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AML1-eto collaborates with the homeobox gene meis 1 in inducing acute leukemia in the mouse bone marrow transplantation model [Elektronische Ressource] / submitted by Mahalakshmi Naidu, Vegi

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From the Department of Medicine III, Grosshadern Hospital and Helmholtz Zentrum München, Clinical Cooperative Group “Leukemia” Ludwig-Maximilians-University, Munich Chair: Prof Dr. Wolfgang Hiddemann AML1-ETO COLLABORATES WITH THE HOMEOBOX GENE MEIS1 IN INDUCING ACUTE LEUKEMIA IN THE MOUSE BONE MARROW TRANSPLANTATION MODEL Thesis Submitted for a Doctoral Degree in Human Biology at the Faculty of Medicine Ludwig-Maximilians-University, Munich,Germany Submitted by Mahalakshmi Naidu, Vegi From Anakapalli, India 2009 Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern und Helmholtz Zentrum München, Klinische Kooperations-Gruppe “Leukämie” der Ludwig-Maximilians-Universität München, Vorstand: Prof Dr. Wolfgang Hiddemann DIE GEMEINSAME EXPRESSION DES FUSIONSGENS AML1-ETO UND DES HOMEOBOX-KOFAKTORS MEIS1 INDUZIERT AKUTE LEUKAEMIEN IM MAUSTRANSPLANTATIONSMODELL Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians- Universität zu München, Deutschland Vorgelegt von Mahalakshmi Naidu, Vegi Aus Anakapalli, India 2009 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: PD Dr.med.Christian Buske Mitberichterstatter: Prof. Dr Ernst Schmid Priv.Doz.Dr.Tim M.

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Published 01 January 2009
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From the Department of Medicine III, Grosshadern Hospital and
Helmholtz Zentrum München, Clinical Cooperative Group “Leukemia”
Ludwig-Maximilians-University, Munich
Chair: Prof Dr. Wolfgang Hiddemann



AML1-ETO COLLABORATES WITH THE HOMEOBOX
GENE MEIS1 IN INDUCING ACUTE LEUKEMIA IN THE
MOUSE BONE MARROW TRANSPLANTATION MODEL



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


Submitted by
Mahalakshmi Naidu, Vegi

From
Anakapalli, India
2009






Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern
und Helmholtz Zentrum München, Klinische Kooperations-Gruppe “Leukämie”
der Ludwig-Maximilians-Universität München,
Vorstand: Prof Dr. Wolfgang Hiddemann


DIE GEMEINSAME EXPRESSION DES FUSIONSGENS
AML1-ETO UND DES HOMEOBOX-KOFAKTORS MEIS1
INDUZIERT AKUTE LEUKAEMIEN IM
MAUSTRANSPLANTATIONSMODELL




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


Vorgelegt von
Mahalakshmi Naidu, Vegi


Aus
Anakapalli, India
2009



Mit Genehmigung der Medizinischen Fakultät
der Universität München


Berichterstatter: PD Dr.med.Christian Buske


Mitberichterstatter: Prof. Dr Ernst Schmid
Priv.Doz.Dr.Tim M. Stromm
-------------------------------------


Mitbetreuung durch den
promovierten Mitarbeiter: --------------------------------------


Dekan: Prof. Dr. med. H.C.M. Reiser
FACR,FRCR


Tag der mündlichen Prüfung: 13.02.09
-------------------------------------






With permission from the Faculty of Medicine, University of Munich,
Germany


Supervisor/Examiner: PD Dr. med. Christian Buske
Second Examiner:


Co-Examiners: Prof. Dr Ernst Schmid
Priv.Doz.Dr.Tim M. Stromm


Co-Supervisor:


Dean: Prof. Dr. med. H.C.M. Reiser
FACR, FRCR



Date of Submission: 09.07.09

Date of Oral Examination: 13.02.09











Dedicated to my
Dear Parents……………………….






















CONTENTS
INTRODUCTION....................................................................................1
Normal and Malignant Hematopoiesis............................................................................... 1
AML................................................................................................................................... 2
The leukemia stem cell concept ......................................................................................... 2
Identification of LSCs ........................................................................................................ 4
Mechanisms of transformation in AML............................................................................. 5
Chromosomal translocations.............................................................................................. 6
The CBF family of transcription factors and AML1.......................................................... 7
ETO .................................................................................................................................... 8
AML1-ETO........................................................................................................................ 8
Two-hit model of leukemogenesis ................................................................................... 10
Leukemogenicity of AML1-ETO..................................................................................... 11
Hox genes and Hox co-factors ......................................................................................... 12
Myeloid Ectopic Integration Site 1 (Meis1)..................................................................... 13
MATERIALS ....................................................................................15
Chemicals and Reagents .................................................................................................... 15
Antibodies ........................................................................................................................... 16
Cytokines............................................................................................................................. 16
Enzymes............................................................................................................................... 16
Stock solutions and buffers................................................................................................ 17
Consumables ....................................................................................................................... 18
Oligonucleotides ................................................................................................................. 19
Primers for V-DJ and DJ recombination..................................................................... 20 H H
Plasmids............................................................................................................................... 20
Molecular weight markers................................................................................................. 20
Cell and tissue culture........................................................................................................ 21
Mammalian cell lines ....................................................................................................... 21
Media and supplements.................................................................................................... 21
CONTENTS
Instruments ......................................................................................................................... 22
Patient samples ................................................................................................................... 22
Mice .................................................................................................................................... 22
METHODS...........................................................................................23
Patient samples 23
Cloning of constructs ......................................................................................................... 23
Preparation of high titre stable virus producing cell lines ............................................. 24
Retroviral transduction of primary bone marrow.......................................................... 26
Bone marrow transplantation and assessment of mice................................................... 27
FACS analysis of murine cells........................................................................................... 31
Ex vivo proliferation and CFC assays .............................................................................. 32
Cytospin preparations and Wright-Giemsa staining...................................................... 32
RNA and genomic DNA isolation and cDNA preparation ............................................. 33
Southern blot analysis........................................................................................................ 33
Western blotting (Immunoblotting) ................................................................................. 34
Sample preparation and cell lysis (total cell extract) ....................................................... 34
Determination of protein concentration ........................................................................... 34
SDS PAGE of cell extracts............................................................................................... 35
Protein blotting................................................................................................................. 35
Protein detection on the blotting membrane with HRP-marked antibodies..................... 36
Polymerase chain reactions (PCRs).................................................................................. 36
Quantitative PCR.............................................................................................................. 36
PCR to check gene expression in mice ............................................................................ 37
PCR for V-D-J recombination status ............................................................................... 37
Identification of retroviral integration sites...................................................................... 38
Statistical analysis 38
RESULTS.............................................................................................39
Expression of Meis1 in AML1-ETO positive patient samples........................................ 39
Transplantation of mice with BM transduced with AML1-ETO (AE) and Meis1 ...... 40
Survival curves of transplanted mice ............................................................................... 41
Characterization of diseased mice .................................................................................... 41
WBC and RBC counts...................................................................................................... 41
CONTENTS
Pathology of transplanted mice........................................................................................ 42
Immunophenotyping of transplanted mice...................................................................... 46
Transplantation of secondary recipient mice .................................................................. 47
Southern blot analysis of the proviral integration pattern in leukemic cells from
AML1-ETO+ Meis1 mice .................................................................................................. 48
Frequency of clonogenic cells in leukemic mice - Colony Forming Units (CFU)......... 49
IgH D-J rearrangements can be detected in myeloid populations of cells with AML1-
ETO/Meis1 positive ALL................................................................................................... 50
+The B220 population has the highest frequency of leukemia propagating cells ......... 51
Identification of retroviral integration sites in diseased mice ........................................ 52
AML-ETO L148D mutant does not induce leukemia when co-expressed with Meis1 53
AML1-ETO ΔTAF/NHR1 is critical in inducing leukemia............................................ 54
Detection of spliced variants of AML1-ETO in diseased mice....................................... 55
DISCUSSION........................................................................................58
SUMMARY ..........................................................................................63
ZUSAMMENFASSUNG .........................................................................65
REFERENCES......................................................................................67
ACKNOWLEDGEMENTS.............................................................76

INTRODUCTION 1
INTRODUCTION
Normal and Malignant Hematopoiesis
The human body produces a large number of various kinds of cells during the course of a
lifetime. For instance, blood is composed of red blood cells, white blood cells and platelets.
Blood cell production is a continuous process, keeping the body metabolism constant during
stress or illness or trauma. This process of blood cell production and homeostasis is called
hematopoiesis (Figure 1). In humans, this process begins in the yolk sac in the first weeks of
rd thembryonic development. Between 3 and 7 month of gestation, stem cells migrate to the
fetal liver and then to the spleen where these two organs play a major role in hematopoiesis.
Later on, the bone marrow (BM) becomes the major hematopoietic organ and hematopoiesis
ceases in the liver and spleen. Malfunctioning of normal hematopoietic development can lead
to malignancies like myelo-proliferative disorders, leukemia, aplastic anaemia, lymphoma,
myelodysplasia, and inborn errors of metabolism (Weissman et al., 2001). Leukemia results
from the deregulation of the normal hematopoietic system due to the acquisition of mutations
in hematopoietic progenitors and is characterized by the accumulation of immature blasts that
fail to differentiate (Figure 2). Based on the natural course, leukemia can be subdivided into
acute and chronic leukemia. Evidence shows that many pathways that are deregulated in
cancer may also regulate normal stem cell development (Domen et al., 1998). Other signalling
pathways associated with oncogenesis, such as the Notch, Sonic hedgehog (Shh) and Wnt
signalling pathways are also involved in the regulation of normal hematopoietic cell self
renewal(Taipale and Beachy, 2001).

Acute leukemia is a heterogeneous disease that occurs due to genetic alterations like
translocations involving oncogenes and transcription factors, activation of signal transduction
pathways and alterations of growth factor receptors. However, many in vivo models have
postulated that the development of cancer is a stepwise process where somatic mutations give
rise to a transformed clonal population. One of the most characterized leukemia types is acute
myeloid leukemia (AML) which accounts for about 30% of all adult leukemias (Parkin, 2001;
Parkin et al., 2001a; Parkin et al., 2001b).


INTRODUCTION 2

Figure 1: Hematopoietic and progenitor cell lineages:
The hematopoietic hierarchy consists of the hematopoietic stem cells (HSC), the multipotent progenitors (MPPs)
and the more downstream progenitors, the common myeloid and the common lymphoid progenitor (CMP and
CLP) respectively. Collectively, these give rise to all the mature cells of the hematopoietic lineage (Passegue et
al., 2003).
AML
AML is characterized by the accumulation of large numbers of myeloid blasts arrested at
varying stages of differentiation. AML cell populations are quite heterogeneous and the
heterogeneity is due to the fact that malignant cells have divergent differentiation capacity and
that most probably different target cells are transformed in different patients. In addition, for
the leukemic clone to eventually become dominant, changes that confer a proliferative or self
renewal advantage must occur. A common feature to all AML cases is an arrest in
differentiation leading to an accumulation of more than 20% blast cells in the bone marrow
(Gilliland and Tallman, 2002).
The leukemia stem cell concept
Every functional specialized mature blood cell is derived from a common blood cell termed
the hematopoietic stem cell (HSC). In 1961, Till and McCulloch reported the existence of
HSCs for the first time as a population of clonogenic bone marrow cells capable of generating
myelo-erythroid colonies in spleen of lethally irradiated mice which could also be re-