Role of galectin-3 in melanoma progression [Elektronische Ressource] / vorgelegt von Alexandra Anastasia Mourad-Zeidan

Alexandra Mourad-Zeidan

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 86
• Poids de l'ouvrage: 3 Mo

Extrait

Role of Galectin-3 in Melanoma Progression
A

DISSERTATION


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



Alexandra Anastasia Mourad-Zeidan, B.S., Diplom Biol. M.S.

Munich, Germany

2008


Aus dem Institut für Immunologie

der Ludwig-Maximilians-Universität München

Vorstand: Prof.Thomas Brocker






Role of Galectin-3 in Melanoma Progression

Dissertation


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


vorgelegt von


Alexandra Anastasia Mourad-Zeidan, B.S., Diplom Biol. M.S


aus

Muenchen, Deutschland


Jahr


2008 Mit Genehmigung der Medizinischen Fakultät
der Universität München




Berichterstatter: Prof. Judith P. Johnson

Priv. Doz. Dr. Barbara Mayer
Mitberichterstatter: Prof. Dr. Dolores J. Schendel

Mitbetreuung durch den
promovierten Mitarbeiter:
Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR, FRCR
Tag der mündlichen
12. 12. 2008
Prüfung:
3
Dedication

To my wonderful son,
Joseph Adrian Zeidan,
a present of God.







iii Table of Contents
Dedication..........................................................................................................................iii
Table of Contents...............................................................................................................iv
List of Figures.................................................................................................................... ix
List of Tables .................................................................................................................... xii
Chapter I: ............................................................................................................................ 1
1. Introduction..................................................................................................................... 1
1. 1 Melanoma Incidence and Risk Factors.................................................................... 1
1.1.1 Stages and Diagnosis of Melanoma Development ................................................ 2
1.1.2 Common cutaneous melanomas ............................................................................ 4
1.1.3 Therapy for metastatic melanoma.......................................................................... 5
1.1.4 Molecular changes associated with melanoma progression .................................. 7
1.1.4.1 Inactivation of tumor suppressors INK4A/ARF, p53, PTEN and
activating mutations in BRAF and N-Ras in melanoma............................................. 8
1.1.4.2 Melanoma Progression from RGP to VGP................................................... 11
1.1.4.5 Matrix Metalloproteinases (MMP) .............................................................. 16
1.1.4.6 Angiogenic factors........................................................................................ 16
iv 1.2 Galectin-3................................................................................................................ 18
1.2.1 Gal-3 intracellular functions ............................................................................ 19
1.2.2 Gal-3 extracellular functions 23
Specific Aims.................................................................................................................... 26
Chapter II: ......................................................................................................................... 27
2. Materials and Methods.................................................................................................. 27
2.1 Cell Lines and Culture Conditions.......................................................................... 27
2.2 Three-dimensional Type-I Collagen Gels............................................................... 28
2.3 Animals................................................................................................................... 29
2.4 Cell Preparation for Injection.................................................................................. 29
2.5 Injection and Monitoring of Tumor Cells............................................................... 30
2.6 In Vitro Proliferation Assay.................................................................................... 31
2.7 Antibodies............................................................................................................... 31
2.8 Protein Extraction ................................................................................................... 32
2.9 Western Immunoblot Analysis ............................................................................... 33
2.9 Expression Constructs............................................................................................. 34
2.10 Transient Transfections and Luciferase Activity Assays...................................... 36
v 2.11 Stable Transfection with small hairpin RNA Lentivirus Expression Vectors ...... 36
2.11.1 shRNA expression vector construction.......................................................... 36
2.11.2 Cell sorting with Flow Cytofluorometry........................................................ 37
2.11.3 Recombinant lentivirus production................................................................ 38
2.11.4 Target cell transduction.................................................................................. 42
2.12 Zymography.......................................................................................................... 42
2.13 Invasion Assay through Matrigel.......................................................................... 43
2.14 RNA Extraction .................................................................................................... 43
2.15 RT-PCR................................................................................................................. 44
2.16 Tissue Microarray (TMA)..................................................................................... 44
2.17 Immunohistochemical Analysis............................................................................ 47
2.18 Immunofluorescence Staining .............................................................................. 48
2.19 In situ Terminal dUTP Nick End Labeling (TUNEL) Assay ............................... 49
2.20 Enzyme-Linked Immunosorbent (ELISA) Assay................................................. 50
2.21 cDNA Microarray Analysis .................................................................................. 51
2.22 Chromatin Immunoprecipitation (ChIP) Assay.................................................... 51
2.23 Densitometric Quantification................................................................................ 53
vi 2.24 Statistical Analysis................................................................................................ 53
Chapter III:........................................................................................................................ 55
3. RESULTS ..................................................................................................................... 55
3.1 Specific Aim I:........................................................................................................ 55
3.1.1. Pattern of Gal-3 expression during melanoma progression................................ 55
3.1.1.1 Tissue microarray analysis............................................................................ 55
3.2 Specific Aim II:....................................................................................................... 67
3.2.1 Effect of Gal-3 shRNA on tumor growth and metastasis in vivo........................ 67
3.2.1.1 Expression of Gal-3 in melanoma cell lines ................................................. 67
3.2.1.3 Effect of Gal-3 downregulation on tumor growth in vivo ............................ 70
3.2.1.4 Effect of Gal-3 downregulation on metastasis in vivo.................................. 76
3.2.1.5 Effect of Gal-3 downregulation on in vivo cell proliferation, microvessel
density (MVD) and apoptosis ................................................................................... 80
3.3 Specific Aim III: ..................................................................................................... 87
3.3.1 Identification of novel Gal-3 downstream target genes by Gal-3 small hairpin
RNA .............................................................................................................................. 87
3.3.1.1 Determining novel genes possibly regulated by Gal-3 via a cDNA
microarray analysis ................................................................................................... 87
vii 3.3.1.2 Validation of target genes after Gal-3 shRNA k