Role of the activin-follistatin system during angiogenesis and tumor progression [Elektronische Ressource] / vorgelegt von Jelena Krneta

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Informations

• Publié par: albert-ludwigs-universitat_freiburg
• Publié le: 01 janvier 2004
• Nombre de lectures: 126
• Langue: English
• Poids de l'ouvrage: 15 Mo

Extrait

Role of the Activin/Follistatin system
during angiogenesis and tumor progression









Inaugural-Dissertation

zur Erlangung der Doktorwürde

der Fakultät für Biologie

der Albert-Ludwigs-Universität

Freiburg im Breisgau








vorgelegt von

Jelena Krneta

aus Belgrad, Serbien und Montenegro




April 2004




Dekan der Fakultät für Biologie: Prof. Dr. Georg Fuchs

Promotionsausschußvorsitzender: Prof. Dr. Karl-Friedrich Fischbach

Betreuer der Arbeit: Prof. Dr. Dieter Marmé

rof. Dr. Helmut Augstin
Refrnt: Prof. Dr. ietr Marmé
Koreferent: Prof. Dr. Gunther Neuhaus
rof. Dr. Anet Neubüser






Tag der Verkündigung des Prüfungsergebnisses: 8.7.2004.

















Die vorliegende Arbeit wurde am Institut für Molekulare Onkologie, Abteilung für Vaskuläre
Biologie & Angiogeneseforschung an der Klinik für Tumorbiologie in Freiburg durchgeführt.




Teile dieser Arbeit sind in folgenden Veröffentlichungen enthalten:
Augustin, H.G., Krneta, J., Alves, F., Baumbach, J., Eberhard, A. and Kahlert, S. Archives of
Pharmacology 2001;364 Suppl 3:SR13. Angiogenesis and tumorigenesis: What is the causal
relationship?
Augustin, H.G., Krneta, J., Alves, F., Baumbach, J., Eberhard, A., Kahlert, S. and Dandekar, G.
Ann Hematol. 2002;81 Suppl 2:S68. Quantitating angiogenesis and assessing the causal
relationship between angiogenesis and tumorigenesis: problems and progress.
Krneta, J., Sananbenesi, F., Phillips, D.J., Alves, F., Esser, N., Reiss, Y. and Augustin, H.G.
Dissociation between angiogenesis and tumorigenesis in Activin and Follistatin overexpressing
R30C tumors (manuscript in preparation). Index
Index
Abbreviations IV
1 Introduction 1
1.1 Physiological and tumor angiogenesis 1
1.2 Regulation of angiogenesis 2
1.3 Angiogenesis and tumor progression 5
1.3.1 Control of the angiogenic switch 7
1.4 Activin and Follistatin 8
1.4.1 Activin and Follistatin in the regulation of endothelial cell function 10
1.4.2 Model for the endothelial cell growth regulation by Follistatin/Activin
system 11
1.5 Aim of the study 12
2 Materials and Methods 13
2.1 Materials 13
2.1.1 Specified chemicals, consumables and equipment 13
2.1.2 Antibodies, growth factors and other molecules and kits 14
2.1.3 Reagent kits for methods in molecular biology and enzymes 15
2.1.4 Nucleic acids 16
2.1.5 Media and stock solutions 16
2.1.6 Buffers and stock solutions 17
2.2 Methods 18
2.2.1 Molecular biology methods 18
2.2.1.1 Amplification of DNA fragments via PCR (polymerase chain reaction) 18
2.2.1.2 RT-PCR 18
2.2.1.3 Analysis of Activin receptor expression by PCR 19
2.2.1.4 DNA separation by agarose gel electrophoresis 19
2.2.1.5 Extraction of DNA fragments from agarose gel 19
2.2.1.6 Quantitation of DNA/RNA 19
I Index
2.2.1.7 Labelling of DNA by random oligonucleotide-primed synthesis 20
2.2.1.8 Procedure for separating radioactively labelled DNA from unincorporated
dNTP precursors 20
2.2.1.9 Northern Blotting 20
2.2.1.10 Isolation of total RNA 21
2.2.1.11 Alcohol precipitation of RNA 21
2.2.2 Cell biology methods 22
2.2.2.1 Cell culture conditions 22
2.2.2.2 Generation of endothelial spheroids 22
2.2.2.3 In vitro angiogenesis assay 22
2.2.2.4 Cell cycle analysis by FACS of Propidium Iodide (PI) labelled cells 23
2.2.2.5 Analysis of the population growth 23
2.2.3 Protein biochemical methods 23
2.2.3.1 Immunoassays for FS and AC 23
2.2.4 In vivo methods and immunohistology 24
2.2.4.1 Animals 24
2.2.4.2 Matrigel implant assay 24
2.2.4.3 Tumorigenesis assay 24
2.2.4.4 Paraffin embedding procedure 25
2.2.4.5 Tumor dissociation protocol for FACS analysis 25
2.2.4.6 Staining procedures 25
2.2.4.7 Quantitation of microvessel density (MVD) 27
2.3 Statistical analysis 28
3 Results 29
3.1 Analysis of the effect of Activin and Follistatin on endothelial sprouting
(“angiogenesis in vitro”) 29
3.2 Analysis of the effect of Activin on angiogenesis in vivo 31
3.3 Analysis of the effect of Follistatin and Activin on tumor angiogenesis 33
3.3.1 Amplification of Activin-ßA and Follistatin cDNA probe 33
3.3.2 Analysis of Activin and Follistatin mRNA expression in transfected R30C 34
3.3.3 Analysis of Activin and Follistatin secretion in transfected R30C 34
3.3.4 Verification of bioactivity of overexpressed proteins 36
II Index
3.3.5 In vivo tumorigenesis assay 37
3.3.6 Analysis of Activin and Follistatin expression in tumor explants 38
3.3.7 Characterization of tumor neovasculature 40
3.3.7.1 Characterization of the morphology and Microvessel density 40
3.3.7.2 Microvessel density (MVD) counting 40
3.3.7.3 Analysis of vessel morphology 42
3.3.7.4 Analysis of vessel perfusion 43
3.3.7.5 Analysis of vessel maturation 45
3.3.8 Analysis of Activin receptor II expression in R30C in vitro 47
3.3.9 Analysis of Activin-mediated signalling in R30C derived tumors 48
3.3.10 Analysis of tumor cell proliferation in tumors 50
3.3.11 Analysis of tumor cell apoptosis in tumors 50
3.3.12 Analysis of the cell cycle profile of tumor cells in tumor xenografts 54
3.3.13 Analysis of the cell cycle profile of tumor cells in 54
3.3.15 Analysis of the effect of Follistatin and Activin overexpression on a
population growth in a long term experiment 57
4 Discussion 58
5 Summary 66
6 References 68
III Abbreviations
Abbreviations
AC activin A
ActR activin receptor
ALK activin receptor-like kinase
AMH anti-Müllerian hormone
Ang angiopoetin
bFGF basic fibroblast growth factor
bp base pair
BMP bone morphogenetic protein
BMPR bone morphogenetic protein receptor
BS-I Bandeiraea simplicifolia I
°C degree Celsius
CAM chorioallantoic membrane
CD cluster of differentiation
cDNA complementary DNA
CMV cytomegalovirus
co-Smad common mediator Smad
CSL cumulative sprout length
3D three dimensional
DAB diaminobenzidin tetrahydrochlorid
DEPC diethyl pyrocarbonate
DNA deoxyribonucleic acid
dNTP deoxynucleoside triphosphate
DTT dithiothreitol
EBSS Earl’s balanced salt solution
EC endothelial cell
ECBM endothelial cell basal medium
ECGM endothelial cell growth medium
EDTA ethylendinitrilo-N, N, N’, N’-tetra-acetate
EGF epidermal growth factor
IV Abbreviations
ELISA enzyme-linked immunosorbent assay
FACS fluorescence activated cell sorting
FCS fetal calf serum
FGF-1 fibroblast growth factor 1
FGF-2 fibroblast growth factor 2
FITC fluorescein-isothiocyanat
FP forward primer
FS follistatin
FSH follicle stimulating hormone
HB-EGF heparin-binding epithelial growth factor
HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid
HHT hereditary hemorrhagic telangiectasia
HIF hypoxia inducible factor
HGF hepatocyte growth factor
HPF high power field
HRP horseradish peroxidase
h hour
HUVE human umbilical vein endothelial
IGF-1 Insulin-like growth factor
IgG immunoglobulin G
IL interleukin
I-Smad inhibitory Smad
kb kilobase
kbp kilobasepair
kDa kilodalton
mg miligram
min minute
mM milimolar
M-MLV Moloney murine leukaemia virus
MMP matrix metalloproteinase
MOPS morpholinopropanesulfonic acid
MPI microvessel pericyte coverage index
V Abbreviations
mRNA messenger RNA
MVD microvessel density
MVEC microvascular endothelial cells
µg microgram
µL microlitre
nM nanomolar
PAI plasminogen activator inhibitors
PBS phosphate-buffered saline
PCR polymerase chain reaction
PDGF platelet-derived growth factor
PD-ECGF platelet-derived endothelial-cell growth factor
PFA paraformaldehyde
PI propidium Iodide
PLGF placenta growth factor
r recombinant
RIA radioimmunoassay
RNA ribonucleic acid
RNase ribonuclease
Rpm rotation per minute
RP reverse primer
rRNA ribosomal ribonucleic acid
R-Smad receptor