The role of Cdx genes in normal and malignant hematopoiesis [Elektronische Ressource] / Silvia Thöne. Betreuer: Dirk Eick

Silvia Thöne

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

The Role of Cdx Genes in
Normal and Malignant Hematopoiesis

Silvia Thöne









Munich 2011 Clinical Cooperative Group Leukemia
Helmholtz Center Munich – German Research Center for Environmental Health
and
University Hospital Grosshadern, Department of Internal Medicine III
Ludwig-Maximilian-University, Munich



T HE R OL E OF C DX G ENES
IN NOR MAL A ND MA L IG NA NT HE MA T OP OIE S IS



Thesis Submitted for the Doctoral Degree in Natural Sciences
at the Faculty of Biology
Ludwig-Maximilian-University, Munich, Germany


By
Silvia Thöne


Munich, 24.01.2011Klinische Kooperationsgruppe “Leukämie”
Helmholtz Zentrum München – Deutsches Forschungszentrum für Umwelt und
Gesundheit
und
Klinikum der Universität München - Großhadern, Medizinische Klinik und
Poliklinik III
Ludwig-Maximilians-Universität, München


DIE R OL L E VON C DX G ENEN
IN DE R NOR MAL E N UND MA L IG NE N HÄ MA T OP OE S E



Dissertation eingereicht zum Erwerb des Doktorgrades der
Naturwissenschaften an der Fakultät für Biologie
Ludwig-Maximilians-Universität, München


Vorgelegt von
Silvia Thöne


München, 24.01.2011
















Date of Submission: 24.01.2011

First Examiner: Prof. Dr. Dirk Eick

Second Examiner: PD Dr. Josef Mautner

Date of oral examination: 19.05.2011
Declaration
I hereby declare to have written this thesis myself and without any sources
other than indicated herein. This work has not been submitted to any other
examining body in this or any other form.

Munich, 24.01.2011

……………………………………………
(Silvia Thöne)




The results of this study were partly published in the following publications:

Thoene S, Rawat VPS, Heilmeier B, Hoster E, Metzeler KH, Herold T,
Hiddemann W, Goekbuget N, Hoelzer D, Bohlander SK, Feuring-Buske M,
Buske C (2009) The homeobox gene CDX2 is aberrantly expressed and
associated with an inferior prognosis in patients with acute lymphoblastic
leukemia. Leukemia; 23(4):649-55.

Rawat VP, Thoene S, Naidu VM, Arseni N, Heilmeier B, Metzeler K,
Petropoulos K, Deshpande A, Quintanilla-Martinez L, Bohlander SK,
Spiekermann K, Hiddemann W, Feuring-Buske M, Buske C (2008)
Overexpression of CDX2 perturbs HOX gene expression in murine progenitors
depending on its N-terminal domain and is closely correlated with deregulated
HOX gene expression in human acute myeloid leukemia. Blood; 111(1):309-19. Table of Contents I
TABLE OF CONTENTS
TABLE OF CONTENTS ............................................................................ I
ABBREVIATIONS & NOMENCLATURE ...................... V
1. INTRODUCTION ............. 1
1.1 Hematopoiesis ............................................................................. 1
1.1.1 Blood Formation in the Embryo ...................... 1
1.1.2 Adult Hematopoiesis ...... 4
1.1.3 Hematopoietic Stem Cells .............................................................. 8
1.2 Leukemia .................................................... 11
1.2.1 Acute Myeloid Leukemia .............................. 11
1.2.2 Acute Erythroiemia ............................................................ 15
1.2.3 Acute Lymphoblastic Leukemia ................... 16
1.2.4 Cancer Stem Cells and the Leukemic Stem Cell Model ............... 17
1.3 Homeobox Genes ....................................................................... 21
1.3.1 Hox Genes ................... 22
1.3.2 Cdx Genes ................................................................ 23
2. AIM OF THE STUDY ..... 30
3. MATERIAL .................................................................................. 31
3.1 Reagents ..................... 31
3.2 Consumables ............................................................................. 32
3.3 Media and Supplements for Cell Culture . 33
3.4 Enzymes ..................................................................................... 33
3.5 FACS α-Mouse Antibodies ........................ 34
3.6 Kits .............................................................................................. 34
3.7 Kits, Material and Reagents for Microarray ............................. 35
3.8 Oligonucleotides ........................................................................ 35
3.9 Buffers and Stock Solutions ..................... 39
3.10 Technical Equipment ................................................................. 41
3.11 Bacteria Strains .......... 41 II Table of Contents
3.12 Cell Lines ................................................................................... 42
4. METHODS .................. 43
4.1 Molecular Biology Methods ...................................................... 43
4.1.1 RNA Extraction ............................................ 43
4.1.2 DNA Extraction 43
4.1.3 Quantification of RNA and DNA................... 43
4.1.4 Polymerase Chain Reaction (PCR) ............................................. 44
4.1.5 Agarose Gel Electrophoresis ....................... 51
4.1.6 Purification of PCR Products out of Agarose Gel ........................ 51
4.1.7 Cloning of DNA Fragments .......................................................... 51
4.1.8 Restriction Analysis ..................................... 52
4.1.9 Southern Blot ............................................... 52
4.1.10 Sequencing ................. 53
4.2 Bacteria ...................................................... 53
4.2.1 Preparation of Competent Cells .................. 53
4.2.2 Transformation of E.coli ............................... 54
4.2.3 Isolation of Plasmid DNA (Miniprep, Maxiprep) ........................... 54
4.3 Culture of Eukaryotic Cell Lines .............................................. 54
4.3.1 General Culture Conditions ......................... 54
4.3.2 Freezing and Thawing of Cells .................... 55
4.3.3 Determination of Cell Number and Vitality ................................... 55
4.3.4 Generation of Packaging Cell Line .............. 55
4.4 Mice and Murine Primary Cells ................................................ 56
4.4.1 Mouse Strains and Progenitor Enrichment by 5-FU Injection ...... 56
4.4.2 Collection of Murine BM .............................................................. 56
4.4.3 Cultivation of Murine BM ............................. 57
4.4.4 Retroviral Transduction ............................... 57
4.4.5 Establishment of a Retroviral Packaging Cell Line for Cdx4 ........ 58
4.4.6 shRNA-Mediated Knock Down .................................................... 59
4.4.7 Immunophenotyping by Fluorescence Activated Cell Sorting ...... 60
4.4.8 Proliferation Assay ....................................... 60
4.4.9 Colony Forming Cell Assay ......................... 60
4.4.10 Delta CFC Assay ......................................... 61
4.4.11 BM Transplantation of Mice ................................ 61
4.4.12 Retroviral BM Transplantation Model .......... 62
4.4.13 Peripheral Blood Analysis ............................ 62
4.4.14 Preparation and Staining of Cytospins ........ 62
4.4.15 Histopathology ............................................................................. 63
4.4.16 Analysis of Sacrificed Experimental Mice .... 63
4.5 Patient Samples ......... 63
4.6 Microarray Analyzes .................................................................. 64 Table of Contents III
4.6.1 Preparation of Microarrays ........................................................... 64
4.6.2 Evaluation of Microarray Data ...................... 64
4.7 Statistical Analyzes .................................... 65
5. RESULTS ................................................................................... 66
5.1 CDX2 in Human AML ................................. 66
5.1.1 CDX2 is the Only CDX Member Aberrantly Expressed in Patients
with Normal Karyotype AML ......................................................... 66
5.1.2 CDX2 Is Expressed in 64% of AML with Abnormal Karyotype ..... 67
5.1.3 Is Higher Expressed in AML with Normal Karyotype
Compared to Abnormal Karyotype ............... 68
5.1.4 High HOX Gene Expression Is Common in Patients with Normal
Karyotype ..................................................................................... 69
5.1.5 CDX2 Expression Levels Positively Correlate with Aberrant HOX
Gene Expression in AML ............................. 71
5.1.1 CDX2 Expression Does Not Correlate with Mutations of FLT3 .... 73
5.2 CDX2 Is Able to Deregulate Hox Gene Expression in Adult
Murine BM in vitro ...................................................................... 73
5.3 CDX2 in Human ALL .. 74
5.3.1 CDX2 Is Aberrantly Expressed in the Majority of Patients with ALL75
5.3.2 High CDX2 Expression Levels Are Associa