Interaction between glycogen synthase kinase-3 and estrogen receptor-α [receptor-alpha] in ligand dependent activation of the receptor [Elektronische Ressource] / presented by Jean Grisouard

Grisouard

Sujets

• Gesundheit

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences








Interaction between



Glycogen Synthase Kinase-3 and Estrogen Receptor-α



in ligand-dependent activation of the receptor







Presented by
Jean GRISOUARD
(Born in Dijon, France)




Heidelberg, 2007 I











Interaction between



Glycogen Synthase Kinase-3 and Estrogen Receptor-α


in ligand-dependent activation of the receptor


























Referees Prof. Dr. Doris Mayer

Prof. Dr. Lutz Gissmann


II

To Mathieu Jourd’heuil,

To Felix Bub,

In Their Memory.......










To my Great Grand-Ma’

Bravery, Strength and Will......




To my Family,

My Refuge......




To Sandra,

My Love......












To my Friends,

From my little village, to the next town and all over the world....... Table of contents III
Table of contents

ZUSAMMENFASSUNG ............................................................................................. 1
I - SUMMARY............................................................................................................. 3
II - INTRODUCTION................................................................................................... 5
II - 1) CANCER OVERVIEW....................................................................................................... 5
II - 2) BREAST CANCER........................................................................................................... 6
II - 2.1) Anatomy of the breast ........................................................................................ 6
II - 2.2) Types of breast cancer....................................................................................... 7
II - 2.3) Epidemiology and aetiology............................................................................... 7
II - 2.4) Detection and diagnosis..................................................................................... 8
II - 2.5) Staging.................................................................................................................. 8
II - 2.6) Grading ............................................................................................................... 10
II - 2.7) Treatment strategies......................................................................................... 11
II - 3) ESTROGEN SIGNALLING AND BREAST CANCER.......................................................... 14
II - 3.1) Estrogen biosynthesis and function ............................................................... 14
II - 3.2) Mechanism of estrogen receptors function and activity.............................. 16
II - 3.2.1) Structure and tissue expression of estrogen receptors........................ 16
II - 3.2.2) Cell signalling of estrogen receptors ....................................................... 17
I - 3.2.3) Biological function of estrogen receptors................................................. 19
II - 3.3) Estrogenic activity and breast tumorigenesis ............................................... 19
II - 3.3.1) Regulation of gene expression upon estrogen stimulation.................. 20
II - 3.3.2) Estrogen receptor status in breast cancer.............................................. 20
II - 4) ESTROGEN SIGNALLING-RELATED PROTEIN KINASES AS KEY PLAYERS OF BREAST
TUMORIGENESIS.................................................................................................................... 21
II - 4.1) The protein kinase superfamily....................................................................... 22
II - 4.2) Protein kinases related to breast cancer....................................................... 23
II - 4.3) Phosphorylation sites and protein kinases involved in ERα signal
transduction ..................................................................................................................... 24
II - 4.4) A putative role for the glycogen synthase kinase-3β (GSK-3β)................. 25
II - 4.4.1) GSK-3β substrates..................................................................................... 26
II - 4.4.2) GSK-3β regulation in a variety of intracellular pathways ..................... 29
II - 4.4.3) Early findings concerning possible ERα regulation by GSK-3β.......... 32 Table of contents IV
II - 5) AIMS ............................................................................................................................ 33
III - EXPERIMENTAL PROCEDURES ..................................................................... 35
III - 1) MATERIALS ................................................................................................................ 35
III - 1.1) Equipment ......................................................................................................... 35
III - 1.2) Chemicals and other compounds.................................................................. 35
III - 1.3) Solutions and media for bacterial culture..................................................... 37
III - 1.4) Molecular biology kits ...................................................................................... 38
III - 1.5) Plasmid constructs........................................................................................... 38
III - 1.6) Small interfering RNA (siRNA) sequences .................................................. 40
III - 1.7) PCR primers ..................................................................................................... 40
III - 1.8) Buffers and solutions for agarose gel electrophoresis............................... 41
III - 1.9) Cell culture equipments and chemicals........................................................ 41
III - 1.10) Cell lines.......................................................................................................... 42
III - 1.11) Solutions and media for cell culture............................................................ 42
III - 1.12) Buffers and solutions for western blot 43
III - 1.13) Antibodies ....................................................................................................... 45
III - 1.13.1) Primary antibody ..................................................................................... 45
III - 1.13.2) Secondary/labelled antibody ................................................................. 45
III - 1.14) Buffers and solutions for β-galactosidase activity measurement........... 46
III - 1.15) Buffers and solutions for immunofluorescence analysis ......................... 46
III - 1.16) Buffers and solutions for immunohistochemistry ...................................... 46
III - 2) METHODS ................................................................................................................... 47
III - 2.1) Molecular biology ............................................................................................. 47
III - 2.1.1) RNA isolation and cDNA synthesis ........................................................ 47
III - 2.1.2) Determination of nucleic acid concentration ......................................... 47
III - 2.1.3) Polymerase chain reaction (PCR) .......................................................... 48
III - 2.1.4) Agarose gel electrophoresis .................................................................... 48
III - 2.1.5) Cloning using restriction endonucleases............................................... 49
III - 2.1.6) Cloning of DNA expression vectors with miR for RNAi....................... 49
III - 2.1.7) Single or multiple site mutations ............................................................. 49
III - 2.1.8) Transformation of competent bacteria with plasmid DNA................... 50
III - 2.1.9) DNA mini- and maxi-prep......................................................................... 50
III - 2.2) Cell culture ........................................................................................................ 50 Table of contents V
III - 2.2.1) Cell culture media and treatments.......................................................... 50
III - 2.2.2) Cell proliferation study .............................................................................. 51
III - 2.2.3) Lysis of cells............................................................................................... 51
III - 2.2.4) Preparation of cytoplasmic and nuclear cell extracts .......................... 52
III - 2.2.5) Transient transfection of cells with plasmids DNA............................... 52
III - 2.2.6) Transient transfection