The four and a half LIM domain protein 2 (FHL2) interacts with CALM and is highly expressed in acute myeloid leukemia (AML) with complex aberrant karyotypes [Elektronische Ressource] / submitted by Zlatana Pašalić

Zlatana Pasalic

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• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 42
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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







The four and a half LIM domain protein 2 (FHL2)
interacts with CALM and is highly expressed in acute
myeloid leukemia (AML) with complex aberrant
karyotypes





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


Submitted by
Zlatana Pašali ć

From
Skövde, Sweden
2008  
Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern
und GSF, Klinische Kooperations Gruppe‚’’Leukämie’’
der Ludwig-Maximilians-Universität München,
Direktor: Prof. Dr. med. Wolfgang Hiddemann











Das Four and a half LIM Domänen Protein 2 (FHL2)
interagiert mit CALM und ist hoch experimiert in
akuten myeloischen Leukämien (AML) mit komplex
aberranten Karyotypen.

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

Vorgelegt von
Zlatana Pašali ć

Aus
Skövde, Schweden
2008  






Mit Genehmigung der Medizinischen Fakultät der
Universität München






Berichterstatter: Prof. Dr. Stefan K. Bohlander

Mitberichterstatter: Priv. Doz.Dr.Susanne Schnittger
Prof. Dr. Ania C. Muntau

Mitbetreuung durch den
promovierten Mitarbeiter:

Dekan: Prof. Dr. med. Dr. h.c. M. Reiser, FACR

Tag der mündlichen Prüfung: 16.12.2008  







To my family  
Acknowledgments

I would like to thank,

Prof. Stefan Bohlander for giving me the opportunity to work in this completely new
field for me. I would like to express my deep gratitude for his support and
understanding that were essential to complete this work.

all the colleagues from the CCG leukemia, the AG Bohlander members Belay Tizazu,
Alexandre Krause, Deepak Bararia, Purvi Kakadia, Luciana Fontanari-Krause, Leticia
Fröhlich Archangelo, especially Philipp Greif and Medhanie Mulaw for helpful and
fruitful discussions as well as Marc Weinkauf from Dr. Dreylings group for the
hematopoietic cell lines

my colleague, my close friend and neighbour Pawandeep Kaur sharing many “tea-
evenings“ with discussions, laughs and “tears”

my close friends Zoofa Shahidi, Dr. Soledad Funes as well as my neighbours Selma
and Sead Granulo

my boyfriend Ensar Muratovi ć for always finding a way to give me new energy and
make me laugh

my sister Majida and my brother Senad that always find a way to encourage me

my parents Asima and Salih for the constant encouragement, the unconditional
support, their conviction and belief in me
Abbreviation

Abbreviations
aa amino acids
Ab antibody
AD activation domain
ALL acute lymphoblastic leukemia
AML acute myeloid leukemia
Amp ampicillin
AML-comp AML with complex aberrant karyotype
AML_nk AML with normal karyotype
ANTH domain AP180 N-terminal homology domain
AP-2 clathrin adaptor protein complex 2
APS ammonium persulfate
Aqua dest. water, deionized
BD binding domain
bp base pairs
BSA bovine serum albumine
cDNA complementary DNA, synthetic DNA
transcribed from a specific RNA through the
action of the reverse transcriptase
cfu colony forming unit
CME clathrin mediated endocytosis
CML chronic myelogenous leukemia
CoIP co-immunoprecipitation
Conc concentration
DAPI 4',6-diamidino-2-phenylindole
dCTP Deoxycytosine triphosphate
ddH2O double distilled water
DEPC diethylpyrocarbonate
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethylsulfoxide
dGTP Deoxyguanosine triphosphate
DMF Dimethylformamide
DNA deoxyribonucleic acid
dNTP Deoxynucleotide
DO drop out
DTT dithiothreitol
dTTP Deoxythymidine triphosphate
ECFP Enhanced Cyan Fluorescent Protein
EDTA ethylenediaminetetraacetic acid
e.g. example
EGFP Enhanced Green Fluorescent Protein
EST expressed sequence tags
EtBr Ethidium-bromide
EtOH ethanol
I
Abbreviation

EYFP Enhanced yellow fluorescent protein
FAB French-American-British classification system
for acute leukemia
FBS Fetal bovine serum
FCS Fetal calf serum
g gram
relative centrifugal acceleration g
GAL Galactose
GAL4 yeast transcription factor
GAL4-AD GAL4 transcriptional activation domain
GAL4-DBD GAL4 DNA binding domain
GAL4-UAS GAL4 upstream activating sequence
Gly Glycine
GFP green fluorescent protein
GST glutathione S transferase
HEPES N-(2-Hydroxythyl) piperazine-N’-2-ethan
sulphuric acid
His Histidine
HRP horse radish peroxidase
hr(s) hour(s)
HSC Hematopoietic stem cell
IPTG isopropyl β-D thiogalactoside
KAc Potassium acetate
kb kilobase
KCl Potassiumchloride
kDa kilodalton
KH hnRNP K homology domain
KH PO Potassium dihydrogenphosphate 2 4
l liter
LacZ E. coli gene encoding beta-galactosidase
LB Luria Bertani bacterial medium
Leu = L Leucine
LiAc lithium acetate
LIM Lin11, Isl-1 and Mec-3 domain
Lys = K Lycine
M Molar
-3m milli (1 x 10 )
MCS multiple cloning site
Met = M Methionine
MgCl Magnesium chloride 2
MgSOsulfate 4
min minute(s)
ml milliliter
MLL Myeloid/lymphoid or mixed-lineage leukemia
mM Milli Molar
II
Abbreviation

MOPS 3-N-morpholino-propanesulfonic acid
mRNA messenger RNA
MW Molecular weight
-9n Nano (1 x 10 )
NaAC sodium acetate
NaOH sodiumhydroxide
NaH PO sodiumdihydrogenphosphate 2 4
Na HPO disodium hydrogenphosphate 2 4
(NH ) SO ammoniumsulfate 4 2 4
NLS nuclear localization signal
nt nucleotide
O/N overnight
ºC degree Celsius
OD optical density
ORF open reading frame
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffer saline
PCR polymerase chain reaction
PEG polyethylenglycol
Pen/Strep Penicillin/Streptomycin
PFA paraformaldehyde
pg pico gram
PHD plant homeodomain
Phe = F Phenylalanine
PMSF Phenylmethylsulfonyl fluoride
PtdIns(4,5)P phosphatidylinositol-4,5-bisphosphate 2
RNA ribonucleic acid
RNAse ribonuclease
Rpm revolutions Per Minute
RPMI Roswell Park Memorial Institute culture
medium
RT room temperature
SD synthetic defined Dropout medium
SDS sodiumdodecylsulfate
sec second(s)
Taq Thermus aquaticus (-Polymerase)
TBS tris buffered saline
TE tris-EDTA buffer
TEMED N,N,N’,N’-Tetramethylethylendiamine
Thr = T Threonine
Tris trishydroxymethylaminomethane
Trp = W Tryptophan
tot. total
Tm melting temperature
Tyr = Y Tyrosine
III
Abbreviation

U unit
UTR untranslated region
UV ultraviolet
V volts
Val = V Valine
vector plasmid or phage chromosome used to carry
cloned DNA segment
vol. volume
v/v volume per volume ratio
w/v weight per volume ratio
WB Western blot
2x g gravity (9.81 m/s )
X-gal 5-bromo-4-chloro-3-indolyl- β-D-
galactopyranoside
Y2H yeast two hybrid
YFP yellow fluorescent protein
YPD yeast extract, peptone, dextrose
-6μ micro (1 x 10 )
μl micro liter
μm micro meter
μM micromolar
IV
Table of Contents

Table of contents
Abbreviations.......................................................................................................................I
Table of content ................................................................................................................. V
Table of figures.............................................................................................................. VIII
1 Introduction................................................................................................................. 1
1.1 Hematopoiesis ...................................................................................................... 1
1.1.1 Hematopoietic stem cell................................................................................ 1
1.2 Chromosomal translocation.................................................................................. 4
1.3 The CALM/AF10 fusion gene ............................................................................. 7
1.3.1 CALM, AF10 and the CALM/AF10 fusion ................................................. 7
2 Material and Methods ............................................................................................... 11
2.1 Material 11
2.1.1 Reagents 11
2.1.2 Material and Kits......................................................