Gene expression profiling of patients with polycythemia rubra vera [Elektronische Ressource] : generation of transgenic mice expressing the human PRV-1 gene / vorgelegt von Philipp Stefan Goerttler

Gene Expression Profiling of Patients With Polycythemia Rubra Vera Generation of Transgenic Mice Expressing the Human PRV-1 Gene Inauguraldissertation Zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-UniversitätFreiburg im Breisgau vorgelegt von Diplom-Biochemiker Philipp Stefan Goerttler aus Freiburg im Breisgau Die vorliegende Arbeit wurde im Zeitraum von Juni 2000 bis August 2004 in der Klinik für Tumorbiologie und dem Zentrum für Klinische Forschung in der Abteilung für Experimentelle Anaesthesie der Albert-Ludwigs-Universität Freiburg unter der Betreuung von Frau Prof. Dr. Heike L. Pahl durchgeführt. Vor der Fakultät für Biologie wurde die Arbeit durch Herrn Prof. Dr. Gunther Neuhaus vertreten. Promotionsvortrag / Promotionsgespräch fanden am 26.10.2004 / 13.12.2004 statt. Prüfer: Prof. Dr. Heike L. Pahl (direkte Betreuung der Arbeit) Prof. Dr. G. Neuhaus (Vertretung der Arbeit vor der Fakultät für Biologie) Prof. Dr. C. Peters Priv. Doz. Dr. G. Scherer Prof. Dr. K. F. Fischbach (Prüfungs-Vorsitzender) Ich bestätige hiermit an Eides statt, daß die vorgelegte Arbeit von mir alleine und nur unter Zuhilfenahme der angegebenen Hilfsmittel durchgeführt wurde. Philipp Goerttler Freiburg, den 14.12.2004 1. Introduction.......
Publié le : jeudi 1 janvier 2004
Lecture(s) : 24
Tags :
Source : FREIDOK.UB.UNI-FREIBURG.DE/FREIDOK/VOLLTEXTE/2004/1550/PDF/GENE_EXPRESSION_PROFILING_OF_PATIENTS_WITH_POLYCYTHAEMIA_RUBRA_VERA.PDF
Nombre de pages : 170
Voir plus Voir moins



Gene Expression Profiling
of Patients With
Polycythemia Rubra Vera

Generation of Transgenic
Mice Expressing the Human
PRV-1 Gene




Inauguraldissertation
Zur Erlangung der Doktorwürde der
Fakultät für Biologie der Albert-Ludwigs-Universität
Freiburg im Breisgau







vorgelegt von
Diplom-Biochemiker Philipp Stefan Goerttler
aus Freiburg im Breisgau









Die vorliegende Arbeit wurde im Zeitraum von Juni 2000 bis August 2004 in der
Klinik für Tumorbiologie und dem Zentrum für Klinische Forschung in der Abteilung
für Experimentelle Anaesthesie der Albert-Ludwigs-Universität Freiburg unter der
Betreuung von Frau Prof. Dr. Heike L. Pahl durchgeführt.
Vor der Fakultät für Biologie wurde die Arbeit durch Herrn Prof. Dr. Gunther Neuhaus
vertreten.

Promotionsvortrag / Promotionsgespräch fanden am 26.10.2004 / 13.12.2004 statt.

Prüfer: Prof. Dr. Heike L. Pahl (direkte Betreuung der Arbeit)
Prof. Dr. G. Neuhaus (Vertretung der Arbeit vor der Fakultät für Biologie)
Prof. Dr. C. Peters
Priv. Doz. Dr. G. Scherer
Prof. Dr. K. F. Fischbach (Prüfungs-Vorsitzender)



Ich bestätige hiermit an Eides statt, daß die vorgelegte Arbeit von mir alleine und nur
unter Zuhilfenahme der angegebenen Hilfsmittel durchgeführt wurde.







Philipp Goerttler Freiburg, den 14.12.2004
1. Introduction........................................................................................................... 5
1.1 Chronic Myeloproliferative Disorders................................................................................ 6
1.2 eloid Leukaemia................................................................................................ 7
1.3 Essential Thrombocythaemia ............................................................................................. 9
1.4 Idiopathic Myelofibrosis.................................................................................................... 10
1.5 Polycythaemia Rubra Vera (PV) ....................................................................................... 11
1.5.1 Disease Pattern 11
1.5.2 Diagnosis..................................................................................................................... 12
1.5.3 Therapy ....................................................................................................................... 14
1.5.4 Molecular Characterisation of PV................................................................................ 15
1.5.5 Clonality.......................................................................................................................16
1.5.6 Progenitor Cell Assays ................................................................................................ 17
1.5.7 Growth Factor Sensitivity ............................................................................................ 18
1.5.8 Erythropoietin Receptor (EPO-R) 18
1.5.9 SHP-1 Phosphatase.................................................................................................... 19
1.5.10 STAT Family of Transcription Factors......................................................................... 19
1.5.11 Thrombopoietin Receptor 20
1.5.12 Genomic Alterations in PV .......................................................................................... 21
1.5.13 Familial Polycythaemias.............................................................................................. 21
1.6 PRV-1................................................................................................................................... 22
1.6.1 Discovery and Characterisation of PRV-1................................................................... 22
1.6.2 Expression of PRV-1 mRNA in CMPDs ...................................................................... 23
1.6.3 Correlation Between PRV-1 Expression and EEC Growth ......................................... 23
1.6.4 Diagnostic Assay for the Determination of PRV-1 mRNA Levels ............................... 24
1.6.5 Expression of the PRV-1 Protein................................................................................. 24
1.6.6 A Murine Homologue of PRV-1................................................................................... 25
Aim of this work .............................................................................................................................. 26
2. Methods ............................................................................................................ 28
2.1 Whole Mount in-situ Hybridisation 28
2.1.1 Mating of Mice ............................................................................................................. 28
2.1.2 Killing of the Mice ........................................................................................................ 28
2.1.3 Preparation and Fixation of the Embryos.................................................................... 28
2.1.4 Dehydration ................................................................................................................. 28
2.1.5 Generation of DIG Labelled RNA Probes ................................................................... 28
2.1.6 In-Situ Hybridisation .................................................................................................... 29
2.2 DNA Preparation From Tail-biopsies of Mice.................................................................. 30
2.3 RNA Preparation ................................................................................................................ 31
1®2.3.1 RNA Isolation From Samples in TRIZOL ................................................................... 31
2.3.2 RNA Isolation From Granulocytes in GTC Solution .................................................... 31
2.3.3 Isolation of Total RNA From Mouse Embryos............................................................. 32
2.4 Quantification of Nucleic Acids........................................................................................ 32
2.5 Agarose Gel Electrophoresis............................................................................................ 32
2.5.1 RNA Electrophoresis ................................................................................................... 32
2.5.2 DNA Elecis 33
2.5.3 Cleanup of DNA Fragments From Agarose Gels........................................................ 33
2.6 Northern Blot ...................................................................................................................... 33
2.6.1 Transfer of RNA to Nylon Membranes ........................................................................ 33
2.6.2 Generation of Radio-Labelled Probes......................................................................... 34
2.6.3 Membrane Hybridisation and Autoradiography........................................................... 34
2.7 Reverse Transcription (RT)............................................................................................... 34
2.8 Polymerase Chain Reaction (PCR)................................................................................... 35
2.8.1 Standard PCR Protocol 35
2.8.2 Colony PCR................................................................................................................. 36
2.8.3 Semi-Quantitative RT-PCR ......................................................................................... 36
2.9 FACS Analysis of Mouse Whole Blood............................................................................ 37
2.9.1 Single Colour Analysis (PRV-1) .................................................................................. 37
2.9.2 Multi Colour Analysis................................................................................................... 37
2.10 Sequence Analysis of DNA ............................................................................................... 38
2.11 Ligations ............................................................................................................................. 39
2.11.1 Ligation of Restriction Fragments Into Plasmid Vectors ............................................. 39
TM2.11.2 Ligation of PCR Products Using TOPO TA Cloning ................................................ 39
2.12 Transformation of Competent E. coli Cells ..................................................................... 39
2.13 Preparation of Plasmid DNA ............................................................................................. 40
®2.14 TaqMan Quantitative RT-PCR ......................................................................................... 40
®2.14.1 Standard PRV-1 and GAPDH TaqMan Assay........................................................... 42
TM2.14.2 Assays-on-Demand .................................................................................................. 43
2.15 Isolation of Granulocytes From Blood Samples............................................................. 44
2.16 Restriction Digest of DNA .................................................................................................45
2.16.1 Analytical Digestions.45
2.16.2 Preparative Digestions 45
2.16.3 Digestion of PCR Products.......................................................................................... 45
2.17 cDNA Microarrays .............................................................................................................. 46
2.17.1 Production of cDNA Microarrays................................................................................. 46
22.17.2 Synthesis of Cy3- and Cy5-dUTP Labelled cDNAs .................................................... 46
2.17.3 Annealing of the Oligo dT Primers .............................................................................. 46
2.17.4 Incorporation of Cy3- and Cy5-dUTP During Reverse Transcription.......................... 47
2.17.5 cDNA Cleanup............................................................................................................. 47
2.17.6 Hybridisation................................................................................................................ 47
2.17.7 Washing Steps.. 48
2.17.8 Data Analysis..... 48
3. Materials ............................................................................................................ 50
3.1 Oligonucleotides ................................................................................................................50
3.2 General Equipment 51
3.3 Chemicals ........................................................................................................................... 52
3.4 Radio-Chemicals ................................................................................................................ 53
3.5 Enzymes.............................................................................................................................. 53
3.6 Kits ..................................................................................................................................... 54
3.7 Buffers and Solutions........................................................................................................ 54
3.7.1 General Buffers and Solutions .................................................................................... 54
3.7.2 Buffers for RNA Preparation........................................................................................ 54
3.7.3 Washing Solutions for Northern Blots ......................................................................... 54
3.7.4 Buffers for DNA Gel Electrophoresis........................................................................... 55
3.7.5 Buffers and Solutions for Western Blotting ................................................................. 55
3.7.6 Buffers, Antibodies and Solutions for In-Situ Hybridisations....................................... 55
3.7.7 DNA Preparation from Tail-biopsies............................................................................ 55
3.8 FACS-Antibodies................................................................................................................ 55
3.9 Other Biochemicals ........................................................................................................... 56
4. Results ............................................................................................................57
4.1 In-situ Hybridisation of Mouse Embryos ......................................................................... 57
4.2 Generation of PRV-1 Transgenic Mice............................................................................. 58
4.2.1 PRV-1/Vav Construct .................................................................................................. 59
4.2.2 PRV-1/H2K Construct 62
4.2.3 Generation of Transgenic Founder Lines.................................................................... 64
4.2.4 Single Colour FACS Analysis of Mouse Whole Blood ................................................ 67
4.2.5 Multi Colour FACS Analysis ........................................................................................ 68
4.2.6 Analysis of Haematological Parameters...................................................................... 72
4.3 cDNA Microarray Analysis of PV Patients....................................................................... 75
4.3.1 Methodological Background 76
4.3.2 PV Gene Expression Signature................................................................................... 82
34.3.3 Differential Gene Expression in PV Patients and Healthy Controls ............................ 88
4.3.4 Many Genes Overexpressed in PV are Regulated by SP1......................................... 92
4.3.5 Chromosomal Clustering of Up- and Downregulated Genes...................................... 94
4.3.6 Verification of the Microarray Results.......................................................................... 96
4.4 Molecular Characterisation of ET Patients.................................................................... 104
4.4.1 Patients...................................................................................................................... 105
4.4.2 Analysis of PRV-1 mRNA and c-Mpl Protein in 20 ET Patients................................ 105
4.4.3 Thrombotic Complications in ET Patients ................................................................. 107
4.4.4 Correlation Between PRV-1 mRNA, Clonality and EEC Growth in ET ..................... 109
4.4.5 Microarray Analysis of ET Patients ........................................................................... 112
4.5 Familial Accumulation of Elevated PRV-1 mRNA Levels............................................. 117
4.5.1 Elevated PRV-1 mRNA Levels in a Girl With Budd-Chiari Syndrome ...................... 117
5. Supplemental Data ............................................................................................119
5.1 Figures .............................................................................................................................. 119
5.2 Tables................................................................................................................................ 122
5.3 Functional Classification................................................................................................. 128
6. Summary ...........................................................................................................135
7. Discussion .........................................................................................................137
7.1 Transgenic Mice ............................................................................................................... 137
7.2 cDNA Microarray Analysis of PV Patients..................................................................... 141
7.3 Molecular Characterisation of ET Patients.................................................................... 149
8. References .........................................................................................................152
9. Abbreviations.....................................................................................................166
41. Introduction
All mature haematopoietic cells originate from a small number of pluripotent stem
cells in the bone marrow which give rise to the two basic lineages of the
haematopoietic compartment: myeloid and lymphoid cells. The resulting lymphoid
and myeloid stem cells have the potential to differentiate into all mature cell types of
these two compartments thereby loosing their multipotent abilities step by step.
Lymphoid stem cells can further differentiate into B- and T-cells whereas
thrombocytes, erythrocytes, granulocytes and monocytes display the progeny of the
myeloid stem cells. The transition between the different developmental stages is
regulated by the presence or absence of distinct haematopoietic growth factors.
The term „leukaemia“ covers a large number of neoplastic disorders of the
haematopoietic system. Classification is based on two major characteristics: First, the
developmental stage of the cells which are involved and second, the compartment of
the haematopoietic system which is affected by the diseases.
Acute leukaemias are characterised by the hyperproliferation of immature cells or
blasts. Abnormal, immature cells occur in bone marrow, peripheral blood and
frequently in the liver, spleen, lymph nodes, and other parenchymatous organs. If
untreated, most patients die within a time period of six months. Genetic dispositions
which can lead to acute leukaemias are described in the literature but exposition to
ionising radiation and mutagenic substances seem to be the major reasons for these
disorders.
In chronic leukaemia the proliferating cells appear mature. The clinical course is
relatively indolent, compared with acute leukaemia, and ranges from 2-6 years
depending on the subtype of the proliferating cell.
Further subdivision and classification is more straightforward in acute than in chronic
leukaemias. A first attempt was taken in 1976 when the French-American-British
cooperative group (FAB) proposed a classification system for acute myeloid and
lymphoid leukaemias (15). In 1985 the revised FAB criteria were published which
included seven (M1-M7) subtypes of acute myeloid leukaemias and 3 subtypes of
acute lymphoid leukaemias (16).
As the FAB classification is based primarily on morphological and cytochemical
features it does not always reflect the genetic or clinical diversity of the disease. In
2001 the World Health Organisation (86) proposed a new classification system.
5In these so-called WHO criteria also genetic, immunophenotypical, biological, and
clinical features were taken into account to define specific disease entities. The
categorisation includes many of the FAB criteria for classification of the myeloid
neoplasms. Additionally, guidelines of the Polycythaemia Vera Study Group (PVSG)
were included for classification of the Chronic Myeloproliferative Diseases (CMPDs).
Although the WHO criteria allow a more precise categorisation including prognostic
values, higher technical demands as compared to the FAB classification have
delayed its acceptance.

Figure 1.1: Classification of acute and chronic leukaemias. For acute myeloid and lymphoid
leukaemia the FAB criteria (15) are shown.
1.1 Chronic Myeloproliferative Disorders
As this work deals predominantly with the molecular characterisation of
polycythaemia rubra vera, major attention should be directed to the CMPDs. This
term was firstly used in 1951 by Dameshek to group four clinically and
pathophysiologically related disorders (44). As shown in Figure 1.1 this group
includes chronic myelogenous leukaemia (CML), polycythaemia rubra vera (PV),
idiopathic myelofibrosis (IMF) and essential thrombocythaemia (ET). Hallmark of the
CMPDs is the malignant clonal expansion of a multipotent stem cell. While all three
cell lines (myeloid, erythroid and megakaryocytic) are involved in each disorder, the
predominant cell lines allow a subclassification of the disorders. For instance, in PV
the proliferation is predominately erythroid, but granulocytes and megakaryocytes are
also part of the malignant clone.
6Transitions between the different members of this group are observed in some cases.
For example, patients diagnosed with ET can develop clinical features of PV in later
stages (73,169,177).
All CMPDs show a variable predisposition to
transform into acute leukaemias (about 90% of the
CML patients but only 5-10% of all PV patients
transform to an acute phase during later stages of
the disease).
The CMPDs display a group of phenotypically closely
related diseases. To date, diagnosis is mostly based
on exclusion criteria leading to a relatively high
number of misdiagnosed patients. With the exception of CML where the so-called
Philadelphia (Ph) chromosome is found in almost all cases, no molecular markers are
widely accepted for diagnosis of the other three CMPDs. Thus, a molecular
classification of the CMPDs could provide more insight into pathogenesis and allow
evaluation of appropriate treatment regimens for newly diagnosed patients.
1.2 Chronic Myeloid Leukaemia
CML is the most common CMPD and results in an increase of both, myeloid and
erythroid cells and a marked myeloid hyperplasia in the bone marrow. Mature
neutrophil granulocytes and their precursors are predominant in peripheral blood and
bone marrow. The median age at presentation is 53 years, but also children can be
affected. The most common abnormality is splenomegaly, present in up to half of the
patients. Progression of the disease, if untreated, normally includes a benign chronic
phase, followed by an accelerated phase and a rapidly developing blast crisis within
the first three to five years. After transition into blast crisis the cells appear more
undifferentiated and resemble the myeloblasts and lymphoblasts found in acute
leukaemias.
CML is the only CMPD whose molecular cause is known. In 1960 the so-called
Philadelphia (Ph) chromosome was firstly described as a shortened chromosome 22
(148) then as a t(9;22) (163). In about 95% of all CML patients this translocation can
be found. The remaining 5% have other translocations involving additional
chromosomes which can’t be detected by routine cytogenetic analysis. These
methods are more and more replaced by more convenient ones such as RT-PCR.
7The t(9;22) is not exclusive for CML as between 10 and 20% of adults and 2 to 5% of
children (164,178) with acute lymphoblastic leukaemia (ALL) and some cases of
acute myeloid leukaemia (AML) are also positive for the Philadelphia chromosome.
During progression of the disease and evolution of a blast crisis a range of secondary
chromosomal changes can occur, including duplication of the Ph chromosome and
mutations or deletions of tumor suppressor genes as p16 and p53 (6).
In all these cases, including those with more complex cytogenetic abnormalities, the
chromosomal translocation leads to the formation of two hybrid genes which involves
the BCR gene (breakpoint cluster region) on chromosome 22 and the ABL gene
(Ableson leukaemia virus) on chromosome 9. The resulting BCR-ABL fusion gene on
the Ph chromosome encodes a constitutively active tyrosine kinase (47) which is the
molecular cause of CML as mice transgenic for the BCR-ABL fusion gene show a
CML-like phenotype (42,43). The role of the reciprocal fusion gene ABL-BCR on
chromosome 9q+ remains unknown (134,135). The size of the resulting BCR-ABL
fusion gene varies between 185 and 230 kd which depends on the portion of the
BCR gene which is involved whereas the breakpoint region in the ABL gene is more
or less invariable. In recent studies correlations between size of the fusion gene and
progression of the disease have been found, suggesting that fusion proteins of
different size have different tyrosine kinase activities (124,199).
CML is the only member in the group of the CMPDs where a molecular cause is
known. Discovery of the BCR-ABL fusion gene has focussed some efforts on the
development of causative therapeutic agents. In different studies it has been shown
® ®that Imatinib (Gleevec , STI 571), a 2-phenylamino-pyrimidine derivate can inhibit
the tyrosine kinase activity of BCR-ABL by occupying its ATP binding site. In the
®treatment of CML in chronic phase Imatinib yields much better results than
interferon- α with about 70% of complete responses compared with 7% after
interferon treatment. After transition to the accelerated phase these responses are
sustained in a smaller percentage of the patients but still higher than under treatment
with interferon. In blast crisis the response rates are comparable to those under
chemotherapy with a smaller number of patients showing durable response.
®Combination therapies including both, chemotherapy and Imatinib treatment are
being explored (53).
8

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.