Characterisation of the leukemic stem cell in a murine model of CALM-AF10 positive myeloid leukemia [Elektronische Ressource] / submitted by Aniruddha Jayant Deshpande

Aniruddha Jayant Deshpande

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

To

Aai and Baba, need I say more ?



















Cover picture: Adaptation of the hematopoietic scheme, with kind permission from Prof.Larry Rohrschneider
(Fred Hutchinson Cancer Research Centre)


ISBN: 3-89963-397-0
From the Department of Medicine III, Grosshadern Hospital and
GSF, Clinical Cooperative Group ‘Leukemia’
Ludwig-Maximilians-University, Munich
Chair: Prof. Dr. med. Wolfgang Hiddemann







Characterisation of the leukemic stem cell in a murine
model of CALM/AF10 positive myeloid leukemia






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




Submitted by
Aniruddha Jayant Deshpande



From
CBD (Navi Mumbai), India

2006






Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern
und GSF, Klinische Kooperations Gruppe ‘Leukämie’
der Ludwig-Maximilians-Universität München,
Vorstand: Prof. Dr. med. Wolfgang Hiddemann







Charakterisierung der leukämischen Stammzelle in einem
murinen Modell der CALM/AF10 positiven myeloischen
Leukämie





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




Vorgelegt von
Aniruddha Jayant Deshpande



Aus
CBD (Navi Mumbai), Indien

2006







Published with Permission from the Faculty of Medicine
University of Munich



Supervisor/Examiner: Prof. Dr. med. Stefan Bohlander

nd 2 Co-Examiner: Prof.Dr.rer.nat. Peter B. Becker
Co-examiners Prof.Dr.phil. Judith P. Johnson
Priv.Doz. Dr.rer.nat. Johannes Herrmann


Co-Supervisor: PD. Dr. med. Christian Buske

Dean: Prof. Dr. med. Dietrich Reinhardt


Date of Oral Exam: 23.01.2006


Gedruckt mit Genehmigung der Medizinischen Fakultät
der Universität München



Berichterstatter: Prof. Dr. med. Stefan Bohlander

2. Berichterstatter: Prof.Dr.rer.nat. Peter B. Becker
Mitberichterstatter Prof.Dr.phil. Judith P. Johnson
Priv.Doz. Dr.rer.nat. Johannes Herrmann

Mitbetreuung durch den
promovierten Mitarbeiter:
PD. Dr. med. Christian Buske

Dekan: Prof. Dr. med. Dietrich Reinhardt


Tag der Mündlichen Prüfung:
23.01.2006 I n d e x

Introduction

1.1 Hematopoiesis 2

1.1.1 Normal hematopoiesis 2
1.1.1.1 General overview 2
1.1.1.2 The hematopoietic hierarchy 2

1.1.2 Leukemic hematopoiesis 3
1.1.2.1 Differentiation arrest 4
1.1.2.2 Increased proliferation 4
1.1.2.3 Inhibition of programmed cell death/apoptosis 5
1.1.2.4 Telomere maintenance 5
1.1.2.5Enhanced self-renewal 5

1.2 Stem cells and cancer 6

1.2.1 Stem cells and ‘stemness’ 6
1.2.1.1 Self-renewal 6
1.2.1.2 Multipotency 8
1.2.1.3 Quiescence 8

1.2.2 The cancer stem cell model 9

1.2.3 The need for identification of the leukemic stem cell 11

1.3 Translocations in leukemia 12

1.3.1 The t(10;11)(p13;q14) translocation in leukemia 13
1.3.2 CALM, AF10, and the CALM/AF10 fusion 14

1.4 Acute biphenotypic leukemias 17

1.4.1 ABLs in humans 17
1.4.2 ABL models in mice 17
1.4.3 Theories explaining biphenotypic character in ABL 18
1.4.3.1 Lineage infidelity 18
1.4.3.2 promiscuity 18

1.5 Mouse models of leukemia 19

1.6 Aim of the study 20
I n d e x

Materials

2.1 Mice and related reagents and equipment 21

2.2 Mammalian cell lines 22

2.3 Oligonucleotides 22

2.4 Plasmids 23

2.5 Antibodies 24

2.6 Reagents, media and apparatus 25

2.6.1 Molecular biology 26
2.6.2 Cell and tissue culture 27
2.6.3 Miscellaneous 28
2.6.4 Software 29

Methods

3.1 Cloning of constructs 30

3.2 Preparation of high titre stable virus producing cell lines 31

3.3 Retroviral transduction of primary bone marrow 32

3.4 Bone marrow transplantation and assessment of mice 33

3.5 FACS analysis of murine cells 35

3.6 Ex Vivo proliferation and CFC Assays 35

3.7 Cytospin preparations and Wright Giemsa staining 36

3.8 Colony-Forming Unit–Spleen (CFU-S) Assay 37

3.9 Quantification of leukemia propagating cell frequency 37

3.10 RNA and genomic DNA isolation and cDNA preparation 38

3.11 Southern and Western (immuno) blotting 38
I n d e x

3.11.1 Southern blot analysis 38
3.11.2 Western blotting (immunoblotting) 39

3.12 PCRs 42

3.12.1 PCR for transcriptional profile analysis 42
3.12.2 PCR for V-D-J recombination status 42


3.13 Statistical analysis 43

Result

4.1 Cloning and expression of CALM/AF10 44

4.2 CALM/AF10 expression enhances the short-term engraftment potential of bone
marrow progenitors 46

4.3 CALM/AF10 causes an aggressive acute leukemia in mice 47

4.4 Leukemic CALM/AF10 mice exhibit hyperleukocytosis and anemia 48

4.5 Spleens of leukemic CALM/AF10 mice are typically enlarged 49

4.6 Leukemic blasts infiltrate multiple organs of CALM/AF10 mice 50

4.7 Leukemic blasts in CALM/AF10 mice stain for myeloid markers 51

4.8 Cells from hematopoietic organs of leukemic CALM/AF10 mice are
predominantly myeloid in appearance with a high number of infiltrating blast
like cls 52

4.9 A subset of leukemic blasts from CALM/AF10 mouse bone marrow coexpress
lymphoid and myeloid markers 53

4.10 The CALM/AF10 induced leukemia is derived from several transformed clones
54


4.11 Identification of the leukemia propagating sub-fraction in the leukemic bone
marow poulation 55
I n d e x

4.12 B population cells have a higher proliferative potential at the single cell level
compared to the B/M and the M population 56

4.13 The frequency of leukemia propagating cell is the highest in the B population
as compared to the B/M population and the M population 57

4.14 Transformed CALM/AF10 B population blasts can differentiate into B/M and
functionally myeloid M population cells in vitro 58

4.15 Clonal D-J rearrangements can be detected in all sub-populations derived H
from B population cells 59

4.16 IgH D-J rearrangements can be detected in myeloid populations of cells from
leukemic CALM/AF10 mice 60

4.17 B population cells are Pax5 negative but express the early B cell factor gene
EBF 62

4.18 The B population cells express various early B lineage markers 63

4.19 The expression of CALM/AF10 in bone marrow progenitors enhances the
recovery of day 12 CFU-S 64

4.20 The leucine zipper-octapeptide motif domain is critical for the hematopoietic
activity of CALM/AF10 65

Discussion 66

Sumary 71

Zusammenfassung 72

Refrnces 73

Acknowledgements 86

Curriculum vitae 88

Publicatons 91 C h a p t e r I - I n t r o d u c t i o n





Leukemia is the neoplastic transformation of hematopoietic cells and is amongst the
most common forms of cancer in humans. Despite advances in the understanding of the
mechanisms involved and therapeutic approaches, most leukemia-affected individ