiben poteozomal olarak parçalanmasi ile

düsük seviyelerde tutulur. b -katenin parçalanmasindaki bozukluklar, sIklIkla kansere

neden olnan siradisi b -katenin birikimi ile sonuçlanir. Ayni dogrultuda birçok

kanserde siradisi b -katenin birikimi yüksek bir sIklIkla gözlenmektedir. Bazi

tümörlerde bu birikim ya CTNNB1 (b -katenin)’in mutasyonal olarak aktivasyonu ya

da APC veya Aksin1’in mutasyonal olarak inaktive edilmesi ile iliskilidir.Birçok
tümörde ise bahsi geçen genlerde herhangi bir mutasyon olmadigi halde b -katenin

birikimi gözlenmektedir. Bundan dolayi, kanser hücrelerindeki siradisi b -katenin

bitirikimine katkisi olan baska ek nedenler olmalidir. Bu çalismada normal b -katenin

tasiyan hepatoselüler kanser hücrelerindeki siradisi b -katenin biriminin p53 genin

mutasyonel olarak inaktive edilmesi ile iliskili oldugu deneysel olarak gösteriyoruz.
Ayrica, dünya çapindaki p53 ve b -katenin mutayonlarinin ters oratili gidisatini

göstermekteyiz. Bu veriler p53 inaktivasyonunun kanser hücrelerinde gözlenen
siradisi b -katenin birikimine önemli bir katkisi olduðunu düsündürmektedir.

iv
ACKNOWLEDGMENT

“The best part of a scientific research is that it has a joyful, game-like nature in
itself. We were saying "we're gonna have fun", we had actually and we have been
continuously to have it “ - J. Dausset, 1983-

Thanks first of all and most of all to my advisor, Prof. Mehmet Öztürk who’s
been unfailingly brilliant to work with. Every time we’ve talked I’ve come away
with new ideas and enthusiasm and whenever I’ve lost the plot completely he’s
steered me gently back in the right direction. I wish to express my thanks to him for
making it possible to have an excellent working environment in Turkey.

My special thanks go to Dr. Uður Yavuzer for her support and always being
there when I needed, and being one of few people who made my time here
memorable.

Thanks to Dr. Iþýk G. Yuluð for her guidance during my callow young-
science days, and being a good listener in my murmurous -mood.

I appreciate moral support by research assistants of Department of Molecular
Biology and Genetics in Bilkent University, especially to Arzu , Cemo, Esra, Hani,
K.Tuba and NÖztürk for their endurance toward any trouble that I caused in course
of close interactions. Thanks to my laboratory partners Sayans for their reciprocal
warm feelings.

Very special thanks to T.K Fountain and Tuba Dincer Erdemir for their
presence, their patience and their constant love. I greatly appreciate their support and
optimism during both the good and difficult times encountered throughout my
studies in Bilkent.

Last, but certainly not least, my ultimate thanks to my family for always
giving their unconditioned interest and support. They gave me the chance to explore
my own limits.
v
TABLE OF CONTENTS

SIGNATURE PAGE ii
ABSTRACT iii
ÖZET iv
ACKNOWLEDGMENTS v
TABLE OF CONTENTS vi
LIST OF FIGURES xii
LIST OF TABLES xv
ABBREVIATIONS xvi
CHAPTER 1. INTRODUCTION 1
1.1 Liver Cancers 1
1.2 General Mechanism of Hepatocellular Carcinogenesis 2
1.3 Pathogenesis of Hepatocellular Carcinoma 4
1.3.1 Significance of Viral Hepatitis to Hepatocellular Carcinoma 4
1.3.2 Alcohol and Cirrhosis 6
1.3.3 Hepatotoxic Chemicals 6
1.3.4 Hemochromatosis and Iron 7
1.4 Molecular pathogenesis of Hepatocellular Carcinoma 7
vi
1.4.1 Oncogenes in HCC 8
1.4.2 Tumor Suppressor Genes in HCC 10
1.4.3 Genetic mechanism of Hepatocellular Carcinoma 14
1.5 The p53 Tumor Suppressor 16
1.5.1 Activation of p53 16
1.5.2 Biochemical and physiological Functions of p53 18
1.5.3 p53 and Oncogenic Stress 20
1.5.4 The p53 Tumor Suppressor in HCC 20
1.6 The Canonical WNT/Wg (Wingless) Signaling 22
1.7 Canonical WNT signaling in Cancer 23
1.7.1 Ligand and Receptor 23
1.7.2 Dishevelled (Dsh) 24
1.7.3 Glycogen Synthase Kinase 3â (GSK-3â) 25
1.7.4 Axin 1 25
1.7.5 The Adenomatous Polyposis Coli (APC) 26
1.7.6 Beta Catenin (â-catenin) 26
1.7.7 T cell factor/lymphocyte enhancer binding factor (TCF/LEF) 32
family of transcriptional factors
1.8 The p53 –WNT cross talk 33
1.9 WNT/â-catenin Signaling in Liver Cancer 34

CHAPTER 2. OBJECTIVES AND RATIONALE 37

CHAPTER 3. MATERIALS AND METHODS 39
3.1 MATERIALS 39
vii
3.1.1 Reagents 39
3.1.2 Bacterial Strain 39
3.1.3 Enzymes 39
3.1.4 Nucleic Acids 40
3.1.5 Oligonucleotides 40
3.1.6 Electrophoresis and Photography 40
3.1.7 Tissue Culture Reagents and Cell Lines 41
3.1.8 Tumor Specimens 41
3.1.9 Radioisotopes 41
3.1.10 Antibodies and Chemiluminesence 41

3.2 SOLUTIONS AND MEDIA 42
3.2.1 General Solutions 42
3.2.2 Microbiological Media and Antibiotics 43
3.2.3 Tissue Culture Solutions 44
3.2.4 Single Strand-Conformational Polymorphism (SSCP) Solutions 45
3.2.5 SDS (sodium deodecyl sulfate)-PAGE (Polyacrylamide gel 45
electrophoresis) Solutions
3.2.6 Immunoblotting Solutions 46
3.2.7 RNA Study Solutions 47
3.2.7 Immunofluorescence solutions 47

3.3 METHODS 48
3.3.1 General Methods 48
3.3.1.1 Transformation of E.coli 48
viii
3.3.1.2 Growth and Storage of Bacterial Strains 49
3.3.1.3 Preparation of Genomic DNA from Cultured Cells 49
3.3.1.4 Quantification and Qualification of Nucleic Acids 50
3.3.1.5 Restriction Enzyme Digestion of DNA 50
3.3.1.6 Gel Electrophoresis of Nucleic Acids 51
3.3.2 Computer Analysis of DNA Sequences 52
3.3.3 Tissue Culture Techniques 52
3.3.3.1 Cell Lines 52
3.3.3.2 Thawing Cell Lines 53
3.3.3.3 Growth Conditions 53
3.3.3.4 Cryopreservation of Cell Lines 54
3.3.3.5 Transient Transfection of Eukaryotic Cells 54
Using BES Method
3.3.3.6 Generation of Stable Cell Lines 55
3.3.4 Amplification of DNA by Polymerase Chain Reaction (PCR) 56
3.3.5 Extraction of Total RNA from Tissue Culture Cells and First 58
Strand cDNA Synthesis
3.3.5.1 Primer Design for Expression Analysis by 59
Semi-Quantitative PCR
3.3.5.2 Fidelity and DNA Contamination Control in First 59
Strand cDNAs
3.3.6 Expression Analysis of a Gene by Semi-Quantitative PCR 60
3.3.7 Mutation Screening Methods 60
3.3.7.1 SSCP 60
3.3.7.2 Direct PCR Sequencing 62
ix