Effect of chromatin modeling by histone deacetylase inhibitors (HDIs) on hematopoietic stem cell (HSC) fate [Elektronische Ressource] / von D. Hilal Gül

Joachim Altschmied

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2006
• Nombre de lectures: 13
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Effect of Chromatin Modeling by Histone Deacetylase-
inhibitors (HDIs) on Hematopoietic Stem Cell (HSC) Fate
Dissertation
Zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main
von
D.Hilal Gül
aus Ankara, Türkei
Frankfurt am Main 2005vom Fachbereich Biochemie, Chemie und Pharmazie der Johann Wolfgang
Goethe-Universität als Dissertation angenommen
Dekan: Prof. Dr. Harald Schwalbe
Gutachter: Prof. Dr. Rolf Marschalek
Prof. Dr. Dieter Hoelzer
Datum der Disputation:
2Acknowledgments:
The present study was carried out in the Department of Hematology of the Johann Wolfgang-
Goethe University Hospital in Frankfurt.
It is a pleasure to thank many people who made this thesis possible.
I wish to express my gratitude to Prof. Dr. Rolf Marschalek for taking the part of
responsibility for this PhD study and being its referee as well as giving valuable advice and
constructive criticism.
I wish to thank Prof. Dr. Dieter Hoelzer for allowing me to do a Ph.D. thesis in his department
as well as for being external referee.
I would like to thank to PD Dr. Martin Ruthardt, my group leader, for giving me the
opportunity to work on a very exciting project and providing me the insight, advice, support,
critics and knowledge in order to perform and complete this thesis project.
I would also like to acknowledge PD Dr. Oliver Ottmann for his correctness, critics and
kindness.
I gladly wish to extend my gratitude to Dr. Joachim Altschmied for his valuable advice, his
kindness as well as his optimism, for which I extend my thanks.
I wish to acknowledge all colleagues from Dr. Ruthardts group as well as colleagues from
different laboratories for being extremely kind and friendly.
Lastly, I wish to express my gratitude to my father, mother, brother and sister-in- law for their
love and never-ending support. I would never complete my studies without them. I dedicate
this work for all of my family members. Thank you!
3Index 4
Index:
1 INTRODUCTION........................................................................................................ 9
1.1 Normal Hematopoiesis...........................9
1.1.1 Hematopoietic Stem Cells (HSCs).........................................................................................................9
1.1.2 Early Hematopoietic Progenitors..............11
1.1.3 Regulation of HSC Fate .......................................................................................................................12
1.1.3.1 Wnt-signalling ................................................................................................................................13
1.1.3.2 Homebox B4, hoxb4 gene ..............................................................................................................15
1.1.3.3 Notch1 Signalling ............................................................................................................................16
1.1.3.4 Sonic Hedgehog (Shh) Pathway......................................................................................................16
1.1.3.5 Stage Specific Cell Cycle Regulation in Early Hematopoiesis......................................................17
1.1.3.6 Chromatin Modeling in Early Hematopoiesis ...............................................................................18
1.2 Leukamia .......................................................................................................................................................22
1.2.1 Chronic Leukemias...............................................................................................................................22
1.2.1.1 Chronic Myeloid Leukemia (CML)................................................................................................22
1.2.1.2 Chronic Lymphoblastic Leukemia (CLL) ......................................................................................23
1.2.2 Acute Leukemias ..................................................................................................................................23
1.2.2.1 Acute Lymphoblastic Leukemia (ALL) .........................................................................................24
1.2.2.2 Acute Myeloid Leukemia (AML) ...................................................................................................24
1.3 Molecular Pathogenesis of AML ................................................................................................................26
1.3.1 Structural Themes.................................................................................................................................26
1.3.1.1 CBF Leukemias ..............................................................................................................................27
1.3.1.2 APL ..................................................................................................................................................29
1.3.2 Functional Themes.......................31
1.3.2.1 Effect on Differentation ..................................................................................................................31
1.3.2.2 Effect on Apoptosis .........................................................................................................................31
1.3.2.3 Effect on Self-renewal.....................................................................................................................32
1.4 Novel Therapeutic Approaches in AML....................................................................................................32
1.4.1 All-trans Retinoic Acid (t-RA).............................................................................................................33
1.4.2 Demethylating Agents (DNA Methylation Inhibitors) .......................................................................33
1.4.3 HDAC Inhibitors (HDIs)......................................................................................................................34
1.4.3.1 Valproic Acid (VPA)35
4Index 5
1.4.3.2 LAQ824 ...........................................................................................................................................36
1.5 Purpose of The Study ...................................................................................................................................36
2 MATERIALS ............................................................................................................. 38
2.1 Instruments....................................................................................................................................................38
2.2 Chemicals.......................................................................................................................................................39
2.3 Antibodies ..................................................................................................................40
2.4 Materials for Mouse Experiments..............................................................................................................41
2.4.1 Mouse Strain .........................................................................................................................................41
2.4.2 Other Materials .....................................................................................................................................41
2.5 Special Materials, Reagents and Cell lines for Cell Culture...................................................................42
2.6 Materials for Molecular Biology....................43
3 METHODS................................................................................................................. 44
3.1 Handling of Animals.....................................................................................................................................44
+
3.2 Enrichment of Human CD34 HSCs........................................................................................................44
3.3 Suspension Cultures of Human HSCs ......................................................................................................45
3.4 Flow Cytometry of Human HSCs..............................................................................................................45
3.5 Colony Assay of Human HSCs...................................................................................................................45
+ -
3.6 Enrichment of Murine Sca1 /lin HSCs...................................................................................................46
3.7 Colony Formation Unit (CFU) Assay, Replating Efficiency of Murine HSCs .....................................47
3.8 Day 12 Spleen Colony Formation Assay (CFU-S D12)............................................................................47
3.9 Competitive Repopulation Assay................................................................................................................48
3.10 Cell Cycle Analysis .......................................................................