Novel approaches of molecular targeting in Philadelphia chromosome positive leukemia [Elektronische Ressource] / by Afsar Ali Mian

Novel Approaches of Molecular Targeting in Philadelphia
Chromosome Positive Leukemia



Dissertation
to obtain the Degree of Doctor of Philosophy
at the Faculty of Natural Sciences



Submitted to the Faculty of Biochemistry, Chemistry and Pharmacy
of the Goethe University
in Frankfurt am Main



By
Afsar Ali Mian
from Swat, Pakistan

Frankfurt am Main, 2009


(D30)

Submitted to the Faculty of Biochemistry, Chemistry and Pharmacy of
the Goethe University in Frankfurt am Main



































Dean: Prof. Dr. Dieter Steinhilber
Examiners:
1. Examiner: Prof. Dr. Rolf Marschalek
2. Examiner: PD. Dr. Martin Ruthardt
Date:




Dedicated to my late father in law
Khurshid Khan (DPO Dir Lower)
who sacrificed his life for
the Nation
Table of Contents 4


1 INTRODUCTION ................................................................................................. 14
1.1 Normal hematopoiesis ............................... 14
1.2 Leukemia .................................................................................................................................................... 14
1.2.1 Acute leukemia .............................. 15
1.2.1.1 Acute lymphoid leukemia (ALL) ........................ 16
1.2.1.2 Acute myeloid leukemia (AML) .......................................................................................... 17
1.2.2 Chronic leukemia ................................ 18
1.2.2.1 Chronic lymphocytic leukemia (CLL) ................................................................................. 18
1.2.2.2 Chronic myelogenous leukemia (CML) .............. 18
1.3 Cytogenetic abnormalities involved in leukemia .................................................................................... 20
1.3.1 The Philadelphia-Chromosome associated translocation products ............... 20
BCR/ABL BCR/ABL1.3.2 Chromosome 22q+: p185 and p210 ........................................................................ 20
1.4 The Philadelphia chromosome ................................................. 22
1.4.1 Breakpoint cluster region (BCR) ................................................................... 22
1.4.2 Abelson murine leukemia virus homology gene (ABL) 23
1.4.3 Breakpoint regions and fusion protein tyrosine kinase . 24
1.4.4 BCR-ABL signaling ...................................................................................................................... 24
1.4.4.1 Stat signaling ....................... 25
1.4.4.2 Ras/MAPK........................... 26
1.4.4.3 PI-3K/Akt ............................................................................................................................ 26
1.5 Molecular therapy of Ph+ leukemia ......................................................................................................... 28
1.5.1 Abl-kinase-inhibitor Imatinib Mesylate ........................................................................................ 29
1.5.2 Resistance towards kinase inhibitors /Resistant BCR/ABL Mutations ......... 30
1.5.3 Mechanisms of Imatinib resistance ............................... 31
1.5.3.1 Imatinib-dependent mechanism ........................................................................................... 31
1.5.3.2 Non-Imatinib-dependent mechanism ................... 31
1.5.4 Strategies to overcome resistance towards kinase inhibitors ......................... 32
1.5.5 Targeting the tetramerization domain of BCR/ABL ..................................................................... 33
1.5.6 Targeting the Coiled coil (CC) enhances the effect of Imatinib and inhibits mutant BCR/ABL .. 34
1.5.7 Allosteric inhibition of BCR/ABL ................................ 34
1.6 Dissertation Hypothesis and Aims ........................................................................................................... 36
2 MATERIALS ....................................................................................................... 39
2.1 Instruments and apparatus....................................................... 39 Table of Contents 5

2.2 Chemicals ................................................................................................................................................... 41
2.3 Special reagents and materials ................. 43
2.3.1 Cell culture medium and reagents ................................................................................................. 43
2.3.2 Chemokines and cytokines ............ 43
2.3.3 Enzymes ........................................ 44
2.3.4 Polymerase Chain Reaction (PCR) ............................................................................................... 44
2.3.5 Antibodies ..................................... 44
2.3.5.1 Primary antibodies used for western blotting ...... 44
2.3.5.2 Secondary antibodies ........................................................................... 45
2.3.5.3 FACS antibodies .................................................. 45
2.3.6 Buffers ........................................... 45
2.3.7 Plasmids and vectors ..................................................................................... 49
2.3.8 Bacterial E.Coli Strain and genotype ............................................................ 50
2.3.9 Medium for bacterium ................... 50
2.3.10 Cell lines ....................................................................................................................................... 50
2.3.10.1 Ph+ cells .............................. 50
2.3.10.2 Other Cell lines .................................................................................................................... 51
2.3.11 Medium for Cell culture ................ 51
2.3.12 Materials for animal experiments .................................................................................................. 52
2.3.12.1 Mice ..................................... 52
2.4 Miscellaneous ............................................................................................................. 53
3 METHODS .......................................................................................................... 54
3.1 Preparation of plasmid DNA .................... 54
3.1.1 Transformation of E.coli ............................................................................................................... 54
3.1.2 Bacterium growth in liquid media . 54
3.1.2.1 Growing an overnight culture .............................................................................................. 54
3.1.2.2 Growing larger cultures ....................................... 54
3.1.3 Miniprep: a small scale preparation of plasmid DNA ... 54
3.1.4 Maxi prep: a large scale preparation of plasmid DNA .................................. 55
3.1.5 Determining of DNA yield and quality ......................................................... 55
3.1.6 Enzymatic Modification of Nucleotide Acids ............................................... 55
3.1.6.1 Restriction digestion of Plasmid DNA ................................................ 55
3.1.6.2 Dephosphorylation of Linear Plasmid-DNA by Alkaline Phosphatase CIP (Calf Intestinal
Phosphatase) ....................................................................................................................................... 55
3.1.6.3 Fill-in of 5’-Overhangs to form blunt ends by Klenow-Reaction ........ 55
3.1.6.4 Ligation of DNA Fragments 56
3.1.6.5 Quick change site-directed mutagenesis .............................................................................. 56
3.1.6.6 Recombination („gateway LR clonase enzyme kit“from Invitrogen) .................................. 56 Table of Contents 6

3.1.6.7 Cloning of Gateway Destination vector ............................................................................... 57
3.1.7 Electrophoretic separation of DNA ............................................................................................... 57
3.1.8 Cloning of the used Plasmids ........ 57
3.1.8.1 Cloning of Eukaryotic expression plasmids ........................................................................ 57
3.1.8.2 Cloning of prokaryotic expression plasmids ........ 59
3.2 Immunoblot ................................................................................................................................................ 60
3.2.1 Lysis of cells (Sambrook et al., 1989) ........................... 60
3.2.2 Determining of protein concentration ................................................................ 60
3.2.3 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) ................................ 60
3.2.4 Transfer of proteins onto a nitrocellulose membrane (Western blot) ............ 61
3.2.5 Immunodetection of specific proteins ........................................................................................... 61
3.3 Characterization of high molecular weight complexes (HPLC) .....................