Role of 3'UTR elements in the regulation of the cyclin D1 proto-oncogene [Elektronische Ressource] / submitted by Anagha Deshpande

Anagha Deshpande

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 44
• Langue: Deutsch

Extrait

From the Department of Medicine III,
University of Munich Hospital Grosshadern
Ludwig-Maximilians-University, Munich and
The Helmholtz Centre Munich
German Research Centre for Environmental Health
Clinical Cooperative Group ‘’Leukemia’’

Chair: Prof. Dr. med. Wolfgang Hiddemann





Role of 3’UTR Elements in the Regulation of the Cyclin D1
Proto-oncogene





Thesis Submitted for a Doctoral degree in Human Biology
at the Faculty of Medicine Ludwig-Maximilians-University,
Munich, Germany




Submitted by
Anagha Deshpande


From
Thane, India

2009

Aus der Medizinischen Klinik und Poliklinik III am Klinikum
Großhadern der Ludwig-Maximilians-Universität München
Und dem Helmholtz Zentrum München,
Deutsches Forschungszentrum für Umwelt und Gesundheit,
Klinische Kooperations Gruppe Leukämie


Direktor: Prof. Dr. med. Wolfgang Hiddemann





Die Rolle von 3’UTR Elementen in der Regulation des Cyclin D1
Proto-Onkogens





Dissertation zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der Ludwig-Maximilians-
Universität zu München, Germany




Vorgelegt von
Anagha Deshpande


Aus
Thane, Indien

2009



With Permission from the Faculty of Medicine
University of Munich



Supervisor/Examiner: Prof. Dr. med. Martin Dreyling


Co-examiners: Priv. Doz. Dr. Fuat Oduncu, M.A., E.M.B.
Prof. Dr. Karl Sotlar


Dean: Prof. Dr. med. Dr.h.c. Maximilian Reiser, FACR, FRCR

Date of Oral Exam: 15.07.2009




Mit Genehmigung der Medizinischen Fakultät
der Universität München



Berichterstatter: Prof. Dr. med. Martin Dreyling

Mitberichterstatter: Priv. Doz. Dr. Fuat Oduncu, M.A. , E.M.B.
Prof. Dr. Karl Sotlar



Dekan: Prof. Dr. med. Dr.h.c. Maximilian Reiser, FACR, FRCR

Tag der Mündlichen Prüfung: 15.07.2009





















To

Aai and Baba
who have continuously showered me with love
and affection…
A b b r e v i a t i o n s


Abbreviations

Ago Argonaut
ALCL Anaplastic large cell lymphoma
AREs AU-rich elements
ATM ataxia talengiectasia mutated
AUBP ARE binding protein
B-ALL B acute lymphoid leukemia
CCND1 Cyclin D1
CDK Cyclin dependent kinase
CLL Chronic lymphocytic leukemia
Deletion del
Fw Forward
GFP Green fluorescent protein
IL Interleukin
ins Insertion
MCL Mantle cell lymphoma
miRNAs MicroRNAs
mRNA Messenger RNA
NF κB Nuclear factor κB
non-Hodgkin's lymphoma NHL
nt Nucleotide
Polymerase chain reaction PCR
PTEN phosphotase and tensin homologue
Rb Retinoblastoma
Rev Reverse
RISC RNA-induced silencing complex
T-ALL T acute lymphoid leukemia
TBP TATA-box binding protein
UTR Untranslated region
YFP Yellow fluorescent protein T a b l e o f C o n t e n t s


Table of Contents

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

1.1 Oncogenesis: ...................................................................................................1
1.2 Mantle cell lymphoma: ..................................................................................2
1.2.1 Clinical characteristics of mantle cell lymphoma ........................................................2
1.2.2 Treatment strategies for mantle cell lymphoma...........................................................3
1.2.3 Cellular biology of MCL..............................................................................................5
1.2.4 Molecular biology of MCL ..........................................................................................5
1.3 Cyclin D 1........................................................................................................8
1.3.1 Cyclin D1 as cell cycle regulator .................................................................................9
1.3.2 CDK independent functions of cyclin D1 ....................................................................9
1.3.3 Cyclin D1 as proto-oncogene.....................................................................................10
1.4 3’UTR mediated gene regulation................................................................13
1.4.1 AU rich elements in the 3’UTR14
1.4.2 MicroRNAs (miRNAs) ..............................................................................................16
1.4.2.2 Biogenesis of miRNAs...........................................................................................17
1.4.2.3 Mechanism of miRNA regulation..........................................................................19
1.4.2.4 MiRNAs in cancer..................................................................................................20
1.4.2.5 MiRNAs in cell cycle regulation............................................................................22
1.4.2.6 MiR-15a and miR-16-1 ..........................................................................................23 T a b l e o f C o n t e n t s


1.4.2.7 MiR-17-92 cluster miRNAs...................................................................................24
1.5 Aim of the study ...........................................................................................26

Materials................................................................................................27

2.1 Mammalian cell lines ...................................................................................27
2.2 Media and reagents......................................................................................27
2.2.1 Cell and tissue culture27
2.2.2 Molecular biology ......................................................................................................29
2.2.2.1 Plasmids .................................................................................................................30
2.2.2.2 Oligonucleotides.....................................................................................................30
2.3 Western blotting...........................................................................................33
2.3.1 Reagents for western blotting.....................................................................................33
2.4 Instruments: .................................................................................................35
2.5 Software ........................................................................................................35

Methods .................................................................................................37

3.1 Cloning of constructs ...................................................................................37
3.2 Mutagenesis (CCND1 mutants)..................................................................41
3.3 Preparation of stable cell lines....................................................................44
3.4 Proliferation assay .......................................................................................45 T a b l e o f C o n t e n t s


3.5 Cell cycle analysis.........................................................................................45
3.6 Western blotting...........................................................................................46
3.7 Reporter assays ............................................................................................48
3.8 Transient transfections and fluorescence microscopy..............................49
3.9 Flow cytometry.............................................................................................49
3.10 Estimation of ectopically expressed cyclin D1 protein .............................50
3.12 RNA isolation and cDNA synthesis ............................................................50
3.13 Real-time RT-PCR.......................................................................................51

Results ...................................................................................................52

4.1 Loss of 3’UTR leads to increased cyclin D1 expression ...........................52
4.2 The cyclin D1 3’UTR can regulate chimeric reporter activity................55
4.3 Effect of the 3’UTR deletion on cyclin D