Thesis fullx 1 - FreiDok plus

Transcription

Biological effects of NF-E2
overexpression in
hematopoietic stem cells
Dissertation
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
Dr.rer.nat
der Biologischen Fakultät
Der Albert-Ludwigs-Universität Freiburg
vorgelegt im März 2012
von Ruzhica Bogeska
geboren in Prilep,
Mazedonien
1
Declaration
I herewith declare that I have prepared the present work without any unallowed help
from third parties and without the use of any aids beyond those given. All data and
concepts taken either directly or indirectly from other sources are so indicated along
with a notation of the source. In particular I have not made use of any paid
assistance from exchange or consulting services (doctoral degree advisors or other
persons). No one has received remuneration from me either directly or indirectly for
work which is related to the content of the present dissertation.
The work has not been submitted in this country or abroad to any other examination
board in this or similar form.
The provisions of the doctoral degree examination procedure of the faculty of Biology
of the University of Freiburg are known to me. In particular I am aware that before the
awarding of the final doctoral degree I am not entitled to use the title of Dr.
The work presented in this thesis was carried out in the Centre for Clinical Research,
Department of Experimental Anaesthesiology, University Clinic Freiburg from June
2008 to March 2012 under the supervision of Prof. Dr. Heike Pahl.
Dekan der Fakultät: Prof. Dr. Gunther Neuhaus
Promotionsvorsitzender: Prof. Dr. Samuel Rossel
Betreuer der Arbeit: Prof. Dr. Heike Pahl
Referent: Prof. Dr. Heike Pahl
Koreferent: Prof. Dr. Christoph Borner
3. Prüfer: Prof. Dr. Thomas Brabletz
Tag der mündlichen Prüfung: 04.05.2012
Ruzhica Bogeska
2
1. Index
1.
Index……………………………………………………………………………………… 1
2.
Introduction……………………………………………………………………….......... 4
2. 1 Myeloproliferative Neoplasms (MPN)……………………….……………………… 4
2.2 MPN clinical symptoms ……………………………………………………….…….... 5
2.3 Molecular lesions and mutations in MPN ………………………………………….. 6
2.3.1 JAK2 (Janus kinase 2)……………………………………………………………... 7
2.3.2 MPL (Myeloproliferative leukemia protein)………………………………………. 7
2.3.3 LNK (lymphocyte adaptor protein)…………………………………………………8
2.3.4 SOCS 1,2,3 (Suppressor of cytokine signaling)………………………………… 8
2.3.5 TET2 (ten eleven translocation)…………………………………………………... 8
2.3.6 EZH2 (Enhancer of zeste homolog 2)……………………………………………. 9
2.3.7 BCL-XL……………………………………………………………………………….. 9
2.3.8 PRV-1 (Polycythemia rubra vera protein -1)…………………………………….. 9
2.3.9 NF-E2 (nuclear factor – erythroid 2)……………………………………………... 9
2.3.9.1 NF-E2 gene structure and expression………………………………..... 10
2.3.9.2 NF-E2 protein structure, interactions
and posttranslational modifications……………………………………... 11
2.3.9.3 NF-E2 function…………………………………………………………… 15
2.4 Hematopoiesis…………………………………………………………………………… 16
2.4.1 The myeloid lineage………………………………………………………………… 20
2.4.1.1 Erythropoiesis……………………………………………………………...21
2.4.1.2 Megakaryopoiesis………………………………………………………… 24
2.4.1.3 Myelopoiesis………………………………………………………………. 25
2.4.2 Altered hematopoiesis in MPNs…………………………………………………... 28
2.4.2.1 Altered NF-E2 expression during hematopoiesis……………………... 29
2.5 Experimental setup……………………………………………………………………… 29
3.
Materials and Methods………………………………………………………………… 31
3.1 Isolation of primary human CD34+ cells…………………………………………….. 31
3.1.1 Peripheral blood processing……………………………………………………….. 31
3.1.2 Density gradient centrifugation and MNCs isolation…………………………….. 31
3.1.3 MNC freezing and thawing…………………………………………………………. 32
3.1.4 CD34+ labeling and purification…………………………………………………….33
3.2 Culture of primary human CD34+ cells……………………………………………… 35
3.2.1 Thawing of CD34+ cell……………………………………………………………… 35
3.2.2 Culture media and conditions……………………………………………………… 36
3.2.3 Cytokine reconstitution……………………………………………………………… 37
3
3.3 Culture of stable cell lines…………………………………………………………… 38
3.3.1 Adherent cell lines…………………………………………………………………… 39
3.4 Isolation of murine bone marrow cells………………………………………………. 39
3.5 Virus mediated gene transfer ………………………………………………………….40
3.5.1 Virus production……………………………………………………………………... 40
3.5.2 Virus titration…………………………………………………………………………. 44
3.5.3 Virus mediated gene transfer in human CD34+ cells ……………………………45
3.6 Fluorescence activated cell sorting (FACS) ……………………………………….. 47
3.7 Apoptosis assays………………………………………………………………………...48
3.7.1 Apoptosis in human CD34+ cells………………………………………………….. 48
3.7.2 Apoptosis in KSL (c-kit+, sca-1+, lin-) and KL (c-kit+, sca-1-, lin-)
murine bone marrow cells…………………………………………………………. 49
3.8 Proliferation assays……………………………………………………………………...52
3.8.1 AlamarBlue assay…………………………………………………………………… 52
3.8.2 Hoechst based proliferation/cell cycle assay…………………………………….. 53
3.8.3 ClickItEdu assay……………………………………………………………………...56
3.9 Differentiation assays…………………………………………………………………... 57
3.9.1 Human hematopoietic progenitor and hematopoietic stem cells assay……….. 57
3.9.2 Human terminal differentiation assays……………………………………………. 58
3.9.3 Human megakaryocytic ploidy assays……………………………………………. 59
3.9.4 Murine megakaryocytic ploidy assays…………………………………………….. 60
3.9.5 Cytospins……………………………………………………………………………...61
3.9.5.1 May-Grünwald Geimsa (MGG) staining………………………………………. 62
3.10 RNA processing…………………………………………………………………….. 62
3.10.1 RNA isolation……………………………………………………………………. 62
3.10.2 cDNA synthesis………………………………………………………………… 62
3.10.3 qRT-PCR ……………………………………………………………………….. 63
3.11 Protein processing…………………………………………………………………. 66
3.11.1 Protein isolation………………………………………………………………… 66
3.11.2 Protein quantification ………………………………………………………….. 67
3.11.3 Protein reduction and alkylation ……………………………………………… 67
3.11.4 SDS Polyacrylamide Gel Electophoresis (SDS-PAGE) …………………… 68
3.11.5 Western Blot…………………………………………………………………….. 69
3.11.6 Detection of Immunocomplexes………………………………………………. 70
3.11.7 Removal of immunocomplexes……………………………………………….. 71
3.12 Statistical analysis…………………………………………………………………. 71
4.
Results…………………………………………………………………………………… 72
4
4. 1 NF-E2 is overexpressed in CD34+ cells derived from PV
patients’ peripheral blood………………………………………………………………... 72
4.2 NF-E2 overexpression in CD34+ cells promotes HSC, MPP,
CMP and GMP expansion while decreasing MEP cell counts.…………………...... 73
4.3 NF-E2 overexpression elevates the number of CD13+CD36+
double positive cells – common myeloid- erythroid progenitors ……………………. 79
4.4 Effect of NF-E2 overexpression on terminal differentiation
along the myeloid lineage ………………………………………………………………. 81
4.5 Cytokine dependent effect of NF-E2 overexpression and silencing
on erythroid and megakaryocytic differentiation. …………………………………….. 83
4.6 Cytokine dependent NF-E2 expression………………………………………………... 90
4.7 Effect of NF-E2 overexpression on apoptosis and
proliferation in CD34+ cells……………………………………………………………… 91
4.8 NF-E2 overexpression in-vivo influences cell cycle in murine HSC
and progenitor cells, but not apoptosis………………………………………………… 93
5.
Discussion………………………………………………………………………………. 96
5.1 NF-E2 expression is 2 fold higher in CD34+ cells
derived from PV patient’s peripheral blood. …………………………………………... 96
5.2 NF-E2 overexpression in HC CD34+ cells promotes
HSC, MPP, CMP and GMP expansion. ……………………………………………….97
Epo-independent erythroid maturation. ……………………………………………….. 100
5.4 Epo-independent erythroid maturation most likely occurs through
another progenitor cell type independently of MEP…………………………………... 101
5.5 NF-E2 overexpression does not affect apoptosis and proliferation
in HC CD34+ cells, but most likely it influences proliferation and
cell cycle in murine HSC and HPC populations. ……………………………………... 103
5.6 Megakaryocytic differentiation is delayed in CD34+ cells
overexpressing NF-E2, as in PV CD34+ cells cultured in-vitro. ……………………. 105
5.7 EPO enhances NF-E2 expression, while TPO has an antagonistic effect. ………. 106
5.8 The endogenous overexpression of NF-E2 in PV CD34+ cells
is not dependent on EPO and TPO signaling cascades. …………………………… 108
6.
Appendix………………………………………………………………………………... 110
6.1 List of abbreviations………………………………………………………………………. 110
6.2 References………………………………………………………………………………… 113
5
2. Introduction
2. 1 Myeloproliferative Neoplasms (MPN)
Myeloproliferative Neoplasms (MPN) are hematopoietic disorders developing
due to the clonal expansion of a mutant hematopoietic stem cell
3
resulting in
overproduction of terminally differentiated cells belonging to the myeloid lineage.
According to a recent classification there are three different subtypes of MPN:
polycytemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis
(PMF) 4. Although the diagnostic hallmarks and symptoms differ between the three
subtypes, they share some common features 5: all three MPN develop due to the
clonal expansion of a mutant hematopoietic stem cell
3, 6, 7
. Furthermore they all
display common markers like the JAK2 kinase point mutation (JAK2V617F) and the
formation of erythropoietin (Epo) independent colonies (endogenous erythroid
colonies - EEC)
8-11
. The major clinical symptom of PV patients is the elevated red
blood cell mass; thrombocytosis is an ET hallmark, while PMF demonstrates
pancytopenia. Often the erythrocytosis in PV is accompanied with elevated white
blood cells and/or platelet counts 12.
Historically, the description of the different MPN subtypes started during the
late 19th century. In 1892 Louis Henry Vaques set the first diagnosis of PV
was followed by the description of a patient with PMF by Gustav Hueck
documented case of ET emerged in 1934
13
, which
14
. The first
15
. During 1951 William Damashek
proposed an interconnection between PV, ET, PMF, Chronic Myelogenous Leukemia
(CML) and erythroleukemia
separate entity
16
. Over the years erythroleukemia was defined as a
17
. After the discovery of the Philadelphia (Ph) chromosome in CML
patients during 1960
18
, the definition of the Ph chromosome as a product of
translocation between chromosome 9 and 22 in 1973
ABL fusion gene
19
and description of the BCR-
20
, the classification of MPNs was redefined. ET, PV and PMF are
now part of a separate group of Philadelphia (Ph) chromosome (BCR-ABL) negative
Myeloproliferative Disorders21.
6
Fig.2.1 Interconnection between the different MPN subtypes and their ability to progress to
another MPN subtype or to AML. The figure was adapted from Passamonti F et al. (2011) 22
Classifying PV, ET and PMF in one group emerged not only because all three
diseases share common clinical symptoms, but also because during follow up
patients show an increased risk of transforming to the other MPN subtypes with the
worse clinical outcome - transformation to acute myeloid leukemia (AML). Clinically
ET can progress to PV where ET patients carrying the JAK2V617F mutation show a
23, 24
higher risk for transforming into PV
. Both PV and ET patients can transform into
PMF (secondary myelofibrosis) 25, 26. All three MPN can progress to AML (Fig.2.1) 22.
Although all three MPN develop due to the clonal expansion of a mutant
hematopoietic stem cell
6, 7
a subset of MPN patients progressing to AML no longer
display the JAK2V617F mutation
27
. This finding suggests that there could be an
activating mutation preceding JAK2V617F.
2.2 MPN clinical symptoms
The classification and diagnosis of BCR-ABL negative MPNs has been revised
in 2008 by the World Health Organization (WHO). The diagnosis criteria are
summarized in Table 2.1
28
. Diagnosis of PV patients requires the determination of 2
major criteria plus one minor, or meeting 1 major criterion with 2 minor. ET diagnosis
is set when all 4 diagnostic criteria are present. In the case of PMF diagnosis
meeting all 3 major criteria and 2 of the minor is required.
7
Table 2.1 MPN diagnostic criteria established by WHO 2008. Reviewed in Tefferi et al. (2008) 28
2.3 Molecular lesions and mutations in MPN
During the last 5-6 decades a lot of research has been done in order to dissect
the molecular defects occurring in MPN. A very recent review
29
(2011) has
summarized most of the molecular lesions and mutations found in the chronic and
acute phase of MPNs up to date (Table 2.2). However disrupted expression of
several molecules like PRV-1, NF-E2 and BCL-XL are not being discussed there.
The following sections in this thesis introduction will shortly describe the main
factors being influenced during the chronic MPN phase with emphasis on NF-E2.
8
Table 2.2. Mutations and alterations in genes involved in MPNs pathogenesis. Reviewed in
Vainchenker et al. (2011) 29
2.3.1 JAK2 (Janus kinase 2)
JAK2 is a protein tyrosine kinase functioning in signal transduction through
type II cytokine receptor family. Members of this family include: EPOR; TPOR (MPL);
GM-CSFR; interferon receptors for IFN-α, IFN-β, IFN-γ; IL-3R; IL-5R and others.
Upon cytokine stimulation receptor bound JAK2 gets phosphorylated and
sequentially
transfers
RAS/RAF/MEK/ERK;
the
signal through
PI3K/AKT/mTOR;
various
signaling
cascades
like:
JAK2/STAT
thereby
regulating
the
transcription of genes involved in cell proliferation and cycling
30
. Recently a novel
function of JAK2 as a histone modifier has been reported 31.
During 2005 the presence of a JAK2V617F mutation in MPNs was described by
several groups
32-35
. The frequency of this mutation is more than 90% in PV, around
50% in ET and PMF
36
. Additional mutations of JAK2 in exon 12 have been detected
in less than 2% JAK2V617F negative MPNs
37
. Both JAK2V617F and exon 12 mutations
render JAK2 constitutively active, thus deregulating physiological cytokine signaling
37, 38
.
2.3.2 MPL (Myeloproliferative leukemia protein)
MPL is a transmembrane protein that functions as a receptor for
thrombopoietin (TPO). TPO signaling has a well established role on megakaryocyte
and platelet development, as well as HSC and early progenitors physiology
39
. In PV
patients, MPL was reported to be down regulated on megakaryocytes and platelets
9
and signaling through JAK2-STAT5 was not detectable
40
. Furthermore a W515L/K
mutation in MPL protein was detected in 3-5% of ET and 8-10% of PMF patients
41,
42
. This mutation constitutively activates the JAK-STAT pathway.
2.3.3 LNK (Lymphocyte adaptor protein)
The main role of LNK in hematopoietic cells is to inhibit cytokine signaling
through the EPOR, TPOR, c-KIT and FMS (Flt3) by negatively regulating JAK2
43-45
.
In ET and PMF two separate loss of function mutations within exon 2 have been
described, thereby resulting in unphysiological amplification of the cytokine signaling
46
. The frequency of these mutations in
pathways activated by phosphorylated JAK2
the chronic phase of MPN is low (detected only in 2 out of 33 investigated JAK2V617F
negative MPN patients), but the frequency of other LNK mutations in MPN patients
that have undergone leukemic transformation was reported to be around 13% 47.
2.3.4 SOCS 1,2,3 (Suppressor of cytokine signaling)
Apart from LNK, other loss of function mutations in proteins acting as negative
regulators of JAK2 signaling
48
have been detected, namely in SOCS 3
49
. However
the frequency of the single mutation described in a PV patient was not studied in
detail and most likely is not very prominent
2
and
3
transcriptional
regulation
49
. On the other hand defects in SOCS 1,
and
function
have
been
described.
Hypermethylated promoter regions resulting in lower transcription were found in ET
and PV patients
50-52
. The molecular function of SOCS in MPNs remains to be further
elucidated. A function of SOCS in proteasomal degradation of JAK2 was proposed
as well 53.
2.3.5 TET2 (Ten eleven translocation)
TET2 is a tumor suppressor protein that acts in nucleotide metabolism. It has a
methylcytosine dioxygenase enzymatic activity and catalyses the conversion of
methylcytosine to 5-hydroxymethylcytosine, believed to have an impact on epigenetic
regulation 54-56.
TET2 frameshift, nonsense, missense mutations and deletions have been
detected in all 3 MPN subtypes 57, 58. The frequency is 16% in PV, 5% in ET and 17%
in PMF 59.
10
2.3.6 EZH2 (Enhancer of zeste homolog 2)
EZH2 is an integral part of a larger protein complex the polycomb repressive
complex 2 (PRC2)
methyltransferase
60
, where it exerts its catalytic function as histone H3 lysine 27
61
. It has been shown to influence stem cell renewal by repressing
62
genes acting in induction of apoptosis and block of differentiation
mutations have been detected in 3% PV and 13% PMF patients
. Loss of function
63
.
2.3.7 BCL-XL
Bcl-x is a member of the evolutionary conserved bcl-2 gene family. Bcl-x is an
apoptosis regulatory gene that encodes two protein isoforms due to alternative
splicing: Bcl-XL and Bcl-Xs
promotes apoptosis
64
. Bcl-XL has an antiapoptotic function, while Bcl-Xs
64
. Exogenous expression of Bcl-XL in a cell line resulted with
Epo independent colony growth
65
, which is a MPN diagnostic marker. Moreover
overexpression of Bcl-XL mRNA and protein was detected in erythroid cells of PV
patients
66
. It is believed that Bcl-XL is upregulated upon EPO signaling during
erythroid differentiation 67.
2.3.8 PRV-1 (Polycythemia rubra vera protein -1)
The function of PRV-1 is not yet fully discovered. A role in epithelial adhesion
and transmigration processes was suggested. Platelet Endothelial Adhesion
Molecule (PECAM-1) was identified as its binding partner
68
. PRV-1 (CD177, NB1 or
HNA-2a) is a glycosyl-phosphatidyl-inositol (GPI)-anchored protein expressed on the
cell
surface
of
megakaryocytes
neutrophils,
neutrophilic
metamyelocytes,
69
myelocytes
, as in some epithelial tissues (normal and tumor derived)
and
70
. Its
expression is elevated during severe infection and inflammation, pregnancy and in
MPN patients.
The overexpression of PRV-1 in PV patients and other MPNs was detected in
Prof. Pahl’s lab and since then it has been routinely used as a diagnostic tool 9, 71.
2.3.9 NF-E2 (Nuclear factor – erythroid 2)
Recently, NF-E2 has been shown to play a role in MPN. Data obtained in Prof.
Pahl’s lab by microarray analysis, quantitative RT-PCR, Northern and Western
blotting revealed an elevated expression of NF-E2 in PV patients. The
11
overexpression of NF-E2 in PV was detected in granulocyte, erythroid and myeloid
precursors as well as in megakaryocytes by immunohistochemistry 72.
2.3.9.1 NF-E2 gene structure and expression
The DNA sequence encoding NF-E2 is located on human chromosome
12q13.13 and mouse chromosome 15F3. The gene for both human and mouse NFE2 is composed of three exons. There are two distinct isoforms detected: the adult αNF-E2 and the fetal - fNF-E2. The two isoforms were first distinguished on the
cDNA level when a cDNA library from human fetal liver cells was screened and
compared to the previously reported cDNA sequence of NF-E2 derived from adult
bone marrow cells
73
. Exon 2 and 3 of the two isoforms were identical, but the
difference was detected in the sequence for exon 1. There are two alternative first
exons of NF-E2 the 1a and 1f (1b) exon (Fig.2.2). The promoter sequence of each
exon was reported to differ, which suggests a tissue specific expression of each
isoform.
Indeed the 1f promoter contained binding sequences for transcriptional
factors like GATA-1, EKLF, SSP and YY-1 that are involved in erythroid
differentiation processes, while the 1a promoter contained TF binding sites
resembling ones found in housekeeping genes. Other studies have detected tissue
specific utilisation of the 1a and 1f promoters
74, 75
. In a mouse model the expression
of NF-E2 through the 1a promoter was detected in eyrthoid, myeloid cells and
megakaryocytes, while the 1f promoter was highly utilized in erythroid and
megakaryocytic cells, but not in myeloid cells
75
. Another study using human cord
blood CD34+ cells reported the preferential utilization of the 1f promoter during
erythroid differentiation and its abrogation during megakaryocytic differentiation
76
.
The first NF-E2 exon does not contain translation initiation site, but most likely has
regulatory elements coordinating translation. The NF-E2 transcript has three ATGs at
nucleotide position 112, 181 and 205 (Fig.2.3). The initial paper describing the ATGs
claims that by Kozaks sequence prediction only the ATGs located at nucleotide 181
and 205 can be used as translational start sites
77
. However, the ATG at position 112
is the first translational site generating a 373aa long protein. The alternative
translation site has never been studied in detail, but in any case the two alternative
translation initiation sites (ATG) located in exon 2, generate two protein variants with
a different size 77.
12
Fig. 2.2 Gene structure of mouse and human NF-E2. Boxes represent exons and connecting lines are
intronic regions. Grey areas in the boxes depict the translated NF-E2 sequence. Arrowheads indicate
a tandem GATA motif. The figure was adapted from Takayama et al. 75
NF-E2 is expressed mainly in hematopoietic precursors and erythrocytic,
granulocytic and megakaryocytic cells
in colon and testis
78
. In lower amounts NF-E2 is also expressed
77
. In the same publication NF-E2 transcripts were detected in
lung, placenta and liver, but the authors argue that this observation could be biased
because of blood infiltration in the samples. Previously NF-E2 was defined as a
lineage specific factor coordinating terminal differentiation, but there is growing
evidence that its expression starts from the HSC
79
and it plays an important role
during various stages of lineage commitment along the myeloid lineage
80-83
(Fig.5.4.1).
2.3.9.2 NF-E2 protein structure, interactions and posttranslational
modifications
NF-E2 is a 45kDa transcription factor composed of 373 AA residues. It
belongs to the bZIP (basic leucine zipper) family, shares homology with the
cap’n’collar subfamily of transcription factors
heterodimer with the small Maf
oncogene) proteins
77
and is active when it forms a
p18 (avian musculoaponeurotic fibrosarcoma
84
. While Mafs are ubiquitously expressed, NF-E2 expression is
tissue specific.
There are few well described functional domains of the NF-E2 protein. The
largest is the transactivation domain (aa1-206) that contains several functional
subdomains ensuring interactions with other proteins or cofactors like heme. Within
the proline rich domain (aa1-79) there are two CP (cysteine/proline sites) that bind to
heme 85. After the proline rich domain follows the PY (proline/tyrosine) domain (aa7913
83). Through the PY domain NF-E2 interacts with other proteins that contain a WW
86
. Some of the identified interactors are WW-1
(tryprtophan/tryptophan) domains
domain of ITCH (or NAPP1)
87, 88
, WW-2 domain of NEDD4 and WW-1 of YAP
Other proteins binding to the transactivation domain of NF-E2 are NAPP2
phosphorylated JNK
90
, CBP
91
and TAF II 130
86
.
89
,
92
.
Fig. 2.3 Nucleotide sequence and predicted amino acid sequence of NF-E2. The DNA binding domain
is in a box and the heptad repeats of leucine or hydrophobic residues comprising the leucine zipper
are in circles. The three potential translation initiation sites (ATGs) are underlined. The figure was
adapted from Chan et al.(1993) 77.
14
The proteins interacting with NF-E2 execute variety of functions. NEDD4 was
identified as an E3 ubiquitin ligase catalyzing the ubiquitination of PTEN
93
and RNA
Polymerase II (Pol II) thus leading to Pol II degradation and transcriptional arrest
94
.
Another function of NEDD4 is to mediate transcriptional activation guided by
progesterone and glucocorticoid signaling
95
. Up to my knowledge NF-E2 has not
been yet identified as a NEDD4 ubiquitination substrate. Another ubiquitin ligase
interacting with NF-E2 is the NEDD4 like ligase ITCH. ITCH catalyzes the addition of
a Lys63 ubiquitin chain to NF-E2, leading to translocation of NF-E2 to the cytoplasm
87
, that most likely is the mechanism by which ITCH acts as transcriptional
corepressor of NF-E2 88.
NAPP2 interaction with NF-E2 also results in transcriptional repression of NFE2 specific genes most likely by recruiting HDAC1 to the NF-E2 – NAPP2 complex
leading to histone deacetylation and repression of transcription
TAF
II
89
. Binding of YAP,
130 and CBP on the other hand leads to activation of NF-E2 target gene
transcription. The mechanism by which YAP assist NF-E2 target gene transcription is
possibly due to its interaction with RNA polymerase CTD (C-terminal domain) 86. TAF
II
130 is a subunit of the general transcription factor TFIID and its interaction with NF-
E2 was reported to be important in regulating globin gene expression in erythroid cell
lines
92
. CREB is another transcriptional factor that binds to TAF
II
130
96
. The
mechanism by which CREB activates transcription is dependent on the elevation of
cAMP concentration in the cell. Elevated amount of cAMP stimulate PKA in the
cytoplasm which leads to its translocation to the nucleus where it phosphorylates
CREB
97
thereby activating the transcription of genes containing cAMP-response
elements (CRE) sequence in their promoter. However, CREB mediated transcription
requires the assembly of a multiprotein complex in which NF-E2 may also be
involved. This assumption is based on a paper published in 1994 reporting that PKA
is necessary for increased NF-E2 – DNA complex formation during erythroid
differentiation of the MEL cell line 98. The phosphorylation of NF-E2 by PKA has been
detected
98, 99
, but this did not affect the NF-E2 – DNA complex formation suggesting
that PKA regulates the complex formation indirectly. Possibly the formation of a
multiprotein transcription complex containing PKA phosphorylated CREB, CBP, NFE2, general transcription factors (like TFIID or TFIIB) and Pol II augments cAMP
induced transcription. Moreover CBP has been identified as a binding partner of both
NF-E2 91 and PKA phosphorylated CREB 100.
15
Other important functional domains of NF-E2 are the DNA binding and beta
zipper domain. Within the DNA binding domain (aa268-287) lays the nucleus locating
sequence (NLS). Experimentally the NLS was reported to be from aa271 to aa273
101
. The bZip domain (aa287-297) functions mainly in dimerisation with the small
Mafs (MafK and MafG) which enables DNA binding to the specific MARE sequence
(TGCTGAC(G)TCAGCA) and transcriptional activation of the complex.
The function and activity, as localization and stability of the NF-E2 protein are
regulated by posttranslational modifications like:
•
Phosphorylation – NF-E2 contains 3 phosphorylation sites reported up to date.
Serine157 (S157) gets phosphorylated by pJNK in undifferentiated MEL cells
which leads to poly-ubiquitination on several lysine (K) residues and
subsequent proteasomal degradation of NF-E2
pJNK
did
not
influence
proteasomal
90
. Phosphorylation of S346 by
degradation
90
.
S169
gets
phosphorylated upon cAMP dependent protein kinase A (PKA-kinase)
activation, however the phosphorylation did not have an effect on DNA binding
or transcriptional ability 99.
•
Ubiquination – there are several ubiquitination sites detected within the NF-E2
protein: K108, K137, K245, K234, K241 and K368
90
. Besides poly-
ubiqitination of these lysine residues leading to proteasomal degradation, NFE2 gets modified by addition of a Lys63 chain by the E3 ubiquitine ligase
ITCH. ITCH dependent Lys63 ubiquitination led to translocation of NF-E2 from
the nucleus to the cytoplasm 87.
•
Sumoylation – only one sumoylation site has been detected up to date: the
K368 which is an ubiquitination site as well. The study investigating the
sumoylation of NF-E2 102 raises few questions. While the data presented in the
study is very clear on the indispensable role of K368 on the transactivation
activity and DNA binding ability of NF-E2 (K368R mutants could not bind DNA
and transactivate the expression of a reporter construct), the sumoylation of
NF-E2 in general and specifically at position K368 was more difficult to prove.
The proposed size of the sumoylated NF-E2 protein was not always consistent
and in general the presented Western Blots were not of best quality.
Immunofluorescence
experiments
within
the
same
paper
show
that
sumoylation of NF-E2 is necessary for its co-localization with SUMO-1 within
the POD type nuclear bodies 102.
16
A schematic representation of NF-E2 protein’s functional domains and groups as
posttranslational modification is represented on Fig. 2.4
Fig. 2.4 NF-E2 protein domains, post-translational modifications and protein-protein interactions.
CNC - Cap’n’collar domain
2.3.9.3 NF-E2 function
The known function of NF-E2 is to regulate the expression of genes acting in
erythropoiesis
103
, megakaryopoiesis and thrombopoiesis
iron and heme metabolism
104
, globin production
92, 105
,
106-109
. In a new publication (2010) a novel function of NF-
E2 was described. The authors report down regulation of cytoprotective genes and
accumulation of reactive oxygen species (ROS) in megakaryocytes, as a result of
competitive displacement of Nrf2 (NF-E2 related factor 2) from the regulatory DNA
110
sequences by NF-E2
been described
. The role of NF-E2 as an epigenetic modulator has also
111, 112
. Up to date a direct enzymatic function of NF-E2 as a histone
modifier has not yet been reported. But by recruiting histone methyltransferases like
MLL2
111
and G9a
113
to the β-globin enhancer HS2 region, NF-E2 is able to induce
activating histone modifications leading to initiation of transcription. Another important
feature in activation of transcription by NF-E2 is its ability to recruit RNA polymerase
II to promoter regions by cooperating with other transcriptional factors like USF
114
17
and GATA-1
115
. By staying attached to the highly condensed chromosomes at the
mouse globin gene cluster during mitosis
116
, NF-E2 acts in gene bookmarking
thereby maintaining cellular memory during erythroid lineage commitment. By
definition gene bookmarking is “a mechanism of epigenetic memory that functions to
transmit through mitosis the pattern of active genes and/or genes that can be
activated to daughter cells. It is thought that, at a point before mitosis, genes that
exist in an open, transcriptionally competent state are bound by proteins or marked
by some kind of modification event. This is thought to facilitate the assembly of
transcription complexes on the promoters in early G1, thereby ensuring that daughter
cells have the same pattern of gene expression as the cell from which they
derived.”117
One way to study the systematic effect of NF-E2 is by generating the NF-E2
knock-out mice (NF-E2-/-). This was done in the laboratory of Stuart Orkin during
1995. Previous research implicated that NF-E2 is indispensable for erythroid
differentiation. Surprisingly the NF-E2-/- mice displayed only mild defects in
erythropoiesis like: anemia with compensatory reticulocytosis and splenomegaly,
slightly decreased haemoglobin synthesis, dysmorphic erythrocytes118. However, the
major systematic defect in the NF-E2-/- mice was the inability to form platelets due to
a block in late stages of megakaryopoiesis leading to perinatal mortality due to
hemorrhage 119.
2.4 Hematopoiesis
The process of generating mature cellular elements in blood is called
hematopoiesis. The etymology of the word comes from ancient Greek “αἷµα“
meaning blood and “ποιεῖν“ – to make. However during the times when the term
emerged, no one could even assume that all blood components are created by a
single pluripotent hematopoietic stem cell (HSC). Only during 1945 the possibility
was acknowledged and experimentally proven in 1961120. In this pioneer study bone
marrow cells were irradiated and transplanted in sublethally irradiated host mice. Ten
days after transplantation the spleens or the recipient mice were investigated and the
formation of CFU (colony forming units) was detected. The amount of CFU correlated
one to one with the number of transplanted cells where 1 in 1x104 transplanted cells
could give rise to a single CFU. This finding and the fact that most of the bone
marrow cells died during increasing doses of radiation suggested that there is a small
18
subfraction of undifferentiated cells surviving higher doses of radiation that can give
rise to CFUs. Histological examination of the CFUs revealed that it is composed of
differentiated cells with mature blood cell phenotype. The major idea of this paper
was that each colony is derived from a single progenitor cell. Later on the clonality of
all the cells in the CFU was proven by several studies
121-123
. All these studies gave
evidence to the existence of hematopoietic stem cells which were phenotyped and
isolated from mice in 1988 124.
Fig. 2.5 The classical model of hematopoiesis and differential expression of specific surface markers
during different stages of mouse (left) and human (right) hematopoiesis. The definition of the
progenitor populations for mouse and human cells are different. This is not only because of the
different research strategies used for detecting surface marker expression, but also because of
differences in the expression of the molecules themselves. For example Flt3 during human
hematopoiesis is expressed on HSC, MPP and oligopotent progenitors 125, 126, while in murine
hematopiesis its expression is limited to MPPs. The expression of Slam markers like CD150 (Slamf1),
CD48 and CD144 on HSC is used for HSC phenotyping of mouse, but not human cells. Mouse cells
positive for CD150 and negative for CD48 and CD144 are defined as LT-HSC. The figure was adapted
from Chao et al. (2008) 127
Since then a lot of research was done on characterizing HSCs and other adult
tissue specific stem cells. But still the HSC and the generation of its progeny stays
the best studied system of stem cell development. The effort of many researchers led
to the creation of the most accepted “classical” model of hematopoiesis that follows a
19
linear scheme: Long term HSC differentiates to short term HSC and thereafter to
multipotent progenitor (MPP)
progenitors (CMPs)
82
128,
129
, which can generate common myeloid
and common lymphoid progenitors (CLP)
130
. The CMP then
commits to megakaryocyte-erythroid progenitors (MEPs) or granulocyte-monocyte
progenitors
(GMPs)
that
generate
granulocytic, monocytic cells
82
erythroid
and
megakaryocytic
and DC (dendritic cells)
131
cells,
or
respectively. The CLP
however has a potential to generate B, T, NK (natural killer) 130 and DC 131 committed
progenitors. Each committed progenitor can generate only specific cell types. Each
defined cell type has a distinct phenotype detected mostly by specific surface marker
expression. The “classical” model of hematopoiesis and the surface markers typically
expressed on each cell type in mouse and human hematopoiesis are depicted on
Fig. 2.5
132
.
However the “classical” model has been disputed recently, mostly by research
done on the lymphoid branch of hematopoiesis. Either a complete new model – the
myeloid model was proposed or the classical model was modified by including novel
progenitors that have a mixed potential 133, 134 (Fig. 2.6)
Fig. 2.6 Suggested models of hematopoiesis. a) Classical model b) Myeloid-based model c) Modified
classical model 83. In this scheme the megakaryocytic lineage for simplicity was combined with the
erythroid and both together are marked with E. Each letter represents the differentiation potential
the cell has where: M – myeloid, E – erythroid, T – T-cell, B – B-cell potential. Combination of letters
represents the mixed potential of a progenitor cell. CMEP stands for common myeloid-erythroid
progenitor where in this case CMEP is equivalent to CMP. CLP – common lymphoid progenitor, CMLP
- common myeloid-lymphoid progenitor, MTP –myeloid – T cell progenitor, MBP –myeloid – B cell
progenitor. The figure was adapted from Kawamoto and Katsura (2009) 134
20
HSCs in human adult hematopoiesis reside in the bone marrow surrounded by
other cell types of the bone marrow stroma like osteblasts and endothelial cells
comprising the stem cell niche
135, 136
. Within this niche the HSC remains dormant
until receiving a signal causing the HSC to go through a process of asymmetric cell
division, thus generating another HSC (self-renewal) and a more committed
progenitor cell
137
. Depending on the physiological requirements different cytokine
signals influence the fate of the progenitor cell and coordinate its differentiation
towards a certain hematopoietic lineage. During the process of lineage commitment,
the cell losses its ability to self-renew and its plasticity (pluripotency), while increasing
the rate of cell cycling 82, 128, 130, 138.
The expression of several lineage specific transcription factors (TF) was
detected in HSC and it was proposed that their activation and effect during/on fate
decision and differentiation is coordinated by combination of cytokine signals
139, 140
.
There is also a model suggesting that the expression of lineage specific TFs in HSC
has a role in suppressing the execution of a specific differentiation program, thus
keeping the HSCs in an undifferentiated state.
However, induction of a specific combination of TF and regulation of their
dosage upon cytokine stimulation or other exogenous and endogenous signals is
crucial for lineage commitment. Some TFs essential for lineage commitment are
listed in table 2.3. As previously mentioned MPN demonstrate trilineage hyperplasia
within the myeloid lineage. An overview of the major cytokines and TFs involved in
myeloid differentiation will be given in the upcoming sections.
Table 2.3 Transcriptional regulators of hematopoiesis. Orkin (2000) 141
21
2.4.1 The myeloid lineage
The most general division within the hematopoietic system is between the
lymphoid and the myeloid lineage. Initially, the separation between the two lineages
was based on anatomical grounds: myeloid were all cells present in the bone marrow
and lymphoid were cells originating from the lymphatic organs. With time this
classification was anatomically and functionally redefined. While terminally
differentiated cells of the lymphoid lineage are functioning mainly in immunity, the
myeloid lineage is more divergent which creates a discrepancy in the terminology
used. While both erythroid and megakaryocytic cells belong to the myeloid lineage,
frequently the terminally differentiated granulocytes and monocytes and their
progenitors are referred as myeloid. In summary the myeloid lineage includes all cells
derived from the CMP – erythroid, megakaryocytic and granulo/monocytic cells, but
the term myeloid cells includes only granulocytes, monocytes and macrophages. The
generation of granulocytes, monocytes and macrophages in this work will be termed
as myelopoiesis.
Within the myeloid lineage divergent lineage commitment is regulated by a
complex network of cytokine signals, cell to cell interactions, spatio-temporal
regulation of TF expression. Crucial for lineage commitment is the dosage of TFs
during a specific stage of hematopoiesis. For example both GATA-1 and PU-1 are
expressed in low amounts in the MPP
142
. During this stage of maturation the cells
get primed, meaning they could execute both the myeloid and the lymphoid program.
However, when PU-1 concentration increases lymphoid differentiation occurs while
the myeloid program is switched off
142
. On the other hand increase of GATA-1
concentration leads to myeloid commitment by the generation of CMPs from the
multipotent MPPs
142
. The CMPs expresses lower amounts of PU-1 than the MPP,
but PU-1 expression is abolished in the MEPs, which show high expression of GATA1. PU-1 up-regulation and GATA-1 downregulation in the CMPs leads to generation
of GMPs. What is crucial in this model of lineage commitment is the threshold point of
TF concentration. Once the threshold concentration is reached the cells lose their
multipotency/oligopotency and get specialized to differentiate only throughout one
lineage (Fig. 2.7) 2.
22
Fig.2.7 GATA-1 and PU-1 dependent priming and how their concentration determines lineage
commitment. See text for explanation. Figure adapted from Iwasaki et al (2007) 2.
2.4.1.1 Erythropoiesis
The
process
of
creating
mature
circulating
erythrocytes
is
called
erythropoiesis. The main function of erythrocytes is to deliver oxygen to all tissues in
the body. The production of erythrocytes is regulated by the amount of O2 in blood
143
. O2 sensors in the kidney cortex can detect the decrease in the partial pressure of
O2 which leads to the release of erythropoietin (EPO) in the blood stream
143
. EPO
than stimulates erythroid precursors in the bone marrow (BM) leading to elevated
erythrocyte production. However the expression of the EPO receptor starts only in
later erythroid precursors: from late BFU-E up to orthochromatic erythroblasts
144
.
The different stages of erythroid differentiation have been detected by two methods:
histological staining enabling morphological distinction of the precursors and the
colony forming ability in semi-solid medium. Later on the expression of different
precursor specific surface marker was described.
The most immature erythroid
precursor identified is the BFU-E (burst forming unit-erythroid) still having high
proliferative capacity and being independent of EPO signaling
144
. The expression of
the EPOR gets upregulated in the more mature CFU-E (colony forming unit-erythroid)
and later erythroid differentiation is highly dependent on EPO
103
. The CFU-E
generates proerythroblasts which are the first recognizable eyrthroid precursors in
the BM detected by histological staining. The proerythroblast sequentially
differentiates into few morphologically distinct cell types which are: basophilic,
polychromatic,
and
orthochromatic
erythroblasts.
During
differentiation
the
23
erythroblasts decrease their size. The orthochromatic erythroblast by extruding the
nucleus creates the retuculocyte, which can then leave the BM and form functional
circulating erythrocytes 145.
The expression of different surface markers gets differentially regulated during
erythropoiesis (Fig.2.8). Markers most commonly used in phenotyping human
erythroid cells are CD71 (transferrin receptor), CD36 (thrombospondin receptor),
CD235a (Glycophorin A). CD71 is expressed starting from the BFU-E up to
reticulocytes. CD36 expression starts at CFU-E and diminishes at the stage of
orthchromatic erythroblast 144. CD235a expression starts at proerythroblast stage and
gets gradually up regulated through erythroid maturation reaching maximal
expression on terminally differentiated erythrocytes 144.
Fig. 2.8 Schematic representation of human erythropoiesis indicating the surface expression of
specific molecules during differentiation. CD34, an adhesive protein that is expressed by primitive
haematopoietic progenitor cells; CD36, an adhesive protein expressed by erythroid progenitor cells;
24
CD41, a membrane protein expressed by early erythroid progenitor cells and megakaryocytic
progenitor cells, megakaryocytes and endothelial cells; CD71, the transferrin receptor; CXCR4, the
stromal-cell-derived factor-1α receptor; CXCR5, the macrophage inflammatory protein-1α receptor;
glycophorin A (GPA), a member of a family of erythrocyte-specific integral membrane proteins; and
KIT, the stem cell factor receptor. Several members of the tumour-necrosis factor (TNF) receptor
superfamily and of the TNF family of cytokines are also shown: TNF-related apoptosis-inducing ligand
(TRAIL), the membrane bound and soluble cytokine receptor ligand for TRAIL-R1 and TRAIL-R2;
TNFR1, the p55 TNF receptor; TNFR2, the p75 TNF receptor; TRAIL-R1, the TRAIL receptor DR4;
TRAIL-R2, the TRAIL receptor DR5; FAS, a membrane-bound and soluble death receptor; and FASL,
the membrane-bound and soluble ligand for FAS. The interferon-γ receptor (IFNγR) is also expressed
during the early stages of differentiation. CFU-e, colony-forming unit-erythroid; EPOR, erythropoietin
receptor. The figure and parts of the figure legend were adapted from Spivak (2005)144
These complex processes of erythroid differentiation are coordinated by a mix
of cytokines acting in an endocrine way (like EPO) or in a paracrine way within the
erythroblastic islands. The erythroblastic islands are comprised by a central
macrophage surrounded with developing erythroblasts
146
. In addition to EPO other
cytokines indispensable in erythroid differentiation are SCF, IGF-1, IL-3, IL-6. Many
of these cytokines activate downstream signaling cascades acing through JAK2.
Other signaling molecules recruited during erythropoiesis are depicted on Fig.2.9.
Fig. 2.9 Schematic representation of human erythropoiesis indicating the cytokines, TFs and
signaling molecules important during erythroid maturation. Deregulated expression or defective
function of a molecule in erythroleukemia is highlighted in red. The expression of hemoglobin
starting in the polychromatic erythroblast is also indicated. Wickema and Crispino (2007) 103
The execution of the erythroid program of differentiation is regulated by a
controlled equilibrium of TF expression and dosage. Some of the factors reported to
be important in determining erythroid commitment are PU-1, GATA-2, GATA-1,
TAL1, STATs, GFI-1B, EKLF, FOXO, NF-E2
147
. These TFs interact with each other,
25
up-regulate genes acting in erythroid cell function or down-regulate genes important
in other hematopoietic lineages.
2.4.1.2 Megakaryopoiesis
Eryhtropoiesis and megakaryopoiesis share common progenitors up to the
MEP where bifurcation to two distinct lineages occurs
148
. The first distinct
megakaryocytic progenitor is the BFU-Mk identified by the colony forming ability of
cells having megakaryocytic potential
149
. Later in differentiation the CFU-Mk forms,
which has an ability to develop to promegakaryoblast. The promegakryoblast can
undergo cell division thus generating more promegakaryoblasts or it can differentiate
to a megakaryoblast that executes a unique program typical for megakaryopoiesis
which includes the process of endomitosis. During endomitosis the amount of DNA
and the cell size duplicate, but cytokinesis does not occur. Thereby a mature
polyploid megakaryocyte is formed. The megakaryocyte can undergo several rounds
of endomitosis reaching up to 256N
150
number of chromosomes. The level of
polyploidy however is not related to megakaryocytic maturity
151
. Only proplatelet
forming megakaryocytes have reached the terminal stage of differentiation and can
produce platelets. Proplatelet formation can occur in megakaryocytes from all ploidy
classes 151.
Megakaryopoiesis occurs in a specialized niche within the bone marrow which
is spatially separated from the erythroblastic islands. This separation could partially
explain the diverse cell fate of the common MEP cells although both MEPs and more
mature erythroid and megakaryocytic cells show overlapping TF profiles. Some of the
TF that show overlapping expression are GATA-1, GATA-2, FOG-1, SCL/TAL,
STATs and NF-E2. The cytokines necessary for early erythroid and megakaryocytic
differentiation also show an overlap (like IL-6 and IL-3). However thrombopoietin
(TPO) is the major cytokine that leads megakaryocytic differentiation. TPO signaling
has a well established role on megakaryocyte and platelet development, as well as
HSC and early progenitors physiology
39
and can act in an endocrine or paracrine
fashion.
One mechanism explaining the diverse linage outcomes (lineage bifurcation)
on a very similar background is the antagonistic action of “lineage specific” TFs. An
example demonstrating how important the dosage and activity of TFs on a
megakaryocyte vs. erythroid bifurcation point is that of EKLF and FLI-1. Elevated
26
expression
of
EKLF
promotes
erythroid,
while
blocking
megakaryocytic
differentiation. The opposite is true for FLI-1; enhanced expression of FLI-1 promotes
megakaryocytic differentiation, while suppressing the erythroid differentiation
program 152, 153.
The process of megakaryopoiesis, the TF and cytokines/chemokines crucial in
for megakaryocytic differentiation and maturation are depicted on Fig 2.10
Fig.2.10 Transcription factors, cytokines and chemokines acting in different stages of
megakaryopoiesis. The middle scheme depicts the classical megakaryocytic differentiation. Violet
arrows on the left side describe TFs acting during different stages of megakaryopoiesis, while the
green arrows on the right side indicate the cytokines and chemokines important during different
stages of megakaryopoiesis. Dashed arrow lines indicate stages during which the expression of a TFs
or the action of a certain cytokine has not been detected. The figure was adapted from Pang et al.
(2005) 104.
2.4.1.3 Myelopoiesis
The formation of mature granulocytes (neutrophils, eosinophils, basophils),
mast cells, monocytes and macrophages starting in BM and completing in the blood
stream and peripheral tissues is referred as myelopoiesis. The myeloid cells function
mainly in immune response. Neutrophils act in host defense against microorganisms
154, 155
, eosinophils attack parasites
inflammatory response
156, 157
, while basophils act in infection and
158
. Monocytes/macrophages and granulocytes act in
immunity by antigen presentation
159
, cytokine production
160, 161
and phagocytosis of
microbes. All myeloid cells act in innate immunity by recognizing the complement
proteins and antigen presentation or recognition of immunoglobins.
27
Fig.2.10 Cytokine guided intermediate and
terminal stages of myelopoiesis.
The figure represents the separate
differentiation stages distinguished on the
bases of the cells ability to form
morphologically different colonies in semisolid media. The formation of the
intermediate CFUs and their differentiation
towards terminally differentiated cells is
guided by a combination of various cytokines
which are depicted for each progenitors
transition.
CFU-GEMM is a type of colony that contains
progenitor cells able to give rise to all lineages
of the hematopoietic system 1. Cells that have
mixed granulocyte-macrophage potential
form CFU-GMs. Progenitor cells committed to
form neutrophils generate CFU-Gs, while cells
with
potential
to
form
monocytes
/macrophages form GFU-Ms. According to
this scheme cells that have eosinophilic or
basophilic potential do not share a common
intermediate progenitor that can be identified
by the colony assays. Different types of assays
have enabled a more precise description of
common myeloid progenitors, emphasized
the complexity of myelopoiesis and made
possible to detect the ability of granulocytemacrophage
and
basophil-mast
cell
progenitors to shift lineage commitment (see
Fig.2.11 and text).
All myeloid cells are derived from the GMP. Sorted and plated in semi-solid
medium GMPs give rise to three distinct types of colony forming units: CFU-GM
(granulocyte-monocyte), CFU-G (granulocyte) and CFU-M (monocyte)
82, 162
. Several
intermediate stages of myeloid differentiation have been identified by histological
staining of BM where the promyelocyte is the most undifferentiated progenitor
identified by this method. The promyelocyte differentiates into committed basophilic,
neutrophilic or eosinophilic myelocytes and later on in metamyelocytes that can
mature into basophils, eosinophils or neutrophils having very distinct morphology.
Another way to detect the stage of the differentiation and maturation of myeloid cells
is by identifying a unique profile of surface markers for each progenitor and terminally
differentiated cell. For example CD13 is specific myeloid marker expressed on
28
myeloid progenitors, granulocytes and monocytes. While CD11b (Mac-1) is
expressed later in differentiation on granulocytes and monocytes.
Some of the cytokines important for coordinating myeloid maturation are:
granulocyte colony stimulating factor (G-CSF)
163, 164
, granulocyte-macrophage CSF
(GM-CSF), macrophage CSF (M-CSF) and IL-3
165
(Fig.2.10). The action of these
cytokines is to maintain survival, proliferation, maturation and functional activation in
their target cells. One of the many molecular events occurring upon cytokine
stimulation is the regulation of the expression and activity of TFs important for
myeloid lineage commitment 166.
Some of the TFs involved in myeloid lineage specification are: PU-1, GATA-1,
GATA-2, NF-κB, Gfi-1, C/EBP α, β and ε
167
. An example of how TF dosage and
regulation of TF expression at different time points affect lineage commitment in
myelopoiesis is that of C/EBP α and GATA-2. When the uncommitted CMP
upregulates C/EBPα it goes through an intermediate stage – the GMP to form the
neutrophile-monocyte progenitor (NMP). The NMP can that generate monocytes and
neutrophils. If during the GMP stage GATA-2 gets upregulated an eosinophilic
progenitor (EoP) is formed. Upregulation of GATA-2 in the CMP or in the GMP
accompanied with downregulation of C/EBPα leads to the generation of a common
basophil-mast cell progenitor (BMCP). If on this stage GATA-2 stays upregulated a
mast cell progenitor (MCP) is generated, but if C/EBPα expression gets higher than a
basophilic progenitor is formed (BaP) 2 (Fig. 2.11).
Fig. 2.11 TF regulated lineage diversification
during myelopoiesis. Uncommitted stands for
CMP where there are comparable amounts of
both C/EBPα and GATA-2. Upregulation of
C/EBPα leads to the formation of GMP. Further
upregulation of C/EBPα and diminishing of
GATA-2 expression results with the formation of
neutrophile-monocyte progenitor (NMP). The
NMP can that generate monocytes and
neutrophils. GATA-2 upregulation results in
eosinophilic progenitor (EoP) formation. On the
other hand increased GATA-2 concentrations in
the CMP or in the GMP accompanied with
downregulation of C/EBPα leads to the
generation of a common basophil-mast cell
progenitor (BMCP). If on this stage GATA-2 stays
upregulated a mast cell progenitor (MCP) is
generated, but if C/EBPα expression gets higher
than a basophilic progenitor is formed (BaP).
Iwasaki and Akashi (2007) 2
29
2.4.2 Altered hematopoiesis in MPNs
For several years now researchers are dissecting the hematopoietic system to
understand the defects occurring at different differentiation stages during MPN
development. Do the molecular defects acquired by the clonally expanding HSC
exert their effect at early or later stages of hematopoiesis? Is there a predominant
lesion and which one acts first? After the discovery of JAK2V617F much research was
done connecting this mutation to the MPN phenotype. In a study done by Jamieson
et al.168 focusing on JAK2V617F effect in PV, elevated counts of circulating
hematopoietic stem cells (HSC), common myeloid (CMP) and megakaryocytic –
erythroid progenitors (MEP) were described in peripheral blood (PB) from PV
patients. The transmission of the mutant JAK2V617F allele from HSC was detected in
all three progenitor populations (CMP, MEP and GMP), although the data
represented shows heterozygosity of JAK2V617F allele in HSC and progenitors.
Interestingly when these populations were sorted and grown in semi-solid medium for
colony formation, not all colonies carried the mutant allele. This could mean that
either the sorted PV HSC and progenitor populations had both JAK2V617F and JAK2wt
cells, or some of the cells heterozygous for JAK2V617F had lost the mutant allele
during the process of colony formation. In any case the amount of erythroid colonies
was higher in PV patients when compared to healthy controls (HC). Here two
questions arise: When JAK2V617F is absent which are the factors promoting an
increase of HSC and progenitor counts and could these factors cause the erythroid
bent observed in PV? Another study published in 2011
169
investigates the JAK2V617F
allele burden in hematopoietic progenitor compartments and terminally differentiated
erythroid and granulocytic cells. While in bone marrow derived cells from ET patients
there was no increase in JAK2V617F allele burden in all populations, in PV JAK2V617F
allele burden increased only in erythroid and granulocytic cells. In PMF the authors
observed an increase in all compartments. This data suggest that the expansion of
HSC and HPC compartments in MPN are most likely affected by other factors rather
than JAK2. Most likely JAK2V617F exerts its major effect during later stages of
differentiation.
30
2.4.2.1 Altered NF-E2 expression during hematopoiesis
Since NF-E2 is expressed starting from the HSC
79
up to the terminally
differentiated cells of the myeloid lineage its function must have an important role in
hematopoiesis. As discussed in the previous sections the concentration of TFs during
lineage commitment is crucial for fate decision and maintenance of HSC, HPC and
terminally differentiated hematopoietic cells. Being up-regulated in MPNs NF-E2 is a
worth candidate to be studied in elucidating the pathogenesis of MPNs. The major
questions addressed in this thesis are:
How the overexpression of NF-E2 in HSC and early progenitor cells affects
hematopoiesis and can it be connected to the pathognomic PV phenotype? Weather
its overexpression keeps the cells in a less differentiated state and/or promotes
terminal differentiation towards a certain lineage? Is NF-E2 overexpression itself
sufficient to alter hematopoiesis or are these changes cytokine dependent?
2.5 Experimental setup
Fig. 2.12 Experimental setup. The major outline of the experiments done in this thesis is
summarized in this scheme.
To study the effect of NF-E2 overexpression the experimental design
represented on figure 2.12 was used. CD34+ cells were isolated from peripheral
blood of PV patients and HC and put in liquid culture. The CD34+ population is a
heterogeneous mix of hematopoietic stem cells (HSC) and early hematopoietic
progenitors (HPC). Three major sets of experiments were done: lenti/retroviral
overexpression of NF-E2 in HC CD34+ cells, liquid culture of PV and HC CD34+ cells
and silencing of NF-E2 in PV by transduction with vectors containing shNF-E2 or
scrambled NF-E2 sequence as control.
31
Depending on the addressed question in each experiment the cells were
stimulated with different combinations of cytokines. The cells were cultured in
expansion medium when HSC and progenitor populations were investigated. This
medium keeps the cells in a less differentiated state longer and later on promotes
myeloid differentiation (Fig.4.4.3). The goal of this study was to observe the effects of
NF-E2 overexpression starting from very early stages of hematopoiesis (HSC and
HPC) up to the terminally differentiated progeny. Moreover PV affects the myeloid
linage. Therefore the expansion medium is very well suited for these experiments,
since the cells get transduced at the HSC and early progenitor stage and then
generate most of the terminally differentiated cells from the myeloid lineage
(erythroid, megakaryocytic cells, granulocytes and monocytes).
32
3. Materials and Methods
3.1 Isolation of primary human CD34+ cells
3.1.1 Peripheral blood processing
Reagents/Instruments
Company
Cat.No.
Dextran 500
Fluka
31392
PBS
Biochrom AG
L182-10
0,9% NaCl
VWR
278102-95
Neubauer chamber
Brand
717805
50ml Falcon tubes
Greiner
227261
*all centrifugation steps were performed at room temperature in Heraeus Megafuge
1.0 centrifuge
Phosphate buffered saline without Ca2+ and Mg2+, ready to
PBS:
use powder for final volume of 10L.
Contents: 130mM NaCl, 2.5mM KCl, 1.5 mM KH4PO4,
8mM Na2HPO2x2H2O
Dextran solution:
3%Dextran in 0.9% NaCl
Anticoagulant-treated peripheral blood (PB) or buffy coats were mixed at a 1:1
ratio with 3% Dextran in 0, 9% NaCl in order to sediment erythrocytes. The
supernatant was transferred to 50 ml Falcon tubes after 20 minutes of incubation at
room temperature (RT) and centrifuged for 10 minutes, 1600 rpm in a Heraeus
Megafuge 1.0. Two or three cell pellets were pooled together in 35ml PBS and
applied slowly (without intermixing) to the top of 15 ml Ficoll-Paque (density gradient
medium).
3.1.2 Density gradient centrifugation and MNCs isolation
Company
Cat.No.
Ficoll-PaqueTM Plus
GE Healthcare
17-1440-03
CliniMACS® PBS/EDTA buffer
Miltenyi Biotec
700-25
20% Immuno Human Serum Albumin Octapharma
10542a/96
PBS
Biochrom AG
L182-10
Neubauer chamber
Brand
717805
50ml Falcon tubes
Greiner
227261
1.0 centrifuge
33
The 50ml Falcons containing the Ficoll-Paque and PB cells in PBS were then
centrifuged for 45min, RT at 1800rpm without break to differentially separate the
blood cells. After centrifugation there are three distinct layers formed. The top layer
contains blood plasma and platelets, the interphase layer mononuclear cells (MNCs)
and under the Ficoll-Paque layer are the sedimented erythrocytes and granulocytes.
The CD34+ cells settle at the interphase between the top layer and the FicollPaque with the MNCs. To isolate the CD34+, cells the interphase was collected and
washed once with 50ml 0,5% HSA in PBS and twice with 50ml 0,5% HSA in
CliniMacs. The centrifugation was done at 1200rpm, 10 minutes, RT. Then the pellet
was resuspended in 10 ml 0,5% HSA in CliniMacs and the cells were counted. Either
the MNCs were frozen in 10%DMSO, 90% FCS or they were immediately used for
CD34+ isolation.
3.1.3 MNC freezing and thawing
DMSO
FCS (Fetal calf serum)
IMDM (Iscove's Modified Dulbecco's Medium)
DNase I
50ml Falcon tubes
15ml Falcon tubes
1,8 ml Cryo tubes
Freezing container
Neubauer chamber
Megafuge 1.0
Thawing medium:
Company
Sigma Aldrich
Gibco
Gibco
Roche
Greiner
Greiner
Nunc
Nalgene
Brand
Heraeus
Cat.No.
D2438
10270-106
21980
11 284 932 001
227261
188271
377267
5100-0001
717805
IMDM (with L-Glutamine, 25 mM HEPES, Phenol Red,
Sodium Pyruvate), 2%FCS and 0,25 mg/ml DNaseI
Freezing medium:
10%DMSO in FCS
34
Up to 1x108 MNCs were frozen in 1ml 10%DMSO in 90% FCS, in a freezing
container filled with isopropanol at -80°C overnight. The cooling rate of the unit is 1°C
per minute. When the MNCs were stored for a longer time period the freezing tubes
were transferred to liquid nitrogen.
The thawing was done at 37°C and the cells were immediately transferred to
Falcon tubes containing thawing medium with the following formulation: 2%FCS in
IMDM medium and DNaseI at final concentration of 0,25 mg/ml. For each vial of
MNCs 10-15ml of thawing medium was used. The cells were then counted with a
Neubauer Chamber. After counting the cells were centrifuged once at 1000rpm, 6
min, RT. The thawing medium was discarded and the MNCs were used in the
desired assay. An additional (second wash in a medium of buffer without DNaseI)
was avoided, because of clump formation.
3.1.4 CD34+ labeling and purification
CliniMACS® PBS/EDTA buffer
20% Immuno Human Serum Albumin
Direct CD34 Progenitor Cell Isolation
Kit, human
Company
Miltenyi Biotec
Octapharma
Cat.No.
700-25
10542a/96
Miltenyi Biotec
130-046-703
Magnetic cell separator
Macs column adaptor
Miltenyi Biotec
130-042-302
Miltenyi Biotec
130-041-407
Nylon membrane filter
with Midi
LS-column
Miltenyi Biotec
130-042-401
antiCD34 PE conjugated antibody
Pharmingen
34375X
FACS Flow
BD Biosciences
340398
PBS
Biochrom AG
L182-10
50ml Falcon tubes
Greiner
227261
15ml Falcon tubes
Greiner
188271
Neubauer chamber
Brand
717805
FACSCalibur
BD Biosciences
1.0 centrifuge
After isolation or thawing the MNCs were pelleted (1000rpm, 6 min.) and then
suspended in 300µl 0,5% HSA in CliniMacs per 1x108 cells. To label the CD34+cells,
100µL blocking reagent and 100µL CD34+microbeads (from Direct CD34 Progenitor
Cell Isolation Kit) were added per 1x108 cells. The cells were incubated for 30 min on
35
a low speed shaker at 4°C in a 15ml Falcon tube. 0,5% HSA in CliniMacs was added
to the tube to a total of 15mL and then centrifuged at 1000rpm for 10 min. The pellet
was usually suspended in 15 ml 0,5% HSA in CliniMacs and applied to a previously
equilibrated LS-column standing in a midi magnet column adaptor. The capacity of
the LS column is up to 1x108 magnetically labeled cells and 2×109 total cells with the
cell density not exceeding 2x108cells/mL. A previously moisturized filter was also
used to remove clumps and prevent clogging of the column.
After the cell
suspension has passed through the LS column by gravity flow, three rounds of
washing were done each with 3ml 0,5% HSA in CliniMacs. Then the LS column was
removed from the adaptor and the cells were eluted by pressing the plunger using
3mL 0,5% HSA. A second elution with 3mL 0,5% HSA in CliniMacs was additionally
done to make sure that all labeled cells are removed from the column. To increase
the purity of the isolated cells an additional LS column was used. Since usually four
buffy coats were purified simultaneously and pooled for performing experiments the
MNCs were mixed after the CD34+ magnetic labeling and the number of columns to
be used was determined by taking into consideration the capacity of the LS column.
Since the amount of labeled CD34+ cells derived from one buffy coat is not more
than 3X106, when pooling together four buffy coats only one additional column was
used and the cells were eluted twice with 5ml 0,5% HSA in CliniMacs.
Fig.3.1.4
Quality
of CD34+
purifications. The top two panels
represent
a
good
quality
purification, while the two bottom
panels depict a low quality CD34+
purification. The red gate in the
forward/sideward scatter plots
defines the CD34+ population. The
green and yellow gates for the bad
quality purification describe an
unspecific (CD34 negative cells) copurification. The specificity of the
purification was asses by staining
with PE labeled antibody against
CD34 cell surface marker.
36
To determine the purity of the isolation 100µl were taken out from the 10 mL
eluate, stained with 4µL antiCD34-PE antibody for 20 min at room temperature and
washed once with PBS. The cells were then FACSed (Fig.3.1.4). When the CD34+
cells were isolated directly on the same day from a buffy coat they were frozen in
10% DMSO in FCS. But when isolated from MNCs they were immediately put in
culture.
3.2 Culture of primary human CD34+ cells
Company
FCS
Gibco
IMDM (Iscove's Modified Dulbecco's
Medium)
Gibco
StemSpan Serum Free Expansion
Medium (SFEM)
Stem Cell Technologies
Penicillin/Streptomycin
Lonza
Primocin (50mg/ml)
Amaxa Biosystems
L-Glutamin (200mM)
Gibco
rhFlt3-Ligand
PeproTech
rhSCF
PeproTech
rhTPO
PeproTech
rhIL-6
PeproTech
rhIL-3
PeproTech
rh EPO (Erypo FS 2000)
Ortho Biotech
Human LDL
Harbor Bio-Products
96 well culture dishes
Greiner
Greiner
Greiner
15ml Falcon tubes
Greiner
Function Line Incubator
Hereaus
Cat.No.
10270 – 106
41966
9650
DE17-602E
VZA-1021
25030
300-19
300-07
300-18
200-06
200-03
AG55700
C8-D0160
655180
665180
662160
188271
3.2.1 Thawing of CD34+ cells
After thawing at 37°C, CD34+ cells from one vial (regardless the count) was
added to 10 ml 20%FCS in IMDM in a 15ml Falcon tube and centrifuged at 1100 rpm
(Heraeus Megafuge 1.0) for 6 minutes. The supernatant was discarded under the cell
culture hood using a suction pump. The pelleted cells were resuspended in an
adequate medium and cultured at standard culture conditions – 5%CO2, 37°C,
humidified atmosphere.
37
3.2.2 Culture media and conditions
The CD34+ cells after thawing or isolation from MNCs were plated in sterile
tissue culture plates. The plates used had different sizes, determined according to
the starting cell number. From 2x104 to 2x105 cells were plated per one well on a 96
well plate in 200µL medium. When the total cell number ranged from 2x105 to 2x106,
a 24 well plate was used and 500µL to 1ml medium per well was added to culture the
cells. More that 2x106 cells were cultured in a 12 well plate in 1 to 2 mL of medium.
Depending on the type of experiment, different types of media were used for culture.
The recipes for their preparation are listed below:
•
Expansion medium
component
final concentration
Medium (SFEM)
Primocin
L-Glutamin (200mM)
rhFlt3-Ligand
rhSCF
rhTPO
rhIL-6
• Modified expansion medium
1%
2 µL/mL (100µg/ml)
1% (2mM)
100 ng/mL
100 ng/mL
20 ng/mL
20 ng/mL
component
final concentration
Medium (SFEM)
1%
Primocin
L-Glutamin (200mM)
1% (2mM)
rhFlt3-Ligand
100 ng/mL
rhSCF
20 ng/mL
rhTPO
200 ng/mL
rhIL-6
20 ng/mL
• Thrombopoietin (TPO) response media
component
final concentration
Medium (SFEM)
1%
Primocin
L-Glutamin (200mM)
1% (2mM)
rhSCF
25 ng/mL
rhTPO
20 ng/mL, 50 ng/mL, or 200
ng/mL
38
•
Vainchenker (erythroid differentiation) medium
component
final concentration
Medium (SFEM)
1%
Primocin
L-Glutamin
1% (2mM)
rhIL-3
5 ng/mL (50IU/mL)
rhSCF
50 ng/mL
rhEPO
1 IU/mL
LDL
40 ng/mL
All media were kept at +4°C and used up within 3 weeks of preparation.
3.2.3 Cytokine reconstitution
Cytokine
Initial amount
Reconstitution Solvent for
Aliquot
concentration reconstruction size
rhSCF
rhTPO
rhIL-6
rhIL3
EPO
10µg
10µg
10µg
10µg
2000 U / 0.5ml
0,1 µg/µl
0,5µg/µl
0,1 µg/µl
0,5 µg/ µl
4 IU/µL
100 µl H2O
20µl H2O
100µl PBS
20µl H2O
10µl
2µl
2µl
1µl
0,5 mL
Prior to reconstitution, the tubes containing the lyophilized cytokines were
shortly spun down in a table top centrifuge and the appropriate volume of solvent
was added. The table above describes the most common cytokine reconstitution. The
recommended solvent sometimes varies from lot to lot, but is always indicated on the
lot specific data sheet. The cytokines were aliquoted in previously autoclaved PCR
tubes and stored at -20°C, except for EPO which was kept on +4°C. The
reconstituted cytokines are stable for at least 3 months.
39
3.3 Culture of stable cell lines
3.3.1 Adherent cell lines
Component
TrypLE Express
Serum Supreme
FCS
DMEM (Dulbecco's Modified Eagle Medium)
PBS
L-Glutamin (200mM)
10 cm culture plate
15 cm culture plate
75 cm2 culture flask
175 cm2 culture flask
Culture medium:
Company
Gibco
Biological industries
Gibco
Gibco
Biochrom AG
Lonza
Gibco
Greiner
CellStar
Greiner
Greiner
Greiner
Hereaus
Cat.No.
12604
04-004-1A
10270 - 106
41966
L182-10
DE17-602E
25030
664160
639160
662160
658975
661175
DMEM (with L-Glutamine, 4500 mg/L D-Glucose, 110
mg/L Sodium Pyruvate), 1% Pen/Str, 10%FCS, 1% LGlutamine
Production medium:
mg/L Sodium Pyruvate), 1% Pen/Str, 10%SerumSupreme,
1% L-Glutamine
Information regarding the cell line used like cell type, morphology, culture
media, optimal cell density etc. can be found at the web page of the “German
Collection of Microorganisms and Cell Cultures (DSMZ)”
(http://www.dsmz.de/human_and_animal_cell_lines/main.php?menu_id=2)
Three adherent cell lines were used in the work done in this thesis:
•
HEK293T
•
Phoenix gp
•
TE-671
The culture medium used for all three of them is composed of DMEM, 10%FCS,
1% Penicillin/Streptomycin and 1% L-Glutamine. When Phoenix gp cells were used
for retrovirus production the medium contained 10%Serum Supreme instead of FCS.
The cell density was maintained as recommended by DSMZ and the cells were split
40
when necessary. To detach the cells and get them in to a single cell suspension
trypsin (TrypLE Express) was added. The volume used was just enough to cover the
cell layer. For example 3 ml trypsin is enough to cover the surface of 175cm2 culture
flask. The culture plate or flask was placed in the cell culture incubator on 37°C, 5%
CO2 for about 2-3 minutes, until the cells detached from the surface. It is better to
keep the trypsinisation time as short as possible, since the cells are sensitive to it and
may lose their survival and proliferation ability. This is especially important for
Phoenix gp and 293T cells when producing virus. To inactivate the trypsin DMEM
culture medium was added, at a ratio trypsin:medium = 1:2. The cells were pipetted
up and down few times and split to get an appropriate splitting ratio or counted in a
Neubauer chamber. The required volume of cell suspension was plated and fresh
pre-warmed DMEM culture medium was supplemented to reach the desired cell
density.
3.4 Isolation of murine bone marrow cells
Component
Red Blood Cell Lysis Buffer
26G syringe 1mL
15ml Falcon tubes
Company
Sigma
BD
Gibco
Greiner
Cat.No.
R7757
305501
41966
188271
After sacrificing a mouse, one femur was dissected. The head and the lower
extremity of the femur were cut off with a scalpel to make the medullary channel
accessible. A syringe was filled with IMDM medium and when the medullary channel
was reached the underlying bone marrow cells were flushed into a 15mL Falcon tube
filled with 7mL IMDM medium. The flushing was repeated several times. After
centrifugation (5 min, 1200rpm, RT) the IMDM was discarded and erythrocyte lysis
was performed by adding 1mL lysis buffer for 30s, RT. By adding 9mL IMDM the lysis
buffer was deactivated. Afterwards the cells were spun down (1200rpm, 5min., RT),
resuspended in 1mL IMDM and counted.
41
3.5 Virus mediated gene transfer
3.5.1 Virus production
•
Retrovirus production
Component
TrypLE Express
DMEM culture medium
DMEM production medium
Phoenix gp cells
PBS
Profection Mammalian transfection system
CaCl2 (2M)
HEPES buffer (2X)
10 cm tissue culture dishes
Company
Gibco
see 3.3.1
see 3.3.1
Cat.No.
12604
Biochrom AG
Promega
Promega
Promega
Greiner
Hereaus
L182-10
E1200
E1200
E1200
664160
The packaging cell line Phoenix gp was used for retroviral production. This cell
line is based on 293T cells, only that it has been modified to express the retroviral
gag-pol genes. Gag-pol is a polyprotein with an acronym derived from Group
Antigens (gag) and polymerase (pol). Gag functions as nucleoprotein, capsid and
matrix protein. Pol has several enzymatic functions: protease, reverse transcriptase
and integrase activity. An envelope (env) protein encoding plasmid, as well as a
plasmid carrying the gene of interest, inserted into a retroviral backbone has to be
introduced to the producer cells in order to generate a fully functional virus (Fig 3.1).
The function of the envelope proteins is to recognize the surface of the host cell
making its infection feasible. For more detailed information about the principle of
retroviral
gene
delivery
system
visit
this
web
page:
http://www.stanford.edu/group/nolan/retroviral_systems/retsys.html
The retroviral backbone used in the experiments performed in this thesis was
pMYsIG (Fig. 3.2) in which the NF-E2 coding sequence was inserted in front of the
IRES-GFP sequence. The transcription of the NF-E2-IRES-GFP transcript was
controlled by the MPSV promoter.
3.2 General structure of pMYsIG. ψ-packaging signal; Δgag - truncated gag sequence, White box MMLV LTR; gray box - PCMV LTR; hatched box - myeloproliferative sarcoma virus (MPSV) promoter ,
LTR – Long Terminal Repeat, MCS - multi-cloning site. After the MCS come IRES (Internal Ribosomal
Entry Site) and a GFP (Green Fluorescent Protein) coding sequence.
42
Fig. 3.1 Retroviral structure and protein
distribution
The Phoenix gp cells were thawed and put in culture approximately one week
before starting the virus production. During this time the cells were regularly split to
maintain the optimal cell density and keep them in the log phase of growth. On the
first day of retrovirus production around 5X106 cells were seeded on a 10 cm tissue
culture dish in 10ml culture medium. The cells were transfected approximately 8
hours later using the Profection Mammalian transfection system kit, when the plate
reached about 80% confluency. Prior to transfection the medium was exchanged with
10ml prewarmed culture medium. The amounts or reagents used for one transfection
are listed in the table below.
Component
pMYsIG-NF-E2 or pMYsIG Empty vector
pGag/Pol (SVgp=SBG-MLV=SV40gagpol)
pEnv (human feline envelope)
CaCl2 (2M)
H2O
Amount
5µg
10µg
3µg
62,5µL
up to 500µL
The components were pipetted in to a 1,5mL eppendorf tube in the following
order water, plasmids, CaCl2 and vortexed briefly. This solution was added drop-wise
while vortexing to 500µL 2xHEPES. When CaPO4 crystals were formed, the
suspension became cloudy. After 15min of incubation at room temperature, the
CaPO4-DNA crystal suspension was vortexed shortly and added drop-wise to the
cells, while swirling the plate gently. The cells were then incubated for 14-16 hours at
37°C, 5% CO2, after which the medium was exchanged with 10mL culture medium. 8
to 10 hours later the culture medium was replaced by 7mL production medium (with
43
SerumSupreme). HEPES buffer was added drop-wise at a final concentration of
20mM. The first viral harvest was done 12 hours after. The production medium was
collected with a syringe, filtered through a 20µm filter unit and flush frozen in liquid
nitrogen. Up to 5 harvests were done every 12 hours. After the last harvest the
transfection efficiency was checked by FACS. (The recipes of the culture and the
production medium are described in Chapter 3.3.1)
•
Lentivirus production
Component
TrypLE Express
DMEM culture medium
PBS
CaCl2 (2.5M)
BES-sodium salt
Na2HPO4x2H2O
NaOH
15 cm tissue culture dishes
Stericup GV PVDF filter units
Millex gv 0.22µm PVDF filter
Polyalomere Ultracentrifugation Tubes
L90 ultracentrifuge
SW28 rotor
2x BES
50mM
Company
Gibco
see 3.3.1
Biochrom AG
Roth
Sigma-Aldrich
Roth
Roth
Greiner
Millipore
Millipore
Beckman Coulter
Hereaus
Beckmann
Beckmann
Cat.No.
12604
L182-10
5239.1
B2831-25G
4984.1
6771.1
664160
SCGV U01 RE
SLGV033RS
326823
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid),
280mM NaCl, 1.5mM Na2HPO4x2H2O, pH=6,96 by adding
NaOH. After pH adjustment the solution was sterile filtered.
2.5M CaCl2
prepared in sterile H2O and sterile filtered before use
For lenti virus packaging the HEK293T cell line was used. The cells were placed
in culture one week prior to transfection. The Lentiviral "gene ontology" (LeGO)
vectors were used for gene expression. The sequence encoding NF-E2 was cloned
3’ of the SFFV (spleen focus-forming virus) promoter, followed by an IRES site and a
GFP coding region. In this case the transcription of one transcript encoding NF-E2IRES-GFP is driven by a single promoter. The expression of NF-E2 correlated with
the amount of GFP expressed. In the experiments where NF-E2 was silenced a
LeGO-G vector containing a human U6 promoter but lacking the IRES site was used
(cloned by Roland Roelz)
170
. In this case the transcription of the shNF-E2 sequence
44
was driven by the hU6 promoter and GFP transcription was driven separately by the
SFFV promoter. Compared to the LeGO-IG vector the intensity of the GFP signal in
LeGO-G vectors was much higher.
LeGO-IG
LeGO-G
Fig.3.3 Structure of LeGO vectors. Self Inactivating Long Terminal Repeat (SIN- LTR), ψ-packaging
signal, Rev-responsive element (RRE), central polypurine tract (cPPT), woodchuck hepatitis virus
regulatory element (wPRE), multi-cloning site (MCS). The marker used GFP.
For transfection of HEK293T the CaPO4 method was used. On the day of
transfection a 100% confluent 15cm plate was split 1:2 in the morning. After 8-10h,
when the confluence of the plate was around 60-80%, the transfection was done.
The amounts used for one 15cm plate are:
Component
pLeGO
gag/pol (pCMV-dR8.74)
env (pMD2 vsvG)
H2O
CaCl2 (2.5M)
2xBES
Total volume
Amount
22.5µg
14.625µg
7.87µg
up to 420µl
105µl
420µl
840µl
First the plasmids and H2O were mixed, then CaCl2 was added and the mix
was applied drop-wise onto the 2x BES solution while vortexing. After 20 minutes of
incubation at room temperature, the CaPO4-DNA crystals were formed and the
solution was placed drop-wise onto the cells while swirling the plate gently. 12 to 16
hours later the medium was exchanged with 15ml prewarmed DMEM culture
medium. The first viral harvest was done approximately 24h later. One to two
additional harvests were pooled together with the first one and stored on +4°C. The
medium containing the virus was ultracentrifuged to pellet the viral particles. Prior to
ultracentrifugation the medium was filtered through a SteriCup filter unit or a PVDF
filter to avoid cross contamination with HEK293T cells. Around 35ml of virus
containing medium were transferred into ultracentrifugation tubes and centrifuged at
19500rpm, 2,5 hours, +4°C in a SW28 rotor. After centrifugation in a Beckmann xL90
ultracentrifuge, the supernatant was completely discarded and the virus was
45
reconstituted in 100-300µL SFEM medium or 0,1% BSA in IMDM. BSA stabilizes the
virus when frozen and thawed. After making 20-50µL aliquots the virus was frozen
and stored on -80°C. One smaller aliquot (5-10µL) was kept for virus titration.
3.5.2 Virus titration
HEK293T or TE671 cells
TrypLE Express
FCS
DMEM (Dulbecco's Modified Eagle Medium)
PBS
L-Glutamine (200mM)
FACS Calibur
Culture medium:
Company
Cat.No.
Gibco
Gibco
Gibco
Biochrom AG
Lonza
Gibco
Greiner
BD
12604
10270 - 106
41966
L182-10
DE17-602E
25030
662160
mg/L Sodium Pyruvate), 1% Pen/Str, 10%FCS, 1% LGlutamine
Determining the virus titer is important in order to have an approximation of the
amount of virus to be used in a certain experiment, thus controlling the amount of
infected cells and the amount of gene copies integrated into the genome of the host
cell. The virus titer actually describes how many viral particles there are in a defined
volume. This determination is largely dependent on the cell line used and the
protocol employed. For titrating retroviruses the TE671 cell line was used and for
lentivirus titration the HEK293T cell line. In both cases 5x104 cell per well were
seeded in a 24-well plate in a volume of 400µL per well. Approximately 8h after
seeding 2, 10, 20 and 50µL of virus containing medium was added to the cells. In the
case of lentivirus titration the virus medium was diluted 1 to 10 prior to adding it to the
cells. 24h after infection the culture medium was exchanged with 500µl fresh
prewarmed medium. After one day (day 4 after seeding and infection) the cells were
trypsinised (the trypsin was inactivated with PBS) and collected for FACS analysis
(FACS Calibur). The percentage of GFP positive cells was used to calculate the virus
titer by the following formula:
46
TV (IU/mL) = N x (%GFP / 100%) x (1mL / Vv)
TV – viral titer, IU/mL – Infectious units per mL, N – cell count at time of infection (5x104), %GFP –
v
positive cells, V – volume viral supernatant used for infection in mL
Using the %GFP value of the lowest volume viral supernatant used for
infection (that had a detectable GFP signal) gives the closes approximation of the
virus titer (Fig.3.5.2).
Fig. 3.5.2 Lentivirus titration. Different volumes (1 to 30µL) of 1 to 10 diluted virus containing
medium was applied to HEK293T cells to test the potency of the produced virus. The amount of
infected cells (GFP positive) was detected by FACS on day 4 after virus supplementation. The lowest
volume (1µL in this example) was used to calculate the viral titer.
3.5.3 Virus mediated gene transfer in human CD34+ cells
After CD34+ isolation or thawing the cells were cultured for one day prior to
viral transduction in the appropriate medium. Then, the cells were retrovirally or
lentivirally transduced.
Fig. 3.5.3 Timeline of retroviral and lentiviral gene transfer in CD34+ cells.
47
• Retroviral gene transfer
Component
FCS
non-tissue culture-treated sterile 6-well plates
Retronectin®
BSA (Bovine Serum Albumin)
PBS Dulbecco without Ca2+ / Mg2+
HBSS (+ CaCl2 + MgCl2)
HEPES
Reconstitution of retronectin
Company
Gibco
Gibco
Greiner
TaKaRA
Sigma
Biochrom
Gibco
Gibco
Cat.No.
41966
10270 - 106
657185
T100B
A-1595
L 182-10
14025-050
15630-056
the retronectin powder was dissolved at final
concentration 1mg/mL in H2O and stored at
-20°C
Coating solution
reconstituted retronectin diluted in PBS at a
final concentration of 50µg /mL
Blocking solution
2%BSA in PBS
Washing buffer
2,5% 1M HEPES in HBSS
One day after placing the CD34+ cells in culture, 2 to 4 wells of a 6-well plate
were covered with 2mL coating solution and incubated overnight at 4°C. The next
day, the coating solution was removed and the plates were blocked by applying
2mL blocking solution and incubating for 30min, RT. The blocking solution was
removed and the plates were washed with 3mL washing buffer. The plates
prepared for the second round of infection were covered with an additional 3mL of
washing buffer and stored at +4°C overnight. It was then proceeded to the first
viral infection cycle. 1mL virus containing medium was applied per well and
centrifuged for 20min, 840g (2000rpm Heraeus Megafuge 1.0), RT. This step was
repeated twice with two additional aliquots of virus. After each centrifugation the
virus containing medium was discarded in a separate glass flask by pipetting it out
of the wells. The flask was autoclaved to destroy any viral particles left in the
medium. After the last centrifugation step the supernatant was completely
aspirated and the CD34+ cells were put on top of the virus coating in 1.5mL
medium. The following day a second round of viral transduction was done the
same way as performed the previous day. On day 6 of culture the CD34+ cells
were FACS sorted. In the work done in this thesis the retroviral gene transfer
48
system was used only for NF-E2 overexpression, but not for shRNA silencing in
human CD34+ cells.
• Lentiviral gene transfer
Lentiviral transduction was used in NF-E2 overexpression and NF-E2 silencing
experiments in human CD34+ cells. The procedure for leniviral transduction is less
time consuming, acquires less material and in general is simpler than the retroviral
transduction. Before infection the CD34+ cells were counted and the amount of
viral particles to be used was calculated. A parameter defining the ratio of
infectious agents (virions) per cell is called Multiplicity of Infection (MOI). In these
experiments the MOI used was around 10 for each round of infection. The
calculation was done as described:
Vv=Nx10 / TV (IU/µL)
v
V
V – volume viral supernatant used for infection in µL, T – viral titer in IU/µL, IU/µL – Infectious
units per µL, N – cell count at time of infection, 10 is the MOI
The calculated Vv was added to the cells one day after putting them in culture.
The medium was mixed by pipetting up and down few times and the cells were
cultured for the next 24h at 37°C, 5% CO2, when the second transduction was
done. The GFP positive cells were sorted on day 5 or 6 of culture. Meanwhile the
medium was exchanged or supplemented when necessary.
3.6 Fluorescence activated cell sorting (FACS)
When the separation of virally transduced cells was necessary, the GFP
positive cells were sorted with MoFlo high speed cell sorter (Beckman Coulter). Prior
to sorting, the cells were collected in an 1,5 ml eppendorf tube and spun down
(13.000rpm, 15s, RT). After discarding the medium they were resuspended in a
minimum of 200µL PBS and stored on ice. The cell density was usually in the range
of 0,5 x106 to 6x106 cell/mL. The maximum density recommended for the MoFlo
sorter is 2,5x107 cell/mL. Obtaining higher cell densities is favorable, because the
rate of event acquisition is higher and the sorting time then is much shorter. To avoid
nozzle clogs while sorting, the cells were passed through a 40µm cell strainer
(Falcon, CatNo.2340). The sorted cells were collected into a 1,5 mL eppendorf tube
containing the appropriate medium. Again the cells were spun down (13.000rpm,
49
15s, RT), resuspended in suitable culture medium and put back in culture (37°C, 5%
CO2).
3.7 Apoptosis assays
Annexin-V is an early apoptotic marker that recognises the exposure of
phosphatidylserine (PS) on the outer leaflet of the plasma membrane, a process that
occurs during apoptosis
171
. However, this assay does not unequivocally distinguish
between apoptotic and necrotic cells, because due to membrane perforation,
annexin-V can enter necrotic cells and stain PS at the inner side of the plasma
membrane
171
. Therefore in these assays the nuclear dye SytoxOrange was used as
a necrotic marker, since it enters the cell only when the plasma membrane is
disrupted. The fluorescent signals of Annexin-V-AlexaFluor 647, SytoxOrange, GFP
or other surface markers were detected by FACS.
3.7.1 Apoptosis in human CD34+ cells
SYTOX Orange nucleic acid stain
Annexin-V-AlexaFluor647
Puromycin
Dimethyl sulfoxide (DMSO)
FACS Calibur
Company
Molecular Probes
BioLegend
Sigma Aldrich
Sigma Aldrich
BD
Cat.No.
S11368
640911
P8833
D2438
Sytox staining solution: the 5 mM stock solution was diluted 1:100 (to 50 µM) in
DMSO, aliquoted and stored at –20°C protected from light
until use.
Annexin V Binding Buffer (pH=7,4)
Final concentration
Molecular weight
10 mM Hepes
238.3012 g/mol
140 mM NaCl2
58.44277 g/mol
2,5 mM CaCl2
110.9 g/mol
H2O
pH was adjusted with NaOH
Amounts for 500ml
1.192 g
4.1 g
0.14 g
Fill up to 500ml
Cultured CD34+ cells were collected daily and the amount of apoptotic and
necrotic cells were determined by staining with Annexin-V- AlexaFluor647 and
SytoxOrange. Depending on how many cells were available in each experiment from
3000 to 10.000 cells were taken out of culture and washed once with 1,5 mL
Annexin-V-binding buffer. 100µL annexin-V-binding buffer were added together with
50
4µL Annexin-V-AlexaFluor647 and 3 µM (1,5µl SytoxOrange staining solution) Sytox
orange. The staining was done while incubating the cells for 20min., RT. The cells
were washed once with 1,5mL Annexin-V-binding buffer and resuspended in 200µL
Annexin-V-binding buffer. The cells were never fixed and fluorescent signals were
detected immediately after staining with the FACSCalibur. Apoptosis was detected in
NF-E2 overexpression experiments and PV or HC derived CD34+ cells cultured in
modified expansion medium.
Fig. 3.7.1 Gating strategy for detecting the amount of apoptotic and necrotic cells in virally
transduced CD34+cells. All acquired events in the forward /sideward scatter plot were plotted to
define the GFP+ (transduced cells). Optionally a gate distinguishing intact cells against cell particles
can be defined, but this does not influence much the later readout, since only intact cells will give a
GFP signal. Afterwards the percentage of viable (double negative), apoptotic (single positive for
AnnexinV) and necrotic (double positive) was detected by staining with SytoxOrange and AnnexinVAlexaFluor647.
3.7.2 Apoptosis in KSL (c-kit+, sca-1+, lin-) and KL (c-kit+, sca-1-,
lin-) murine bone marrow cells
SYTOX Orange nucleic acid stain
Annexin-V-AlexaFluor647
anti lineage cocktail –FITC conjugated
anti sca-1 PacificBlue
anti c-kit APC-eFluor780 conjugated
Dimethyl sulfoxide (DMSO)
FACS tubes
FACS Canto
Company
Molecular Probes
BioLegend
Biolegend
Biolegend
eBioScience
Sigma Aldrich
BD Falcon
BD
Cat.No.
S11368
640911
78022
108120
471171-82
D2438
352052
Sytox staining solution: the 5 mM stock solution was diluted 1:100 (to 50 µM) in
DMSO, aliquoted and stored at –20°C protected from light
until use.
51
Annexin V Binding Buffer (pH=7,4)
Final concentration
Molecular weight
10 mM Hepes
238.3012 g/mol
140 mM NaCl2
58.44277 g/mol
2,5 mM CaCl2
110.9 g/mol
H2O
pH was adjusted with NaOH
Amounts for 500ml
1.192 g
4.1 g
0.14 g
Fill up to 500ml
After bone marrow aspiration or flushing of a femur, the total cell count was
determined by counting in a Neubauer chamber. The volume containing 5X105 cells
was transferred into a 1,5mL eppendorf tube and centrifuged (13.000 rpm, 15s, in a
table top centrifuge). The medium was discarded and the cell pellet was washed with
1,5 mL annexin-V-binding buffer. The supernatant was aspirated and the cells were
resuspended
in
100µL
annexin-V-binding
buffer.
Then
4µl
of
Annexin-V-
AlexaFluor647, 3 µM (1,5µl SytoxOrange staining solution) Sytox orange were
pipetted into the eppendorf tube. To detect apoptosis in the KSL and KL bone
marrow population 1µL of each antibody were added (anti lineage cocktail –FITC
conjugated, anti sca-1 PacificBlue, anti c-kit APC-eFluor780 conjugated), gently
vortexed and incubated 20-30 min, RT in the dark. The cells were washed once with
annexin-V-binding buffer. After washing 250 µl annexin-V-binding buffer were added
and the cells were FACSed as soon as possible (within the next few hours). Fixation
was never done because it damages the plasma membrane and Sytox dyes can
enter freely through the plasma membrane and stain nucleic acid in each cell. This is
why Sytox dyes cannot be used as a necrotic marker when the cells are fixed. When
using tandem dyes for surface marker staining fixation is not recommended since it
destroys the fluorescent properties of the dye.
For detection of the fluorescent
signals the FACS Canto was used.
52
Fig. 3.7.2.1 FACS settings used for detection of apoptotic and necrotic cells within the KL and KSL
populations in murine bone marrow.
Fig. 3.7.2.1 Gating strategy for detection of apoptotic/necrotic cells in the KL and KSL populations
in murine bone marrow.
3.8 Proliferation assays
53
3.8.1 AlamarBlue assay
Company
AlamarBlue
AbD Serotec
SPECTRAmax®GEMINI XS DualScanning Microplate Spectrofluorometer
Flat bottom transparent cell culture plates Greiner
Cat.No.
BUF012A
Alamar Blue is a reagent which contains an oxidation-reduction (REDOX)
indicator called resazurin. When exposed to reducing agents it changes its initial
oxidized state (non-fluorescent and blue) to its reduced form (fluorescent and red).
resazurin
resorufin
When resazurin enters the cytosol the innate metabolic cell activity is able to
reduce it, but not to such extend as in growing and proliferating cells. This is why
detecting the difference in absorbance or fluorescence, gives information about the
cell’s proliferation status.
Before starting the experiment the optimal cell number, cell density and
interval of fluorescence measurements were determined. Depending on the cell
number used and on the proliferation rate of the cell line, the time period for
fluorescence measurements may differ (few days up to one week). Too high cell
numbers can saturate the reaction too fast in which case the eventual difference in
proliferation rates won’t be detected. When optimizing the experiment 1000, 2000,
5000 cells were used per well (for a 96 well plate) in 100µl final volume. When a
larger format was used the volume and cell number were scaled up. Using a larger
format can help if the purpose of the experiment is to follow cell proliferation during a
longer period of time. To precisely determine the optimal amounts a standard curve
was created where a cell range was tested to get the best cell density and time
interval of measurements.
After this the optimal cell number was plated (96-, 24-, 12-, 6-well plates can
be used for measurements in the SPECTRAmax®GEMINI) in appropriate volume
(100µl per 96 well; 500µl per 24well, 1ml per 12well or 2ml per 6well). AlamarBlue
54
was added to 10% of the final volume (for example: 10µl AlamarBlue for 100µl final
volume). Usually a control was Included (for example: 100µl medium + 10 µl
AlamarBlue) to detect possible contamination since bacteria and yeast are also able
to reduce AlamarBlue.
The fluorometer was set at these specifications (SoftMax Pro software):
- Excitation wavelength=560nm,
- Emission wavelength=585nm,
- CutOff filter=570nm,
- PMT(Photo-multiplier Tube)=Medium
- Sensitivity=8
There was condensation on the lid of the tissue culture plate, since this changes
signal detection the lid was removed while measuring. Fluorescence was measured
in Relative Fluorescent Units (RFU) starting from 0h after adding AlamarBlue. The
measurements were repeated after a desired period of time (1h, 6h, 12h or more).
The plate was kept in culture without exchanging or adding new medium for up to 1
week if the cells were viable and the reaction did not get saturated quickly – during
the first days of the experiment. After the end of the experiment a diagram was crated
were the x axis was time and y axis was RFU (See Fig. 4.7.2).
3.8.2 Hoechst based proliferation/cell cycle assay
LIVE/DEAD Fixable Dead cell stain – near IR
Hoechst DNA stain (powder)
Verapamil
anti sca-1 PE conjugated
anti c-kit APC conjugated
PBS
FACS tubes
FACS Canto
LIVE/DEAD stain reconstitution
Company
Molecular Probes
Molecular Probes
Sigma Aldrich
Biolegend
eBioscience
eBioscience
Biochrom AG
BD Falcon
BD
Cat.No.
L10119
H1399
V-106 5MG
78022
12-5981-81
17-1172-82
L182-10
352052
50µl DMSO were added per vial and stored
at –20°C protected from light and moisture.
LIVE/DEAD stain working solution
reconstituted LIVE/DEAD Fixable Dead cell
stain was diluted 1:50 in DMSO
55
Hoechst working solution
[5µg/µl] in PBS stored at +4°C
Verapamil stock solution
5mg Verapamil were suspended in 1mL 95%
ethanol to reach a concentration of 50µM.
1X106 bone marrow cells aspirated or flushed from a femur were put in an
1,5mL eppendorf tube. IMDM medium was added up to 1mL, bringing the cell density
to 1X106cells/mL. The tubes were put in a table top heater at 37°C for 10-15min. 1µL
Hoechst working solution was pipetted in to each tube to reach a final concentration
of 5µg/mL and incubated for 90min at 37°C protected from light. During the
incubation the cells were mixed within the heater 2-3 times. The cells were spun
down at 13.000rpm, 4°C for 15 seconds. After staining with Hoechst, the cell
suspension was always kept on ice and washed once with cold PBS. When working
with many samples simultaneously a staining master mix containing the other
staining components was prepared. For one staining in final volume of 100µL the
amounts used are:
Reagent
PBS
LIVE/DEAD Fixable Dead cell stain working solution
anti sca-1 PE conjugated
anti c-kit APC conjugated
Volume
90µL
5µL
2µL
2µL
2µL
After adding 100µL to each tube, the cells were kept on ice 30-40 min. and
subsequently washed once with cold PBS (13.000rpm, 15s,4°C). The PBS was
aspirated and the cells were resuspended in 250µL PBS. The cells were transferred
to FACS tubes and acquired on medium flow speed with the FACSCanto. In order to
have enough cells to make cell cycle/proliferation analysis 2000 to 5000 events were
acquired in the KSL gate.
When analyzing the acquired data in some samples a subG1 peak was
observed. To test the origin of this peak, whether it is an artifact or cells analogous to
the ones defined as the side population (SP), Verapamil was used. The SP (bone
marrow stem cells mostly) has the propensity to efflux Hoechst more that the other
cell types in the bone marrow 172. Since Verapamil blocks the drug efflux channels 173
and this way enables staining of DNA even in the SP cells, its treatment was used as
a control to confirm the stem cell origin of the cells in the subG1 peak.
56
Fig. 3.8.2.1 FACS settings for detection of the amount of cycling cells within the KL and KSL
Fig. 3.8.2.1 Gating strategy used for detection of the amount of cycling cells within the KL and KSL
3.8.3 ClickItEdu assay
Company Cat.No.
57
ClickIt Edu Alexa Fluor 594 imaging kit
12 reaction field adherent slides
Cover glasess 24x50 mm, No. 1.5H
ProLong gold antifade reagent
ScanR screening station microscope
Invitrogen
Marienfeld
Marienfeld
Invitrogen
Olympus
C10084
09 000 00
0102222
P36934
The ClickÍt Edu kits provided by Invitrogen offer an alternative to BrdU based
detection of proliferation. The principle behind the ClickIt Edu kits is based on the fact
that proliferating cells, synthesizing DNA will incorporate the thymidine analog Edu
(5-ethynyl-2´-deoxyuridine). Detection of the amount of incorporated Edu is based on
the so called ClickIt chemistry. The ClickIT reaction creates a covalent bond between
an azide group bound to AlexaFluor dyes and an alkyne group present in Edu. The
reaction is catalyzed by copper. The protocol contains several steps:
1) ClickIt Edu labeling – CD34+ cells after being infected with NF-E2 or Empty control
virus were counted and mixed with Edu at 10µM concentration. 2x104 cells were then
plated per field on a 12 reaction field adherent slide. The final volume added per field
was 20µL. The slides with the cells were incubated for 4h at 37°C, 5% CO2 in
humidified atmosphere. During this incubation time the cells adhere to the slide.
2) Cell fixation – after the medium was removed from the cells they were fixed by
using 20µl 3,7% formaldehyde in PBS per field. The cells were incubated for 15 min
and subsequently washed twice with 40µL 3% BSA in PBS.
3) Permeabilisation – cells were permeabilasied by adding 20µL of 0,5% Triton X-100
in PBS per field and incubating for 20 min, RT.
4) EdU detection – after removing the permeabilisation solution the cells were
washed twice with 40µL 3%BSA in PBS. Then 20µL of the Click-It reaction cocktail
was added per field and incubated for 30min, RT, protected from light. The cells were
then washed once with 3%BSA in PBS. The Click-It reaction cocktail was prepared
as recommended by the manufacturer:
Amount per reaction field Amount for a 12 field slide
Reaction component
(µL)
(µL)
1X Click-iT reaction buffer
17.20
206.40
CuSO4 (Component H)
0.80
9.60
AlexaFluor azide
0.05
0.58
Reaction buffer additive
2.00
24.00
Total volume
20µL
240µL
* 1X Click-iT reaction buffer, AlexaFluor azide and reaction buffer additive stock
solutions were prepared upon opening the kit as recommended in the manufacturers
manual (http://tools.invitrogen.com/content/sfs/manuals/mp10338.pdf)
58
5) DNA staining – after washing once more with 3% BSA in PBS and once with PBS
only, 20µL of 5µg/mL Hoechst solution was added, incubated for 30 min. at RT,
protected from light. Finally cells were washed twice with PBS, air dried overnight
and covered with a cover slip after adding the mounting solution.
6) Imaging – the Edu –AlexaFluor signal was detected by using the ScanR screening
microscope (part of the ZKF Core Facility). For analysis only the GFP+ cells were
taken. EdU positive cells were defined as proliferating.
3.9 Differentiation assays
3.9.1 Human hematopoietic progenitor and hematopoietic stem
cells assay
Progenitor and HSC populations were determined by FACS staining using the
following antibodies: anti-CD34 PacificBlue (Biolegend, Cat: 343511), anti- CD38
AlexaFluor700 (Biolegend Cat: 303523), anti- CD45RA APC (Abnova Cat:
MAB4624), CD123 PE (BD Cat: 555644), Flt3R (CD135) – biotin (Biolegend Cat:
313312) revealed with Streptavidin-PE/Cy7 (Biolegend Cat: 405206) and Thy-1
CD90) PerCP-Cy5. The staining was done on CD34+ cultured cells in expansion
medium. Two days after the first lentiviral transduction the cells were collected from
culture in an eppendorf tube and the volume was adjusted to 100µL.
[C] for 1x106 µl for 1x106
Stock [C] cells
cells
CD34 PacificBlue
0,5 mg/ml ≤1µg
2 µl
CD38 AlexaFluor700
0,5 mg/ml ≤1µg
2 µl
CD45RA APC
10µl
CD123 PE
0,2 mg/ml ≤0,4µg
2µl
Flt3R (CD135)
0,5 mg/ml ≤2 µg
2µl
Streptavidin-PE/Cy7
0.2 mg/ml ≤0.125 µg
4µl
Thy-1 (CD90) PerCP-Cy5.5
5µl
isotype
Mouse IgG1, κ
Mouse IgG1, κ
Mouse IgG2b
Mouse IgG1, κ
Mouse IgG1, κ
Mouse IgG1, κ
Mouse IgG1, κ
The amounts of Ab listed in the table above were adjusted according to the
cell number used per a staining. First Flt3R (CD135) – biotin Ab was added and the
cells were incubated on ice for 20min. Thereafter the cells were washed once with
PBS. The other Abs including Streptavidin-PE/Cy7 were mixed in 100µL PBS and
added to the cells. The mixture was incubated on ice for 30min., washed once with
59
PBS and resuspended in 200µL PBS. The handling and all centrifugation steps were
done at +4°C. Fluorescence was detected with FACSCanto II (BD Biosciencies).
3.9.2 Human terminal differentiation assays
Component
anti CD11b PE conjugated
CD13 PE conjugated
anti CD13 PE-Cy7 conjugated
anti CD19 PE conjugated
anti CD235a PE conjugated
anti CD36 APC conjugated
anti CD41a PE conjugated
anti CD42b APC conjugated
Company
BD
BD
BD
BD
BD
BD
BD
BD
Cat.No.
30455
555394
338432
30665x
555570
550956
555467
551061
Terminal differentiation of cultured CD34+ cells was detected by staining for
specific surface markers. Detection of cells from the erythroid lineage was done by a
combination
of
CD36-APC
and
CD235a-PE
specific
antibodies.
While
megakaryocytic cells were detected by staining with CD41a-PE and CD42b-APC
fluorophore conjugated antibodies. Double staining with CD13 PE-Cy7 and CD11b
enables the distinction between terminally differentiated granulocytes, monocytes
(CD13+CD11b+) and intermediate myeloid progenitors (CD13+CD11b-). CD19 is a
B-cell marker and it was used as a proof of principle that differentiation in expansion
medium occurs only along the myeloid lineage. The samples were collected at
different time points and stained with the desired combination of antibodies. For all
mentioned antibodies the staining was done the same way. In a final volume of
100µL, 2µL of each antibody was added to the cell suspension and incubated for 20
min. on RT. When the cell count was low the staining was performed directly in
culture medium, without washing with PBS prior to antibody addition. On the other
hand, washing once with PBS after staining is recommended, since it reduces the
background signal. After the wash the cells were suspended in 200µL PBS and
analyzed on a FACS Calibur or a FACSCanto.
3.9.3 Human megakaryocytic ploidy assays
60
Verapamil
PBS
FACS tubes
anti human CD41a PE conjugated
anti human CD42b APC conjugated
FACS Canto
Company
Molecular Probes
Sigma Aldrich
Biochrom AG
BD Falcon
Gibco
BD
BD
BD
Cat.No.
H1399
V-106 5MG
L182-10
352052
21980
555467
551061
Hoechst working solution
[5µg/µl] in PBS stored on +4°C
Verapamil stock solution
5mg Verapamil were suspended in 1mL 95%
ethanol to reach a concentration of 50µM.
Previously cultured CD34+ cells with appropriate cytokines were collected and
counted. 5X104 cells or more when available were added to 1,5mL eppendorf tube
containing enough culture medium to reach a final volume of 1mL. The eppendorf
tubes were placed in a table top heater on 37°C for 10-15min. Afterwards 1µL of
Hoechst working solution was added per tube to reach final concentration of 5µg/mL.
The cells were incubated for 90 to 120min at 37°C protected from light. Verapamil
could be added during the incubation to increase the efficiency of the Hoechst
staining, but is not absolutely necessary. However, if used, the final concentration of
Verapamil should be 0,25µM (diluting the stock solution 1 to 200) and added directly
into the medium together with Hoechst. The tubes were shaken 2 to 3 times in the
heater during the incubation. After the Hoechst staining was finished the cells were
pelleted (13.000rpm, 4°C, 15s), resuspended in cold PBS and stained with CD41aPE and CD42b-APC antibodies (30min, on ice, protected from light). After washing
with PBS once the cells were resuspended in 200µL PBS, transferred into FACS
tubes, and kept on ice until acquired on FACSCanto on medium flow speed.
3.9.4 Murine megakaryocytic ploidy assays
61
Verapamil
PBS
FACS tubes
anti mouse/rat CD41 PE conjugated
anti mouse CD42d APC conjugated
FACS Canto
Company
Molecular Probes
Sigma Aldrich
Biochrom AG
BD Falcon
Gibco
Biolegend
eBioScience
BD
Cat.No.
H1399
V-106 5MG
L182-10
352052
21980
133905
17-0421-80
The principle and the protocol of this assay is exactly the same as described in
3.9.3. The difference is that the bone marrow cells were not cultured, but stained
directly after isolation and the antibodies used are mouse specific. In general, 1x106
bone marrow cells were suspended in IMDM medium in final volume of 1mL. Hoechst
staining was done for 90min., at 37°C where Verapamil was optionally added to the
mixture. After one wash with PBS the cells were labeled with CD41 and CD42
fluorescent antibodies, washed one more time with PBS and acquired with a
FACSCanto. It is important to keep the flow speed on medium and never on high, so
that a better resolution is obtained. When using lower flow speeds the diameter of the
flow stream is lower and the cells flow through the flow cell in a single cell
suspension. Excluding cell clumps is important since this would give a false signal.
For example a doublet would be detected as a 4n cell.
Fig. 3.9.4.1 FACS settings for detection of the distinct ploidy classes in populations single or double
positive for two megakaryocytic markers (CD41 and CD42) in murine bone marrow.
62
Fig. 3.9.4.2 Gating strategy used to detect the distinct ploidy classes in populations single or double
positive for two megakaryocytic markers (CD41 and CD42) in murine bone marrow. Note that there
are only few polyploid cells within the CD41+CD42- cell population, which means that CD41 alone is
not only expressed on megakaryocytic cells in mBM. The granularity and the size of the
megakaryocytic cells can vary, where many of the CD41+CD42+ cells are big and granular. This is why
a gate defining a population with defined forward/sideward properties was not used for the analysis.
3.9.5 Cytospins
Component
Slides (SuperFrost 76x26mm)
Filter cards (Shandon Filter Cards)
Cytospin Clips (CytoClip-Slide Clips)
Cytospin Funnels (TPX Sample
Chambers)
Cytospin centrifuge (Shandon CytoSpin 3)
Company
Roth
Thermo Electron Corp.
Cat.No.
1879
5991022
59920063
Thermo Electron Corp. A78710018
For histological evaluation of differentiation cytospins combined with MayGrünwald Geimsa staining were used. The number of cells used per cytospin was
2x104, while the cell density was always adjusted to 1x105 cells/mL. After counting
the cells were suspended in a final volume of 200µL PBS and applied to the
assembled cytospin device. A 3min. long centrifugation at 800rpm at low speed
acceleration in the cytospin centrifuge was done. The slides were then dried shortly
for 15 min or left to dry overnight. Thereafter the cells were stained.
63
3.9.5.1 May-Grünwald Geimsa (MGG) staining
Component
May-Grünwald eosine-methylene blue solution
Geimsa azur methyleneblue solution
Buffer tablets (pH=6.8)
Entellan
Cover slip (24x24mm) thickness 1
ddH2O
Company
Merck
Merck
Merck
Merck
Marienfeld
Cat.No.
Hx107432
Hx000080
TP1077274113
Hx 624397
0101060
Geimsa staining solution
1 to 20 diluted stock Geimsa solution in H2O
Buffered water
1 buffer tablet dissolved in 1L of ddH2O
After spinning the cells down and drying the slide they were stained with
undiluted May-Grünwald for 5 min., washed twice in buffered water and transferred to
a staining container with Geimsa staining solution for 20 min. Afterwards the slides
were washed few times until the excess stain was completely dissolved and let to dry
overnight. For microscopy the cells were embedded in entellan and covered with a
cover slip.
3.10 RNA processing
3.10.1 RNA isolation
The RNeasy® mini kit (QIAGEN, CatNo. 74104) was used for RNA
purification, because when working with CD34+ cells the amount of available cells is
usually low. The kit is suitable for not more than 1x107 cells and the lower range is
5x104 cells to get high quality RNA. The isolation was done as recommended in the
producer’s manual. The RNA’s quantity and quality was detected by using the
NanoDrop (PeqLab) spectrophotometer. The absorption of the solution was acquired
at three wave lengths: 230, 260 and 280nm. Nucleic acids absorb at 260 nm.
Proteins and phenol absorb light strongly at 280nm. Other contaminants left from the
purification buffers may absorb light at 230nm. This is why the ratios 260/280 and
260/230 are used as a quality control. The 260/280 ratio for pure RNA is from 1,8 to
2,0 and 260/230 is 1,8 to 2,2.
3.10.2 cDNA synthesis
The One Step TaqMan reverse kit (Applied biosystems, Cat.No. N808_0234)
was used to perform the reverse transcription of total RNA to complementary DNA
(cDNA). The amounts used for a single reaction are listed below:
64
Component
RT buffer
MgCl2
dNTPs
Random Hexsamer Primers
RNAse Inhibitor
Multiscribe Reverse Transcriptase
RNA
H2O and RNA
Total volume
Concentration
10X
25mM
2,5mM each
50µM
20U/µL
5U/µL
Amount
5µL
11µL
10µL
2.5µL
1µL
1.25µL
50ng
19.25µL
50µL
After the RNA and the reverse transcription mix were pipetted into RNAse free PCR
tubes, the cDNA synthesis was done in a thermocycler with the following program:
25°C 10min 48°C 30min 95°C 5min 8°C ∞
The samples were stored at -20°C until used for qRT-PCR.
3.10.3
qRT-PCR
(quantitative
Reverse
Transcription-Polymerase
Chain
Reaction)
2x TaqMAn Universal master mix
20x Gene expression mix
96 well TaqMan Amplification Plates
ABI Prism 7000 Sequence Detection System
Company
Cat.No.
Applied Biosystems 4324020
Thermo
AB110
Applied Biosystems
A singleplex (detection of one gene per one tube) TaqMan system was used
for all gene expression quantifications done in this thesis. This system is based on a
fluorescent reporter probe method. The probe is designed to be complementary to a
specific DNA sequence and has a reporter fluorophore (FAM) attached to its 5’end
and a quencher at its 3’ end. As in every PCR reaction the sequence specific primers
anneal to the cDNA and the polymerase starts catalyzing the reaction. The TaqMan
polymerase also has a 5’ to 3’ exonuclease activity and degrades the probe during
the process of amplification. This leads to the separation of the fluorophore from its
quencher making the detection of a florescent signal possible upon excitation. The
intensity of the signal directly correlates to the number of RNA transcripts within the
sample that was reverse transcribed.
65
•
qRT-PCR reaction
Component
2x TaqMan Universal master mix
20x Gene expression mix
cDNA
H2O
Amount for 1 reaction
12,5µL
1,25µL
5ng=5µL
6,25µL
A 53 fold master mix was prepared for the gene of interest and the housekeeping
gene, which was B2M in all experiments. 25µL of the master mix were added per
well, where 5µL cDNA solution were pipetted in addition. When there was not
enough cDNA available, 2.5ng (2,5µL) cDNA per well were used. The PCR
reaction and the fluorescent signals detection were done with the ABI Prism 7000
Sequence Detection System using the following program:
Initial denaturation:
95°C, 10min
Denaturation:
95°C, 15s
Annealing and elongation:
60°C, 1min
The 20x Gene expression mixes specific for the gene of interest were ordered
directly for the company or previously designed.
66
Assay-On-Demand
Human NF-E2
Human β-globin
Human B2M
Company
Applied Biosystems
Applied Biosystems
Applied Biosystems
Cat.No.
Hs00232351_m1
Hs00758889_s1
Hs00187842_m1
The distinction between JAK2wt and JAK2V617F and their quantification in patient
samples was done by using the primers and probe listed in the table below. The
detection of JAK2V617F was possible by modifying the reverse primer. The other
components of the reaction were equal for both WT and mutant JAK2.
Nucleotides
JAK2 forward primer
JAK2 reverse primer
JAK2V617F reverse primer
Probe
•
Sequence
5'-CAGCAAGTATGATGAGCAAGCTTT-3'
5'-TGAACCAGAATATTCTCGTCTCCAC-3'
5'-CCAGAATATTCTCGTCTCCACTGAA-3'
5'-FAM-TCACAAGCATTTGGTTTT-MGB-3'
qRT-PCR data analysis
The intensity of the fluorescent signal increases with each PCR cycle until the
availability of the reaction components becomes limiting. On a plot presenting the
number of PCR cycles on the x-axis and the normalized reporter fluorescence
(∆Rn) as a Log function of X at the y-axis, four different phases of amplification
can be distinguished: linear ground phase, early exponential phase, log-linear and
plateau phase. To evaluate the expression of a gene, a threshold line is set within
the exponential phase of amplification. The cycle number where the threshold is
set is called the threshold cycle (Ct) and its value is used for estimating the
amount of gene expression.
Fig. 3.4 qRT-PCR amplification plot. The figure was adapted from Ahmed (2005) 174 (http://www.cgpjournal.com/2005%20issue%206/Quantitative%20Real-time.html)
There are two ways to evaluate the data acquired by qRT-PCR: by creating a
standard curve or by the ∆∆Ct method. The advantage of the standard curve
67
method is the possibility of determining the exact copy number of the gene of
interest, while the ∆∆Ct method gives only relative changes in gene expression.
To create a standard curve linearized pCR 2.1 or pCRII TOPO plasmids carrying
the specific sequence recognized by the primers and probe were used. The exact
copy number of the standard was determined and by creating serial dilutions
within the range of the sample’s gene expression a standard curve was plotted.
The x-axis represented the copy number and the y-axis was the Ct value at a
certain threshold. The copy numbers of the samples were calculated by using a
linear equation and the data was normalized to the copy number of the
housekeeping gene.
When using the ∆∆Ct method, the difference between the Ct values of the
gene of interest and the housekeeping gene was calculated. Then a Ct value of
the gene of interest was chosen as a calibrator to which the other samples were
compared to. The formula used to calculate the relative expression of a certain
gene compared to a calibrator sample is: 2- ∆∆Ct.
3.11 Protein processing
3.11.1 Protein isolation
To prepare whole cells extracts the RIPA buffer supplemented with protease
inhibitors was used.
RIPA buffer:
Reagent
NaCl
NP-40
Sodium deoxycholic acid
SDS
Tris pH 8.0
ddH2O
Lysis buffer
Final concentration
150mM
1%
0.50%
0.10%
50mM
Company
VWR
Roche
Sigma-Aldrich
Roth
Roth
Cat.No.
278102-95
11332473001
D6750
4360.2
4855.3
RIPA supplemented with 2xComplete (Roche,
Cat.No.04693116001)
CD34+ or other tissue cultured cells were collected, counted, washed once
with PBS and lysed. The volume of the lysis buffer was 50µL for up to 1x105 cells,
60µL for 1x105 to 1x106 and 90µL for more that 3x106 cells. The samples were kept
for 20min. on ice. Thereafter they were either frozen at -80°C or it was proceeded
68
directly to protein isolation. To disrupt all cellular membranes the samples were
sonicated with an ultrasonic probe (SonoPulse, Bandelin) with power of 40% for 10s
and 2x10% cycles. After sonication samples were centrifuged 13.000rpm, 15 min.,
4°C and the protein containing supernatant was collected for protein reduction and
alkylation and for protein quantification.
3.11.2 Protein quantification
Reagent
Lowry assay
BSA
96-well microtiter plates
Company
BioRad
Fermentas
Greiner
Cat.No.
500-0116
B9001S
655160
A small amount of the protein supernatant was taken out and diluted 1 to 10
(1,2µL protein+10,8µL H2O). In a 96 well microtiter plate 5µL of the diluted protein
supernatant and the standard solutions was added per well. Standard protein
solutions were prepared by diluting BSA to get concentrations of [0,05];[ 0,1]; [0,25];
[0,5]; [0,75] and [1] µg/mL. All samples were pipetted in duplicates. The protein
concentration was detected as described in the Lowry assay manual (BioRad,
Cat.No. 500-0116). According to the measured absorbance normalized automatically
by the software to the blank (including only water instead of protein) a standard curve
was ploted. The x axis was the concentration of the BSA protein solutions and the y
axis was absorbance. The protein concentration of the samples was calculated as a
linear function of the standard curve y=mx+b.
3.11.3 Protein reduction and alkylation
5XSDS sample buffer:
Reagent
Final concentration
1M Tris (pH=6,8) 0.25M
SDS
0.1 g/mL
Bromphenol-blue 5mg/mL
100% Glycerol
1/2 of final volume
ddH2O
up to desired volume
Company
Roth
Roth
Roth
Fluka
Cat.No.
4855.3
4360.1
A512.1
49780
Iodoacetamide (IAA)
1M in H2O (SigmaAldrich Cat.No. I1149), stored on -20°C
Dithiothreitol (DTT)
1M in H2O (SigmaAldrich Cat.No. 43817), stored on -20°C
69
To denature and negatively charge the proteins, the protein sample was boiled
in 1XSDS sample buffer (Lämmli buffer) 10min, 95°C. Prior to boiling, 50mM DTT
was added to the protein sample to obtain reduction of the disulfide bonds within the
proteins. The samples were cooled down to RT and IAA at a final concentration of
120mM was added. The samples were incubated for 20min. in the dark. To quench
the excess IAA additional 20mM DTT was added and the samples were kept for
20min in the dark at RT. IAA alkalizes the sulphide residues within the protein, which
makes the folding of the protein unlikely. The samples were stored on -80°C.
3.11.4 SDS Polyacrylamide Gel Electophoresis (SDS-PAGE)
Tris
SDS
Glycin
Rotiphorese
TEMED
APS
Isopropanol
Byotinylated Protein ladder
Precision Plus Protein Standard (Kaleidoscope)
Mini PROTEAN II Cell
Company
Roth
Roth
Roth
Roth
Roth
Fluka
Roth
Cell Signaling
BioRad
BioRad
Cat.No.
4855.3
4360.2
3908.2
3029.1
2367.3
9913
6752.3
7727
161-0375
165-2926
4x separating gel buffer
1,5M Tris (pH=8,8) and 0,4% SDS
4xStacking gel buffer
0,5M Tris (pH=6,8) and 0,4% SDS
5xSDS running buffer
0,125M Tris (pH=8,7), 1M Glycin and 0,5% SDS
Rotiphorese
30% Acrylamide and NN Methylenbisacrylamide solution
APS solution
10% APS in H2O
The separating SDS-PAGE gel was cast between two clean glass plates using
the Mini PROTEAN assembling system. The concentration of the gel was prepared
according to the size of the proteins to be detected. To achieve a better resolution at
the range between 10 and 60kDa a 12% gel was prepared. For separating 30 to
120kDa proteins a 10% gel was used and for proteins ranging between 50 and
200kDa an 8% gel was run. After the gel was poured 0,5 to 1mL isopropanol were
put on top to ensure a horizontal and flat distribution of the gel’s top border. When
the separation gel was polymerized the isopropanol was poured off and the stacking
gel was cast on top. The concentration of the stacking gel was always 3,9%. The gel
70
cassette was then placed into the electrophoresis tank in 1xSDS running buffer. The
samples were loaded and ran with 100V and 65mA electrical current. When the
separation of the proteins was sufficient (visible by the Kaleidoscope marker)
electrophoresis was stopped and the separating gel was blotted.
3.11.5 Western Blot
Tris
Methanol
Glycin
Ponceau S
Trichloracetic acid
Gel Blotting Paper
Immobilion PVDF membrane
Mini-Trans Blot Module
Transfer buffer
Company
Roth
VWR
Roth
Fluka
Roth
Schleicher and Schuell
Millipore
BioRad
Cat.No.
4855.3
20.847.295
3908.2
81460
7437.1
10426694
IPVH00010
170-3935
25mMTris, 192mM Glycine, 20%Methanol, H2O to desired
volume
Ponceau S solution
2% Ponceau S, 3% Trichloracetic acid, H2O up to desired
volume
To make the detection of specific proteins possible by using antibodies, the
sample proteins are transferred to a PVDF membrane. The membrane was
previously swelled by putting it in Methanol for 15s, 2 min. in H2O and 5min in
Transfer buffer. Also, the separating gel was incubated for 2min. in transfer buffer
prior assembling the gel sandwich into the blotting device. It is important to pay
attention to the order of putting the gel and the membrane. Since the negatively
charged proteins will move towards the anode the membrane has to be on the anode
side and the gel on the cathode side. The order of assembly from cathode to anode
is as follows: fiber pad, gel blotting (filter) paper, separating gel, PVDF membrane,
gel blotting paper, fiber pad. The transfer of proteins to the PVDF membrane was
done by using 100V, 350mA electrical current for 3h. The efficiency of the transfer
was confirmed by soaking the membrane into Ponceau S solution. By washing few
times with ddH2O the staining was removed from the membrane.
71
3.11.6 Detection of Immunocomplexes
Tris
NaCl
Tween-20
Non fat dry milk
BSA
ECL Western Blot Analytical System
ECL Plus Western Blot Detection system
Chemiluminescence Detection System
Company
Roth
VWR
Roth
Fluka
Sigma Aldrich
GE
GE
INTAS
Cat.No.
4855.3
278102-95
9127.1
70166
A-9647
RPN2109
RPN2132
TBS(10X)
0,2M Tris (ph=7,5); 150mM NaCl
Wash buffer
0,1%-0,15%(v/v) Tween-20 in 1xTBS
Blocking buffer A
0,1%(v/v) Tween-20, 5%(w/v) dry milk in 1XTBS
Blocking buffer B
0,1%(v/v) Tween-20, 5%(w/v) BSA in 1XTBS
Primary antibody
anti- Biotin
anti-JAK2
anti-NF-E2
anti-NF-E2
β-actin
Company
Cell Signaling
Cell Signaling
generated in Pahl lab
Sigma Aldrich (ATLAS)
Sigma Aldrich
Secondary antibody
anti- Rabbit IgG HRP linked
anti- mouse IgG HRP linked
Company
GE
GE
CatNo
7075
3230
1089
HPA001914
A5441
LotNo
R00723
CatNo
NA934V
NA931V
Usually after Western blotting the membranes were blocked for 1h at RT, by
adding around 20mL blocking buffer A in a 50mL Falcon tube. The incubation with
the primary antibody was done over night at +4°C (or 1h, RT when applicable) in
10mL blocking buffer with the recommended Ab dilution. The membrane was washed
3 times with washing buffer to get rid of the unbound Ab. Thereafter, the membrane
was incubated for 1h, RT with 1 to 4000 diluted secondary Ab in 10mL blocking
buffer A or B. Again the membrane was washed twice with wash buffer and once with
1XTBS alone. The detection of the immunocomplexes was done by detecting the
chemiluminescent signal.
72
3.11.7 Removal of immunocomplexes
Stripping buffer
Reagent
Tris (pH=6,8)
SDS
β -mercaptoethanol
ddH2O
Final concentration
50mM
2% (w/v)
100mM
up to desired volume
Company
Roth
Roth
Sigma Aldrich
Cat.No.
4855.3
4360.2
M7522
When it was necessary to detect several immunocomplexes the PVDF
membrane was incubated at 50°C in 40mL stripping buffer for 20-30min within a
water bath. Afterwards, the membrane was washed several times with 1xTBS until
the smell of β –mercaptoethanol was gone. Then the membrane was used for
detection of other immunocomplexes as described before.
3.12 Statistical analysis
For statistical data analysis and plotting graphs either the GraphPadPrism 5
(http://www.graphpad.com) or the SigmaPlot 11.0 (Systat Software Inc.) software
was used. Paired t-test was used when the same sample received different
treatments. For example, when comparing the phenotype of CD34+ cells originating
from same donors transduced with ether Empty or NF-E2 vector. When two unrelated
groups were compared like PV and HC, unpaired t-test was used. One way ANOVA
was applied to compare multiple parameters simultaneously.
73
4. Results
4. 1 NF-E2 is overexpressed in CD34+ cells derived from
PV patients’ peripheral blood.
To determine the amount of NF-E2 in CD34+ cells originating from PV or HC,
the cells were cultured in modified expansion medium and RNA samples were
collected daily (from day 4 to day 8 of culture). The level of expression was detected
by qRT-PCR. PV cells expressed 1,5 to 2 fold more NF-E2 than the HC from day 4
up to day 8 of differentiation in-vitro (Fig.4.1.1, b). The expression of CD34 was
confirmed by FACS daily. On the starting day of culture (day 1) the amount of CD34+
cells was from 90-100% depending on the efficiency of CD34 isolation. After 4 days
in modified expansion medium the percentage of CD34 positive cells dropped by only
10-20% and was gradually reduced to 40% on day8 of culture. Both PV and HC
CD34+
cells
showed
the
same
pattern
of
CD34
expression
(Fig.4.1.1,
a).
Fig.4.1.1 CD34+ cell surface expression and NF-E2 overexpression in in-vitro differentiating PV and
HC CD34+ cells. a) CD34 surface expression was detected daily (from day4 to day8) by FACS in
peripheral blood isolated CD34+ cells from healthy controls (HC) and PV patients cultured in modified
expansion medium. The data is represented as % positive cells within the viable cell population. b) An
RNA sample was collected daily form day4 to day8 of culture and the amount of NF-E2 transcripts in
PV and HC cells was detected by qRT-PCR. Evaluation of qRT-PCR data was done by the ΔΔCt method.
Statistical analysis was performed by using unpaired two-sided t-test, where * p≤0.05.
To investigate the effect of NF-E2 overexpression on hematopoiesis, CD34+
cells isolated from HC were transduced with virus where an equal amount of NF-E2
overexpression as in PV patients was achieved ranging from 1.5 to 2.5 fold (Fig
4.1.2, c). The amount of NF-E2 expression in HC or empty vector transduced cells
was taken as a reference value. The expression levels in HC and PV cells on day 8
of culture were detected by qRT-PCR for HC and PV cells (Fig.4.1.2,a) and for NFE2 or empty vector transduced cells (Fig.4.1.2,b).This overexpression was also
74
detectable on protein level (Fig.4.1.2, d).
Thus, the experimentally achieved
exogenous NF-E2 expression reflected the levels of expression observed in PV
patients.
Fig.4.1.2 NF-E2 expression in in-vitro differentiating CD34+ cells. The expression of NF-E2 was
detected as number of mRNA transcripts per 100.000 B2M. After cDNA synthesis the amount of NFE2 transcripts was detected by qRT- PCR and analysed by the standard curve method in a) healthy
control (HC) or PV cells and b) HC cells transduced with pLeGO-iG-NF-E2 (NF-E2) or empty pLeGO-iG
(Empty) on day 8 of culture in modified expansion medium. c) Fold exogenous NF-E2 expression in
retrovirally transduced HC CD34+ compared to the endogenous levels of NF-E2 expression in CD34+
cells differentiated for 8 days in modified expansion medium. The amount of NF-E2 transcripts in HC
cells or cells transduced with the empty vector were taken as reference value. d) Western blot from
NF-E2 transduced and control cells on day 14. Statistical analysis was performed by using unpaired a)
or paired b) two-sided t-test.
4.2 NF-E2 overexpression in CD34+ cells promotes HSC,
MPP, CMP and GMP expansion while decreasing MEP
cell counts.
After isolation, the CD34+ cells were lentivirally transduced on day 2 and 3 of
culture with pLeGO-iG-NF-E2 (NF-E2) or empty pLeGO-iG (Empty). On day 4 and 5
of culture the cells were collected and stained with a combination of fluorescently
labeled antibodies to detect HSC and progenitor populations. Only the viable and
transduced (GFP+) cells were included in the analysis. The combination of surface
specific markers was chosen as previously described
125, 175
. The FACS gating
strategy used to distinguish the different populations is described in Fig.4.2.1. The
75
surface marker expression typical for HSC and progenitor populations is summarized
in Table.4.2.1.
Population
Phenotype
HSC
CD34+CD38-CD45RA- CD90+Flt3+
MPP
CD34+CD38-CD45RA- CD90-Flt3+
CMP
CD34+CD38+CD45RA- IL-3Ra+ (orFlt3+)
MEP
CD34+CD38+CD45RA- IL-3Ra- (or Flt3-)
GMP
CD34+CD38+CD45RA+ IL-3Ra+
Table 4.2.1 Phenotypic characterization of HSC and hematopoietic progenitor cells
Fig.4.2.1 Human HSC and progenitor analysis in NF-E2 overexpressing and control PB derived
CD34+ cells differentiated in-vitro. The cells were collected on day 4 and 5 of culture labeled for
surface specific markers and analyzed by FACS. The gating strategy is depicted in a). b) Represents
the initial gating for CD34+CD38- and CD34+CD38+ populations. Representative measurements for
HSC and MPP determination are shown in c) and for progenitor distinction in d). The upper panels in
c) show CD45RA depletion and the lower ones MPP and HSC population distribution as percentages
of CD34+CD38-CD45RA- population. On the upper two and the lower two panels in d) two alternative
strategies are depicted for CMP, MEP and GMP analysis as a percent of CD34+CD38+ cells.
FACS measurements showing the percentage of HSC and MPP of the
CD34+CD38-CD45RA- population are presented in Fig.4.2.1 c) (two bottom plots).
NF-E2 overexpression caused an increase in the percentage of HSC and MPP
populations. Representative FACS data for progenitor distinction is depicted in
76
Fig.4.2.1 d). The upper two dot plots represent the most commonly used way of
CMP, MEP and GMP distinction, while the bottom dot plots show a gating strategy by
an alternative progenitor definition. In both cases, an increase of the percentage of
CMP and GMP population in the NF-E2 overexpressing cells was observed, at the
expense of MEPs that became less.
To confirm that these observations are genuine and do not reflect changes in
the CD34+CD38- or CD34+CD38+ population due to NF-E2 overexpression their
percentage within the GFP+ cells was compared. There was no significant difference
detected between NF-E2 and empty transduced cells for neither CD34+CD38(Fig.4.2.2 a and c) or CD34+CD38+ population (Fig.4.2.2 b and d) on day4 and 5 of
culture. Around two fold decrease in the percentage of CD34+/CD38- population
was detected from day4 to day5 of culture in both NF-E2 and empty transduced cells
(Fig. 4.2.2 a and c). On the other hand the frequency of CD34+/CD38+ cells stayed
constant reaching a mean value of around 50% of all viably transduced cells.
Fig.4.2.2 Frequency of CD34+CD38- and CD34+CD38+ populations among the viable virus
transduced (GFP+) CD34+ cells. The percentage distribution with mean and SEM values of
CD34+CD38- population are depicted on panel a) day4 of culture and c) day 5 of culture. Panels b)
and d) represent the CD34+CD38+ population for day 4 and day5 of in-vitro culture respectively.
Paired t-test was used for statistical analysis where no statistical difference was detected between
77
the Empty control and NF-E2 vector transduced cells. n is the number of independent experiments
included in the analysis.
To evaluate the ability of NF-E2 overexpressing CD34+ cell to generate and/or
maintain cells with HSC phenotype the percentage of HSC within the CD34+/CD38population on day4 and day5 was compared to the empty control transduced cells.
On day 4 of culture there was not a significant difference in the amount of HSC (Fig.
4.2.3 a), but on day 5 there were significantly more cells with HSC phenotype in the
NF-E2 overexpressing cells (Fig.4.2.3 b). The absolute number of HSC on day 5 of
culture was also elevated when NF-E2 was overexpressed (Fig.4.2.3 c).
Fig.4.2.3 Increase of HSC frequency in NF-E2 overexpressing CD34+ cells. Graphs a) and b) show the
percentage HSC within the CD34+CD38-CD45RA- population on day 4 and day 5. The absolute
number of HSCs existing within 100.000 transduced (GFP+) CD34+ cells is depicted in c). Statistically
significant difference between the groups was determined by paired t-test.
NF-E2 overexpression also caused expansion of the MPP pool (Fig.4.2.4).
The increase in percentage MPPs within the CD34+/CD38+ population was detected
on both day 4 (Fig.4.2.4 a) and 5 (Fig.4.2.4 b) of culture. The same observation was
valid when the absolute numbers MPPs were compared (Fig.4.2.4 c).
78
Fig.4.2.4 Increase in MPP frequency in NF-E2 overexpressing CD34+ cells. The expansion of MPP
population represented as percentage of CD34+CD38-CD45RA- population was measured on day 4 a)
and day 5 b). The total number of MPPs generated within 100.000 transduced (GFP+) cells was
calculated for each experiment and is depicted in c). The data distribution with mean of n=4
independent experiments is plotted. Statistically significant difference between the groups was
determined by paired t-test.
NF-E2 overexpression also affected the later progenitor compartments. The
percentage of CMP and GMP compartments were significantly elevated, while MEPs
counts were lower (Fig.4.2.5 a and b), reflecting the changes observed as absolute
cell numbers (Fig.4.2.5 c). The shift in later progenitor population distribution was
present on day 4 and day 5 of culture and was detected by two different gating
strategies. Fig. 4.2.5 a represents the most commonly used way (classical definition)
of CMP, GMP and MEP distinction, while Fig. 4.2.5 b represents an alternative way
of progenitor distinction. The classical definition uses the expression of IL-3Rα and
CD45RA expression on CD34+ CD38+ cells, while the alternative definition utilizes
the expression of Flt3 and CD45RA. The difference between the two gating
strategies was that on day5 the percentage of CMPs in both NF-E2 and Empty vector
transduced cells was higher than the percentage detected by the classical definition,
while the percentage of MEPs was in general lower. However the increase within the
79
CMP and GMP compartments on the expense of MEPs was also confirmed by
determining the absolute numbers of cells within each population (Fig.4.2.5 c).
Fig.4.2.5 Effect of NF-E2 overexpression in CD34+ cells on hematopoietic progenitor compartments.
The amount of CMP, MEP and GMP as percentage of CD38+CD34+ population was detected by two
alternative strategies. a) Gating strategy using IL-3Rα and CD45RA expression. b) gating strategy
based on Flt3 and CD45RA expression. c) Absolute cell number of CMP,MEP and GMP in 100.000
GFP+ cells on day 5 of culture in expansion medium. Statistical significance was determined by using
paired t-test. N is the number of experiments used in the analysis. * p=0.01 to 0.05, ** p= 0.001 to
0.01, ***p≤0.001.
80
4.3 NF-E2 overexpression elevates the number of
CD13+CD36+ double positive cells – common myeloiderythroid progenitors
A single study done by Chen et al.
176
describes a cell type positive for both
CD36 and CD13 that has a combined myeloid and erythroid potential. By
definition, CD36 is expressed during earlier stages of erythroid differentiation,
while CD13 is expressed on myeloid cells (from myeloid precursors to terminally
differentiated cells). The sorted, double positive population was able to generate
both erythroid and myeloid colonies when cultured in semisolid medium.
Unfortunately, the dual nature of the progenitor was not assed in-vivo and only
the effect of cytokines acting predominantly in erythroid and granulocytic
differentiation was tested.
When NF-E2 is overexpressed the frequency of CD36+CD13+ cells is
significantly elevated (Fig.4.3.1 a and b). This difference however was present
only in 2 (UPN:10166 and 2388) out of 5 PV patients (Fig.4.3.1 c and d). The
increase in the CD13+CD36+ population was not dependent on the JAK2V617F
presence, since one of the patients showing the increase (UPN2388) was
negative, while the other patient (UPN 10166) was positive for the JAK2V617F
mutation. The PV patients that had comparable amount of CD13+CD36+ to
healthy controls had more CD36 single positive cells, while the amount of CD13
single positive cells was reduced. In total the amount of cells expressing CD36
was always more in CD34+ cells derived from PV patients and differentiated for 8
days (Fig.4.3.1 e). The UPN numbers of all patients tested for both CD13+CD36+
and CD36+ population frequency with PRV-1 and JAK2V617F status is included in
Fig. 4.3.1 d.
81
p=0,03
b)
NF-E2
n=3
50
CD13
%CD13+CD36+
Empty control
CD13
a)
40
30
20
10
day 8
c)
CD36
PV patients
Healthy control
p=ns
d)
UPN:10166
PV n=5
HC n=6
50
%CD13+CD36+
CD13
NF
-
Em
pt
CD36
E2
y
0
40
30
20
10
day 8
UPN:10174
PV
C
CD36
H
CD13
0
UPN
Age
PRV-1
JAK2
V617F
10166
51
(+)
2388
49
(+)
(+)
(-)
10174
50
(+)
(+)
2710
72
(+)
(+)
1820
66
(+)
(+)
CD36
e)
P=0,038
100
%CD36+
80
60
40
20
day8
PV
H
C
0
Fig.4.3.1 Proposed human common myeloid-erythroid progenitor in NF-E2 overexpressing and
control PB derived CD34+ cells differentiated in-vitro. A representative FACS measurement of
CD34+ cells differentiated in-vitro for 8 days either for a) NF-E2 or Empty control vector transduced
cells or for c) HC and PV cells. A significant increase in the CD13+CD36+ double positive population in
NF-E2 overexpressing CD34+ cells of n=3 independent experiments is represented in b). The
percentage of double positive cells in PV compared to HC as well as details for the cohort of PV
patients investigated is depicted in d). Plot e) represents the percentage of cells expressing CD36.
Bars represent mean with standard error. Paired two sided t-test in b) and unpaired two sided t-test
in d) were used for statistical evaluation.
82
4.4
Effect of NF-E2 overexpression on
differentiation along the myeloid lineage
terminal
To investigate how the delay of maturation observed during earlier days of
culture in expansion medium (section 4.2), affects fate decision and terminal
differentiation in NF-E2 overexpressing cells; the expression of several lineage
specific markers was measured. Cells expressing CD36 and CD235a were defined
as erythroid, while CD41 and CD42 double positive cells as megakaryocytic. CD13 is
a marker expressed on early myeloid progenitors, granulocytes and monocytes, while
CD11b (Mac-1) is only expressed on terminally differentiated granulocytes and
monocytes.
After 8 days in culture the cells were stained with fluorochrome labeled
antibodies against the markers mentioned above. NF-E2 overexpression promoted
Epo-independent erythroid differentiation (Fig.4.4.1 a), while delaying megakaryocytic
differentiation (Fig.4.4.1 b).
Fig.4.4.1 Ectopic NF-E2 expression in CD34+ cells promotes EPO-independent erythroid
differentiation and delays megakaryocytic differentiation during culture in expansion medium. a)
The dot plot represents a CD36 and CD235a (GpA) staining on day 8 of culture, where the double
positive population contains terminally differentiated erythroid cells. The lower panel describes the
percentage of CD36+CD235a+ measured in n=4 independent experiments. b) Top - representative
FACS staining with two megakaryocytic markers and bottom – graphical representation of
percentage double positive cells for CD41a and CD42b measured in n=5 independent experiments on
day 8 of culture. Statistical analysis was done by using paired t-test.
83
The number of cells expressing markers specific for other myeloid cell types,
apart from erythroid and megakaryocytic, was slightly elevated when NF-E2 was
overexpressed (Fig.4.4.2 a and b). Statistically, the surface expression (detected as
mean fluorescent intensity MFI) of both CD13 and CD11b was not significantly
different (Fig. 4.4.2 c), which may be due to the low number of experiments included
in the analysis (n=3).
Fig.4.4.2 Increase in myeloid and granulocytic surface marker expression in NF-E2 overexpressing
CD34+ cells. a) Histogram overlays of cells transduced with NF-E2 or Empty vector stained for CD13
respectively CD11b and unstained controls. b) Statistical evaluation of percentage CD13 (left panel)
or CD11b (right panel) positive cells. c) Intensity of CD13 (left) or CD11b (right) surface expression per
cell represented as MFI. Paired one tailed t-test was used for statistical analysis of n=3 independent
experiments.
84
To confirm the differentiation status detected by FACS, cytospins were
prepared and stained with May-Grünwald-Geimsa (Fig.4.4.3) Indeed on day 5 of
culture the cells had an immature/progenitor phenotype (Fig.4.4.3 a) as confirmed in
section 4.2) and on day 8 the cells had already differentiated towards a certain
myeloid type – megakaryocytic, erythroid, granulocytic or monocytic (Fig.4.4.3 b).
The presence of cells belonging to the lymphoid lineage was not detected, since IL-7
is not part of the expansion medium and therefore lymphoid cells which are highly IL7 dependent could not be generated 177, 178.
Fig.4.4.3 Histological evaluation of in-vitro differentiating CD34+ cells. 2x104 CD34+ cells cultured in
expansion medium were collected on day 5 a) or day 8 b), cytospined and stained with MayGrünwald Geimsa. Dark blue arrow lines in a) mark early progenitor cells, while purple arrows mark
later myeloid progenitors (myelocytes). Pictures were acquired with an Axiovert microscope.
4.5 Cytokine dependent effect of NF-E2 overexpression
and silencing on erythroid and megakaryocytic
differentiation.
Since NF-E2 has an important role in HSC and progenitor physiology, as well
as in terminal differentiation processes its regulation must be under a tight control of
cytokine signals. Also, PV cells are known to be hypersensitive to certain cytokines
179-182
. The rationale for performing the experiments described bellow was: to see if
NF-E2 overexpression in PV happens because of abnormal cytokine stimulation,
whether the level of NF-E2 expression can be regulated by adjusting the
concentration of cytokine stimulation and what will be the effect of NF-E2
overexpression when the cells are stimulated (and therefore programmed) with a
lineage specific cytokine. The interest was put mainly on erythroid and
megakaryocytic differentiation, since as previously described PV patients carry
85
lesions in both Epo and Tpo signalling. Epo and Tpo are two very potent cytokines
that guide terminal erythroid and megakaryocytic differentiation respectively.
To see how changes in Tpo and SCF concentrations affect terminal
differentiation, CD34+ cells were cultured in modified expansion medium. The
modified expansion medium contains 10 times more Tpo and 5 times less SCF than
the expansion medium. This change in cytokine concentration elevated the amount
of erythroid cells generated in culture. Cells overexpressing NF-E2 responded by
promoting Epo – independent erythroid differentiation by increasing the numbers of
cells with erythroid phenotype around 2 fold (Fig.4.5.1 a). This observation was
present in CD34+ cells from PV patients that also had a 2 fold increase in Epo –
independently generated erythroid cells (Fig.4.5.1 b). Details regarding the PV
patients studied in Fig. 4.5.1 are summarized in Table 4.5.1. Judging by the amount
of JAK2V617F allele burden in the total cell population on day 8 of culture (Table 4.5.1)
it seems that the JAK2V617F mutation does not correlate to the amount of erythroid
cells generated independently of EPO in the PV patients investigated here.
Fig.4.5.1 Increased TPO and decreased SCF concentrations enhance EPO-independent erythroid
differentiation. CD34+ cells were differentiated in-vitro in expansion (20T) and modified expansion
medium (200T) for 8 days, labeled with CD36 and CD235a markers and analyzed by FACS. The
percentage of double positive cells transduced with pLeGO-iG-NF-E2 (NF-E2) or empty pLeGO-iG
(Empty) differentiated in both media is presented in a). In b) is Epo independent erythroid
differentiation of PV PB derived CD34+ cells compared to HC differentiated in modified expansion
medium. c) Effect of NF-E2 silencing in PV CD34+ cells on erythroid differentiation in modified
expansion medium. The cells were transduced with pLeGO-G-shNF-E2 or pLeGO-G-scrambled (scr)
86
control vector. Paired t-test analysis was performed in a) and c) while unpaired t-test was used to
detect differences between healthy control (HC) and PV cells in b).
Silencing of NF-E2 in PV cells decreased erythroid cell number (Fig.4.5.1 c). It
is worth to notice that the scrambled control never exceeds 10% of CD36/CD235a
double positive cells while both untreated PV and HC cells overexpressing NF-E2
reach 40% and respectively 20% of double positive cells. This observation, however,
could be due to altered differentiation caused by the lentiviral transduction itself,
since transduction with LeGO-G vectors lacking the IRES site leads to accumulation
of high amounts of GFP. Moreover, the amount of cells double positive for CD36 and
CD235a was very variable in the experiments performed (Table 4.5.2).
To see whether the overexpression of NF-E2 in PV derived CD34+ cells
differentiating in-vitro is functional on transcriptional level, the amount of β-globin
transcripts was detected on day 8 of culture. β-globin is a well proven NF-E2 target
gene 92, 105, 183 and its expression is crucial for erythroid cell function, since the protein
it encodes is a subunit of hemoglobin. In PV cells the expression of β-globin was
around 4 fold higher than in HC derived cells (Fig. 4.5.2). The PV patients with the
following UPN numbers were included in for the analysis: 2710, 10166 and 1891.
Fig.4.5.2 β-globin expression is elevated in CD34+ cells from PV patients differentiating in-vitro.
CD34+ cells differentiated for 8 days in modified expansion medium were collected to extract mRNA.
After cDNA synthesis the amount of β-globin transcripts was detected by qRT-PCR. Data analysis was
performed with the ΔΔCt method. Statistical significance was calculated with unpaired t-test.
Megakaryocytic differentiation was also affected by changes of TPO and SCF
concentrations. In expansion medium (20ng/mL TPO, 100ng/mL SCF, Flt3L, IL-6)
NF-E2 overexpression delayed megakaryocytic differentiation, while in modified
expansion medium (200ng/mL TPO, 20ng/mL SCF, Flt3L, IL-6)
the effect was
reversed - there were more megakaryocytic cells when NF-E2 was overexpressed
(Fig. 4.5.3 a). There was no difference between the number of megakaryocytic cells
generated from HC or PV CD34+ cells cultured in modified expansion medium (Fig.
87
4.5.3 b). Silencing of NF-E2 in PV cells had no statistically significant effect on
megakaryocytic differentiation (Fig. 4.5.2 c). Details about the PV patients included in
these experiments are summarized in Table 4.5.1 and Table 4.5.2.
UPN:
2710
Age: PRV-1
72
(+)
%CD36+CD235+
37
%CD41+CD42+
24
hNF-E2/ 100.000 B2M
60841
V617F
% JAK2
allele burden
41
10166
10174
51
50
(+)
(+)
48
68
14
6
98329
63209
83
3
1820
66
(+)
32
7
38439
22
Table.4.5.1 PV patients analyzed for erythroid and megakaryocytic marker expression in modified
expansion medium.
Fig.4.5.3 Increased TPO and decreased SCF concentrations exert an effect on megakaryocytic
differentiation of CD34+ cells. CD34+ cells differentiated in-vitro in expansion (20T) or modified
expansion medium (200T) for 8 days, were labeled with CD41a and CD42b markers and analyzed by
FACS. a) Depicts the percentage of double positive cells (CD41+CD42+) transduced with pLeGO-iGNF-E2 (NF-E2) or empty pLeGO-iG (Empty) differentiated in both media. b) Megakaryocytic surface
marker expression in PV and HC CD34+ cells differentiated in modified expansion medium for 8 days.
c) The effect of NF-E2 silencing in PV CD34+ cells on megakaryocytic differentiation. Statistical
analysis in a) and c) was done with paired t-test, while unpaired t-test was used to analyze the data in
b). * p=0.01 to 0.05, ** p= 0.001 to 0.01.
UPN:
1971
10166
1647
1728
Age:
65
51
68
76
PRV-1
(+)
(+)
NA
NA
V617F
JAK2
status
(+)
(+)
(+)
(+)
%CD36+CD235+
shNF-E2/scrambled
4.6 / 5.5
10.7 / 16.5
5.9 / 10
8.2 / 4.3
%CD41+CD42+
shNF-E2/scrambled
7.3 / 5.3
4.6 / 9.2
3.9 / 8.9
1.7 / 2.15
Table.4.5.2 PV patients analyzed for shNF-E2 effect on erythroid and megakaryocytic marker
expression in modified expansion medium.
88
Both the expansion and the modified expansion medium contain a mix of
cytokines that activate multiple signaling cascades and promote multilineage
differentiation within the myeloid branch of hematopoiesis. To investigate whether the
altered commitment of NF-E2 overexpressing HC and PV cells toward the erythroid
and megakaryocytic lineage is specifically influenced by certain cytokines, CD34+
cells were cultured in media containing different concentrations of Tpo or Epo only or
both Tpo+Epo. In all cases, SCF was supplemented to sustain cell proliferation. On
day 12 of culture the cells were collected and analyzed for lineage marker
expression. In these media there was no elevation in erythroid marker expression
when NF-E2 was overexpressed in HC CD34+ cells (Fig. 4.5.4 a) nor in PV CD34+
cells (Fig.4.5.4 b). NF-E2 silencing in PV CD34+ cells did not reduce the amount of
erythroid cells in culture either (Fig.4.5.4 c). Later NF-E2 quantification in cells
stimulated with EPO shows that the PV cells transduced with a scrambled shNF-E2
vector do not reach the level of expression detected in untransduced cells (Fig.4.6.1).
Therefore an eventual effect of NF-E2 silencing on erythroid differentiation in PV
cannot be excluded.
Fig.4.5.4 The effect of: different TPO concentrations, TPO and EPO in combination, and EPO alone
on later erythroid differentiation. The CD34+ cells were cultured in SFEM medium containing
50ng/mL TPO (50T), 200ng/mL TPO (200T), 50ng/mL TPO with 1IU/mL EPO (E+T) or 1IU/mL EPO (Epo)
in each case 25ng/mL SCF was added to the medium. On day 12 of culture the cells were collected,
stained with CD36 and CD235a and analysed by FACS. The y axis in all three graphs represents the
89
percentage of cells double positive for CD36 and CD235a. a) cells transduced with pLeGO-iG-NF-E2
(NF-E2) or empty pLeGO-iG (Empty), b) HC and PV cells and c) PV cells transduced with pLeGO-GshNF-E2 or pLeGO-G-scrambled (scr) control vector.
While erythroid differentiation was not influenced by TPO and EPO signaling
alone or in combination, TPO had an effect on the megakaryocytic differentiation of
NF-E2 overexpressing HC and PV CD34+ cells (Fig.4.5.5 a and b). In both cases,
the amount of cells double positive for CD41/CD42 was decreased. Interestingly,
silencing of NF-E2 in PV cells also reduced the number of CD41/CD42 double
positive cells (Fig. 4.5.5 c) but only in one cytokine condition (50ng/mL TPO,
25ng/mL SCF) where the strongest silencing was achieved (Fig. 4.6.1).
Fig.4.5.5 The effect of: different TPO concentrations, TPO and EPO in combination, and EPO alone
on later megakaryocytic differentiation. The CD34+ cells were cultured in SFEM medium containing
all supplemented with 25ng/mL SCF. On day 12 of culture the cells were collected, stained with
CD41a and CD42b and analyzed by FACS. The percentage of double positive cells for CD41a and
CD42b is depicted in all three graphs. a) cells transduced with pLeGO-iG-NF-E2 (NF-E2) or empty
pLeGO-iG (Empty), b) HC and PV cells and c) PV cells transduced with pLeGO-G-shNF-E2 or pLeGO-Gscrambled (scr) control vector. * p=0.01 to 0.05, ** p= 0.001 to 0.01.
V617F
UPN:
2388
1703
10393
Age:
49
57
58
PRV-1
(+)
(-)
NA
JAK2
UPN:
Age:
PRV-1
JAK2
status
(-)
(-)
NA
V617F
status
Table.4.5.3 PV patients analysed for erythroid and
megakaryocytic marker expression in different
concentration of TPO, EPO and TPO and Epo in
combination. NA – not available, (+) – positive, (-) negative
Table.4.5.4 PV patients analysed for shNF-E2 effect on
erythroid and megakaryocytic marker expression in
different concentration of TPO, EPO and TPO and Epo90
in combination. NA – not available, (+) – positive, (-) negative
2388
1703
10393
1892
49
57
58
52
(+)
(-)
NA
(+)
(-)
(-)
NA
NA
During the process of maturation the megakaryocytic cells undergo a process
called endomitosis thus becoming fully functional megakaryocytes able to shed
platelets. During endomitosis the amount of DNA and the size of the megakaryocyte
are doubled but the cell does not divide. By measuring the quantity of DNA the ploidy
state can be determined, which is a hallmark of megakaryocytic maturation and
function.
Megakaryocytes derived from PV cells in-vitro increased their ploidy to a
higher level than the HC (Fig.4.5.6 a). In PV there were more megakaryocytes with
8N and more than 8N class of ploidy. Silencing of NF-E2 in PV reversed the
phenotype by promoting the generation of more immature megakaryocytes (Fig.4.5.6
b). Mainly by increasing the number of megakaryocytes with 2N ploidy and reducing
the number of ones with 8N. Higher than 8N plody class was not affected by the
silencing. This finding may suggest that NF-E2 is more important in initial stages of
megakaryocyte development.
Fig.4.5.6 Megakaryocytic ploidy. CD34+ cells cultured for 12 days in SFEM medium supplemented
with 50ng/mL TPO and 25ng/mL SCF were stained with Hoechst and anti-CD41a/anti-CD42b
antibodies. Large cells (with high FSC value), double positive for CD41a and CD42b were analyzed for
their DNA content (Hoechst fluorescent intensity) with FACS. a) n=3 for PV and n=5 for HC
independent experiments were performed. b) PV cells transduced with pLeGO-G-shNF-E2 or pLeGOG-scrambled. Statistical significance was determined by using unpaired t-test in a), while in b) paired
t-test was used. Where * p=0.01 to 0.05, ** p= 0.001 to 0.01, ***p≤0.001.
91
4.6 Cytokine dependent NF-E2 expression
The amount of NF-E2 mRNA expressed in CD34+ cells cultured in different
cytokine conditions was detected by qRT-PCR. To evaluate the level of expression
the ∆∆Ct method was used. Since this method offers only relative comparison
between the analyzed samples, the sample with lowest expression of NF-E2 was
used as a common calibrator. Within the analyzed samples the lowest expression
was detected when NF-E2 was silenced in PV cells cultured in 50 ng/mL TPO and 25
ng/mL SCF. Fig. 4.6.1 depicts the relative expression of NF-E2 influenced by
different cytokine stimulation. In healthy controls EPO induced NF-E2 expression
while TPO had an antagonistic effect. EPO caused around 10 fold expression, but
addition of TPO together with EPO decreased NF-E2 expression to levels not much
higher than when only TPO was used. The lowest expression of NF-E2 was detected
when cells were cultured in expansion medium (Tpo, Flt3, SCF, IL-6). NF-E2
expression was elevated in PV cells in all culture conditions.
Fig.4.6.1 Cytokine dependent NF-E2 expression. CD34+ cells originated from either healthy controls
(HC) and PV patients, or PV cells transduced with pLeGO-G-shNF-E2 and control pLeGO-G-scrambled
vector were cultured in different cytokine conditions. The basic medium was SFEM containing
all supplemented with 25ng/mL SCF and modified expansion medium (mix). Cells were collected for
mRNA extraction on day12 of culture, except when cultured in modified expansion medium where
NF-E2 expression was scored on day8 of culture. After cDNA synthesis the amount of NF-E2
transcripts was detected by qRT-PCR. The ΔΔCt method was used for data analysis where the sample
with lowest NF-E2 expression in the silencing experiments was set as a common calibrator. The
dashed line represents the reference value which equals 1.
92
4.7 Effect of NF-E2 overexpression on apoptosis and
proliferation in CD34+ cells
Since NF-E2 overexpression affects the expansion of both HSC and
progenitor cells, and terminally differentiating cells within the myeloid lineage, an
important question to address is whether the expansion observed is because of
alterations in the processes of cell apoptosis and proliferation. To study eventual
induction of apoptosis the amount of outer membrane phosphatidylserine exposure
was detected by staining with AnnexinV. Approximately 24h and 48 h after the first
viral transduction with pMYSiG-NF-E2 or pMYSiG-Empty vector the CD34+ cells
were collected and stained with Annexin V. There was no difference in the amount of
apoptotic cells between NF-E2 and Empty vector transduced cells (Fig. 4.7.1 a and
b). When the cells were cultured in modified expansion medium the percentage of
apoptotic cells was from 10% to 15% for both time points (Fig. 4.7.1 a). However,
when the cells were cultured in erythroid differentiation promoting medium containing
EPO (Vaincheker medium), as expected the amount of apoptotic cells was lower
(Fig.4.7.1 b). The observed decrease in apoptosis happens due to upregulated BclXL expression as a result of EPO stimulation 65.
Fig.4.7.1 NF-E2 overexpression does not affect apoptosis in CD34+ cells. After one day in culture
the cells were retrovirally transduced with pMYSiG-NF-E2 or pMYSiG-Empty vector. Two and three
days after transduction the cells were collected and the apoptotic status was determined by
Annexin-V/Sytox Orange staining. Single positive cells for Annexin-V were taken as apoptotic cells.
The cells were cultured in modified expansion medium a) or Vainchenker medium b). Bars represent
mean with standard error. The groups were compared to each other by using paired t-test, but
statistical significance was not detected.
To estimate the status of proliferation in NF-E2 or empty vector transduced
cells two separate methods were used. One of the methods is based on the ability of
proliferating cells to reduce a substrate contained in the AlamarBlue reagent which
changes its fluorescence properties. 24h, 48h and 72h after viral transduction 1000
93
CD34+ cells were seeded out and cultured in modified expansion or Vainchenker
medium with 10% AlamarBlue. Fluorescence detected as relative fluorescent units
(RFU) was measured daily. An example plot from one time point comparing NF-E2
and empty vector transduced cells proliferation status is shown in Fig. 4.7.2 a. As
depicted in the plot (Fig. 4.7.2 a) NF-E2 overexpression did not affect proliferation of
CD34+ cells in either modified expansion or Vainchenker medium (Table 4.7.1).
A more sensitive method (ClickItEdu) was used to evaluate cell proliferation
and cell cycling of NF-E2 and empty vector transduced CD34+ cells. The cells were
incubated for 4h with Edu (a thymidine analog), during which time only the cells going
through cell division could incorporate Edu into their DNA. Afterward the cells were
fixed, permeabilised and fluorescently stained against Edu. Hoechst was used to
stain DNA. The detection of the fluorescent signals for Edu and Hoechst was done
with the ScanR screening microscope. Only the GFP+ (transduced) cells were
analysed for Edu incorporation. There was not a significant difference detected for
Edu incorporation between the NF-E2 and Empty control transduced CD34+ cells
during day 1 to 3 after the first viral transduction (Fig. 4.7.2 b).
Fig.4.7.2 NF-E2 overexpression does not affect proliferation of CD34+ cells. The ability of CD34+
cells to reduce the AlamarBlue reagent or to incorporate Edu was used to test their proliferation
status. a) Around 1000 pMYSiG-NF-E2 or pMYSiG-Empty vector transduced CD34+ cells were cultured
up to 1 week in modified expansion or Vainchenker medium including 10% AlamarBlue reagent.
Fluorescence was measured daily as relative fluorescence units (RFU). b) 20.000 CD34+ cells
transduced with pMYSiG-NF-E2 or pMYSiG-Empty vector were incubated on an adherent slide in
modified expansion medium containing Edu for 4h. Fluorescently labeled Edu and the GFP signal
were detected microscopically. N is the number of independent experiments included for statistical
analysis. Statistical significance was evaluated by using the paired t-test.
When using the AlamarBlue method proliferation is monitored during a longer
time window (around 5 days long until the reaction gets saturated). This enables
detection of changes in cell proliferation during the process of differentiation. On the
other hand the ClickIt Edu method gives information about the ability of the cells to
94
proliferate within a short time frame (4h in the experiments done), but it is more
sensitive that the AlamarBlue method.
Table.4.7.1 CD34+ cells proliferation is not affected during differentiation in modified expansion
and Vainchenker medium. The time points when the cells were seeded out and put in culture with
AlamarBlue are indicated in the top column. NS indicated non significant difference in proliferation
for pMYSiG-NF-E2 or pMYSiG-Empty vector transduced cells for each time point. An example plot for
measurement performed for one time point is depicted in Fig.4.7.2
4.8 NF-E2 overexpression in-vivo influences cell cycle in
murine HSC and progenitor cells, but not apoptosis.
To study the effect of NF-E2 overexpression in-vivo, a transgenic mouse
overexpressing human NF-E2 in the hematopoietic system was generated in our lab.
A vav promoter regulated the transcription of the hNF-E2 transgene, ensuring its
expression in HSC and progenitors up to their more differentiated progeny. The
transgenic mice displayed a complex phenotype reflecting many features typical for
MPNs like: thrombocytosis, leukocytosis, expansion of the HSC and progenitor pool
(mainly CMPs and MEPs).
To see whether the expansion in the HSC and progenitor compartments is
caused by disrupted cell cycle regulation or a defect in apoptosis, bone marrow cells
were specifically stained and analyzed by FACS.
The KSL (kit+sca-1+Lin-)
population containing HSCs and the KL (kit+sca-1-Lin-) population composed of
hematopoietic progenitors were analysed for DNA content and Annexin-V exposure.
Less of the transgenic (TG) KSL cells were in cycle (Fig. 4.8.1 a), while on the other
hand more cells of the KL population were cycling (Fig. 4.8.1 c) when compared to
wild type (WT) mice. The amount of apoptotic cells in both KL and KSL was equal for
both TG and WT mice (Fig.4.8.1 c and d).
95
Fig.4.8.1 Cell cycle and apoptosis status in HSC (KSL population) from hNF-E2 transgenic and wild
type mice. Total BM was stained with surface specific antibodies against lineage markers, sca-1 and
c-kit. The KSL (Lin-, sca-1+, c-kit+) and the KL (Lin-, sca-1 -, c-kit+) population were analyzed for DNA
content, detected by Hoechst staining a) and c) and for Annexin-V exposure on the outer membrane
b) and d). Cell containing more than 2n are defined as cycling cells, while cells positive for Annexin-V
and unstained by Sytox (necrotic marker) are defined as apoptotic. Statistical significance was
determined with t-test, bars represent mean values with standard deviation.
Since some MPN patients respond well to therapy with vorinostat (HDAC
inhibitor) and reduce the levels of NF-E2 expression during the therapy, we have
treated the TG mice with this drug to see whether it could reverse the MPN
phenotype observed. The treatment reduced hNF-E2 expression and platelet
numbers in the TG mice
79
. Possibly it also reverses the phenotype observed within
the TG KSL and KL population, by normalizing the cell cycling (Fig.4.8.2 a and c),
while not affecting apoptosis (Fig.4.8.2 b and d).
96
Fig.4.8.2 Cell cycle and apoptosis status in hematopoietic stem cell and progenitor populations
from hNF-E2 transgenic and wild type mice treated with Vorinostat (SAHA). Total BM isolated from
Vorinostat (SAHA) treated animals was stained with surface specific antibodies against lineage
markers, sca-1 and c-kit. The KSL (Lin-, sca-1+, c-kit+) and the KL (Lin-, sca-1 -, c-kit+) population were
analyzed for DNA content, detected by Hoechst staining a) and c) and for Annexin-V exposure on the
outer membrane b) and d). Cell containing more than 2n are defined as cycling cells, while cells
positive for Annexin-V and unstained by Sytox (necrotic marker) are defined as apoptotic. Statistical
significance was determined with t-test, bars represent mean values with standard deviation.
97
5. Discussion
5.1 NF-E2 expression is 2 fold higher in CD34+ cells
derived from PV patient’s peripheral blood.
The overexpression of NF-E2 in PV patients was first detected and quantified
in granulocytes, where the overexpression was 2 to 40 fold higher than in healthy
controls
72
cell types
. The overexpression was also confirmed in other terminally differentiated
77, 78
. However fluctuations in TF expression during hematopoiesis are
important for lineage commitment and/or maintenance in primed (still not fully
committed) stages of differentiation. This is why the concentrations of hematopoietic
TFs are different in early and late stages of hematopoiesis. Already few decades ago
the expression of NF-E2 has been detected in terminally differentiated cells of the
myeloid lineage (erythroid, megakaryocytic, granulocytic)
78
. With the development of
more sensitive and sophisticated techniques during the years, the expression of NFE2 in less differentiated progenitors became feasible. There are few publications
describing the expression of NF-E2 during earlier stages of hematopoiesis. A study
done by Edvardsson et al.
80
reports the expression of NF-E2 in CMPs, its sequential
0,5 fold down-regulation in GMPs and around 2 fold up-regulation in MEPs. CD34+
cells derived from healthy donors bone marrow were used for these experiments. In
another study the same group investigated the expression of NF-E2 during erythroid
and neuthrophil differentiation of bone marrow healthy donor derived CD34+ cells
cultured in-vitro
81
. During neutrophil maturation the expression of NF-E2 was down-
regulated, while during erythroid culture NF-E2 was up-regulated gradually as the
cells were becoming more erythroid.
It was of great importance to confirm that the overexpression of NF-E2 is
present during early stages of differentiation in PV patients derived cells. For this
reason CD34+ cells isolated from peripheral blood were cultured in-vitro and the
amount of NF-E2 expression was detected by qRT-PCR from day 4 to day 8 of
culture. During this time frame the overexpression of NF-E2 in PV CD34+ cells
remained constantly elevated by 1.5 to 2 fold (Fig. 4.1.1 b). Also there weren’t any
changes in the amount of NF-E2 expression between the different days. The CD34+
population is a heterogeneous population containing HSCs and HPCs. During the invitro culture in expansion medium the cells maintain the immature HSC and HPC
98
phenotype until day 5 (Fig. 4.2.3; 4.2.4; 4.2.5), after which the cells get differentiated
and gradually lose CD34 expression (Fig. 4.1.1 a).
In conclusion peripheral blood derived CD34+ cells from PV patients
ovrexpress NF-E2 by around 2 fold during early stages of differentiation (HSCs and
HPCs). The same level of overexpression (2 fold more than healthy controls) is
maintained also in the more differentiated progeny generated in-vitro regardless of
the cytokines included in the culture medium (Fig.4.6.1).
HSC, MPP, CMP and GMP expansion.
Once the overexpression of NF-E2 in CD34+ cells from PV patients was
confirmed the next question addressed in this thesis was to determine what influence
a 2 fold NF-E2 overexpression has on early hematopoiesis. When PB CD34+ cells
isolated from healthy donors were transduced with lentivirus encoding NF-E2 and 2
fold level of overexpression was achieved, the amount of HSC, CMP, GMP and MEP
were quantified. The amount of HSCs, MPPs, CMPs and GMPs was significantly
elevated while MEPs number decreased (Fig. 4.2.5). Exactly these compartments
are being affected in PV and moreover reported to be elevated in peripheral blood of
PV patients
168
. This observation in human CD34+ cells overexpressing NF-E2 fits
with our hNF-E2 transgenic mouse model where we have also observed an increase
in the frequency of HSC and CMP populations
79
. The difference between the mouse
model and the human CD34+ in-vitro system is that the GMP frequency is not
affected in the mouse, while being expanded in the human system. Although bone
marrow cells from the transgenic mice were able to form more CFU-GMs and had
elevated neutrophil counts in peripheral blood. Also MEP frequency is increased in
the NF-E2 transgenic mice (accompanied with an elevation in CFU-E and BFU-E
formation), but is decreased in the human system. The discrepancies between the
two experimental systems may be due to differences between mouse and human
physiology, which is also reflected in the surface marker expression used for defining
the progenitor populations (Fig.2.5). The human CD34+ in-vitro system was designed
to study early EPO independent stages of hematopoiesis in a controlled cytokine
environment , while the mouse model studies the broad physiological effects of NFE2 overexpression. The different level of NF-E2 overexpression achieved in the
human CD34+ cells (2 fold) and the two hNF-E2 transgenic mouse strains studied (3
99
fold and 30 fold) could also play a role in the expansion of MEPs and GMPs. Despite
the slight discrepancies between the two experimental systems we can conclude that
NF-E2 plays an important role in the physiology of HSC and progenitor
compartments and its overexpression is connected to MPN’s pathology.
Another question that arises here is whether JAK2V617F mutation is the dominant
mutation responsible for PV pathology or there is a preceding event affecting
hematopoiesis in MPN . Indeed JAK2V617F has an effect on PV development, but
most likely other molecular defects acting earlier in hematopoiesis have to be
acquired prior JAK2V617F in order to develop PV. A recent study demonstrates that
JAK2V617F influences later stages of hematopoiesis and does not affect the expansion
of HSC and HPC compartments in MPN patients 169. Moreover expansion of the early
hematopoietic compartments is observed even in the absence of JAK2V617F.
Aberrations in the function of other molecules linked to MPN and leukemia also lead
to an increase in HSC numbers. For example, TET2 loss of function mutations led to
an increase of the LSK population in a murine model, prior development of myeloid
leukemia phenotype in the mice
184
. All together these findings strongly suggest that
aberrations in NF-E2 expression during early stages of hematopoieis are a crucial
event in the development of MPNs. What are the exact molecular mechanisms
triggered by NF-E2 overexpression leading to expansion of the HSC and early
progenitor compartments remain to be further elucidated.
Following the pattern of NF-E2 expression during normal human hematopoiesis
elevation in NF-E2 expression in CMPs should lead to the generation of MEPs, while
the decrease of expression in CMPs should result in GMPs generation
80
. Logically it
would be expected that NF-E2 overexpression starting from HSC would eventually
promote MEP generation while reducing GMP generation. Elevated MEP numbers
could offer an explanation for the erythrocytosis typical for PV patients, although the
frequency of the MEP population is not elevated in PV patients
168
. However,
unexpectedly the amount of MEPs was lower while the number of GMPs was higher
in CD34+ cells overexpressing NF-E2 in the culture conditions used (expansion
medium) for the experiments performed in this thesis (Fig. 4.2.5). A possible
explanation for this observation would be the concentration of NF-E2 within the
differentiating CD34+ cells. As discussed before, transcription factor concentration
regulated by cytokine signaling is crucial for lineage commitment and fate decision.
The cytokine mix used in these experiments (Flt3, TPO, IL-6, SCF –expansion
100
medium) keeps the expression of NF-E2 on a lower level (Fig. 4.6.1). Lower levels of
NF-E2 might be enough to prime the CMP towards both MEP and GMP
differentiation, but higher expression of NF-E2 within the MEP might be necessary to
sustain their survival, proliferation and further maturation. NF-E2 expression and
functional activity during different maturation stages in hematopoiesis is regulated by
cytokine stimulation and depends on the differential expression of cytokine receptors
on cells differentiating along a certain lineage. EPO is a cytokine that regulates NFE2 expression and activity and has a indispensable role in erythroid differentiation.
Although EPO is not the only factor regulating NF-E2 expression and its expression
occurs even in the absence of EPO, there are few studies providing evidence on how
EPO affects NF-E2. In erythroid cell lines the stability of NF-E2 mRNA was higher
than in myeloid cell lines and the stimulation of the erythroid cell line with EPO
induced the expression of NF-E2
185
. Also in CD34+ cells differentiating in-vitro the
expression of NF-E2 protein increased 3 days after EPO stimulation, but the
occupancy of HS sites within the α-globin locus by NF-E2 was present before EPO
stimulation
186
. The recruitment of Pol II on the same HS sites in the α-globin locus
increased upon EPO stimulation
186
. Moreover the expression of the EPO receptor
was detected to be moderate in CMPs, high in MEPs and low in GMPs
80, 187
, which
fits with the expression level of NF-E2 in these progenitors 80 (Fig.5.4.1).
These findings would argue that NF-E2 is able to prime immature hematopoietic
cell types, but the later lineage outcome (expression of lineage specific genes) is
highly dependent on cytokine signaling. However in the absence of EPO a slight (1.5
to 2 fold) increase in the amount of NF-E2 in HSCs and HPCs delays hematopoietic
maturation and promotes GMP expansion (chapter 4.2). Regarding PV hematopoieis
a possible model uniting both NF-E2 overexpression and JAK2 mutations leading to
abnormal amplification and/or independence of cytokine signals would be the
following:
-
NF-E2 ovrexpression increases the pool of HSCs, MPPs , CMPs in the
patients
-
The CMPs (possibly the MEPs and the GMPs) are primed by NF-E2 and other
cooperating factors making the transcription
regulatory elements from a
variety of lineage specific genes accessible and prone to activation after the
cell receives the proper cytokine signal.
101
-
After cytokine stimulation the lineage specific genes “primed” by NF-E2 get
transcribed and lineage commitment occurs.
-
Most
of
the
cytokines
identified
to
guide
myeloid,
erythroid
and
megakaryocytic lineage commitment and differentiation activate JAK2
signaling cascades.
-
Inappropriate signaling through mutant JAK2 (cytokine independent or
unphysiologically amplified) in CMPs, MEPs, GMPs and lineage committed
cell boosts the generation of terminally differentiated cells (erythrocytes,
platelets and granulocytes).
-
NF-E2 overexpression combined with JAK2 mutations leads to trilineage
hyperplasia in PV patients
Epo-independent erythroid maturation.
The ability of PV cells to mature as erythroid in the absence of EPO is a well
described feature and it is used as a diagnostic tool
188
. PV patient’s derived cells
from bone marrow or peripheral blood can form EPO independent colonies
(endogenous erythroid colonies - EECs) when cultured in semisolid medium 189-192. In
these early studies either total bone marrow or the mononuclear cell fraction from
peripheral blood was used. Ugo et al. have confirmed that CD34+ cells from PV
patients become more erythroid than healthy controls when cultured in liquid serum
free medium without EPO (the medium contained IL-3 and SCF) 193. We were able to
enhance the observation done by Ugo et al. in a culture medium containing Flt3,
TPO, IL-6, SCF (modified expansion medium). In these conditions EPO independent
erythroid differentiation was present in both healthy donor and PV patient CD34+
cells differentiating in-vitro, but the amount of erythroid cells was 2 fold higher in PV
cells (Fig.4.1.2).
Up to my knowledge there is only one publication describing the formation of
EECs induced by NF-E2 overexpression. In this study NF-E2 overexpression in
primary hematopoietic progenitors from mouse liver induced EEC formation
185
. NF-
E2 overexpression in human CD34+ cells was tested in our lab and in our
experimental system NF-E2 overexpression in healthy human CD34+ cells enhanced
EPO-independent eyrthroid differentiation by 2 fold. Thus recapitulating the PV
102
phenotype we have observed when culturing the cells in modified expansion
medium.
5.4 Epo-independent erythroid maturation most likely
occurs through another progenitor cell type
independently of MEP
Since NF-E2 overexpression in healthy donor CD34+ cells promotes HSC, MPP,
CMP and GMP expansion at the expense of MEPs, the next question to ask was how
this shift influences terminal differentiation. The amount of myeloid and granulocytic
cells were slightly elevated when NF-E2 was overexpressed (Fig. 4.4.2), which
corresponds to the slight increase in the frequency of GMPs. On the other hand the
amount of megakaryocytic cells was less, while the amount of erythroid cells was
more (Fig. 4.4.1).
The fact that there are less MEPs and less megakaryocytic cells when NF-E2 is
overexpressed in CD34+ cells cultured in expansion medium, while there are more
erythroid and myeloid cells made us consider an alternative hierarchy of
hematopoiesis (Fig. 5.4.1). The most accepted “classical” model of hematopoiesis
follows a linear scheme while the new myeloid model follows a less hierarchical
scheme (see introduction chapter 2.4).
Our experimental data is not sufficient to prove either of these models but it
supports the notion that erythroid cells can be generated through an alternative way
than through the MEP. MEPs upregulate the expression of the EPO receptor and
their further development is highly dependent on the EPO signaling cascade. Since
in our experimental system EPO was not included, the generation of erythroid cells
should go through progenitors which do not depend on EPO signaling. These
progenitors may be a subset of cells within the CMP population that have a mixed
myeloid-erythroid potential and could be defined as common myeloid-eyrthroid
progenitors (CMEP). A cell type in human hematopoiesis having both eyrthroid and
myeloid potential has been described only in one study up to date 176. These cells are
double positive for CD36 and CD13. NF-E2 overexpression in healthy control CD34+
cells led to doubling the amount of CD36+CD13+ cells (Fig. 4.3.1 b). In the cohort of
PV patients investigated in this thesis 2 out of 5 patients showed a doubling increase
of the CD36+CD13+ (Fig. 4.3.1 d). Although more experimental evidence is
necessary to characterize the CD36+CD13+ population as a separate entity
103
functioning as an intermediate of both erythroid and myeloid differentiation, it is likely
that the enhanced EPO-independent differentiation (formation of EECs) in PV is due
to increased numbers of progenitor cells that can become erythroid without EPO.
Fig. 5.4.1 Alternative model of hematopoiesis and the influence of NF-E2 overexpression
during hematopoietic development. Black arrows show already established and experimentally
proven transitions from more immature to committed cell types. Dashed arrow lines show a possible
transitions or increase in frequency of a certain cell population that need stronger experimental
evidence. Red arrow lines indicate how NF-E2 overexpression influences the frequency of a certain
cell type - upward arrows indicate an increase and downward arrows a decrease. Side boxes contain
information about the relative mRNA expression of TPOR, EPOR and NF-E2 during the different
stages of hematopoiesis along the myeloid lineage. HSC-hematopoietic stem cell, MPP – multipotent
progenitor, CMP – common myeloid progenitor, CMEP – common myeloid-erythroid progenitor, MEP
– megakaryocite-erythroid progenitor, GMP – granulocyte-monocyte progenitor, TPOR –
thrombopoietin receptor (c-mpl), EPOR – erythropoietin receptor, NF-E2 – nuclear factor-erythroid 2.
Fig. 5.4.1 describes how NF-E2 influences hematopoiesis in our liquid culture
system and offers an alternative to the classical model of hematopoiesis. The
differential expression of the TPOR
80, 187
, EPOR
187
and NF-E2
79-81
(additionally see
Fig.4.6.1 for NF-E2 expression in erythroid and megakaryocytic cells) during human
hematopoiesis is also described. Another novelty in this scheme is the expression of
NF-E2 that starts from the HSC, which was not acknowledged before.
104
5.5 NF-E2 overexpression does not affect apoptosis and
proliferation in HC CD34+ cells, but most likely it
influences proliferation and cell cycle in murine HSC
and HPC populations.
A possible explanation for the expansion of the HSC, MPP, CMP and GMP
populations in CD34+ cells transduced with NF-E2 would be enhanced proliferation
and survival or impaired apoptosis caused by NF-E2 expression. However, this was
not the case. NF-E2 overexpression did not influence the rate of DNA synthesis in
CD34+ cells nor did it affect their survival (Fig. 4.7.2). This was proven by two
different methods that asses metabolic activity and rate of DNA synthesis. Also
apoptosis was not influenced by NF-E2 overexpression in both modified expansion
medium and erythroid differentiation (Vainchenker) medium (Fig. 4.7.1). As expected
EPO (Vainchenker medium) lowered the amount of apoptotic cells in both control and
NF-E2 overexpressing cells. Moreover, the cell counts were equal in Empty vector
and NF-E2 transduced healthy donor CD34+ cells. Because of technical limitations it
was not possible to detect the effect of NF-E2 overexpression within separate cell
populations (HSC, MPP, CMP, GMP, MEP and lineage committed cells).
In the hNF-E2 transgenic mice (TG) the findings were different. Here we have
studied the amount of cells in cycle (in S and G2M phase of the cell cycle) in the KSL
and KL population. The KSL population is enriched in HSC and MPPs, while the KL
population contains oligopotent progenitors (CMPs, MEPs, GMPs). The KSL
population which was significantly elevated in the TG mice had less cycling cells and
more cells that had the ability to efflux Hoechst (Fig.4.8.1 a). Cells able to efflux
Hoechst through the ABCG2 transporters (mdr type of cell surface transporters) have
been defined as the side population (SP)
194
. The SP cells express surface markers
typical for HSC and are able to reconstitute the hematopoietic system in lethally
irradiated mice
172
. The detection of the SP is done by plotting blue against red
fluorescence of Hoechst. In our experimental setup we could only detect blue
fluorescence of Hoechst, where a sub G1 peak was always present only in the KSL
population (Fig. 3.8.2.1). Treatment with Verapamil (a drug blocking Ca++ channels
and ABCG2 transporters, thus blocking Hoechst efflux)
173
diminished the sub G1
peak meaning that the cells in the sub G1 peak are mostly cells that could be defined
as the SP. Still the detection of the red fluorescent signal of Hoechst is necessary to
increase the sensitivity of the method. An increase in the amount of SP cells and a
105
decrease in cycling cells within the KSL population would mean that the hNF-E2
overexpression in mice increases the amount of quiescent HSCs. However, a more
sensitive method should be used to confirm this observation. An alternative would be
to use RNA and DNA staining in combination. Quiescent cells (G0 of the cell cycle)
have low amounts of both RNA and DNA 195.
On the other hand hNF-E2 overexpression in the TG mice leads to an increase in
the amount of cycling cells within the KL population (Fig. 4.8.1 c). This can explain
the increased number of cells with KL phenotype and would mean that NF-E2
overexpression increases the proliferation rate in oligopotent progenitors.
Indeed NF-E2 overexpression has an effect on cell cycle regulation of the KSL
and KL populations in mice, but to explain the opposite effect in both populations a
more detailed study should be done. It would be interesting to search for genes
involved in cell cycle regulation transcriptionally regulated by NF-E2 and compare
their differential expression in KSL and KL populations.
As in the human system, apoptosis was not affected by hNF-E2 overexpression
in both KSL and KL population in the TG mice (Fig. 4.8.1 b and d).
SAHA (Vorinostat) is a chemical compound that acts like a broad HDAC (histone
deacethylases) inhibitor. It has gone through several Phase I and II clinical trials for
treatment of advanced solid tumors and hematological malignancies
196-198
. 17% of
the patient suffering from advanced leukemias or myelodisplastic syndromes showed
complete recovery or hematologic improvement
199
. Givinostat is another type of
broad HDAC inhibitor that has been used in a Phase II trial for treatment of MPN
patients
200
. The expression of NF-E2 in Givinostat treated patients decreased 84
days after the start of the treatment
79
. Vorinostat had the same effect on NF-E2
expression in our TG mice. Treatment with HDAC inhibitors completely alters the cell
biology of both normal and malignant cells. It affects crucial processes like apoptosis
201
, autophagy
201
, cell cycle
202, 203
and metabolic enzymatic activity
204
. It was
previously reported that SAHA treatment of various cell lines induces apoptosis
205, 206
and regulates cell cycle progression
201,
202, 203
. However, SAHA treatment did not
influence apoptosis in KL and KSL populations. The amount of apoptotic cells stayed
at the same level before and after SAHA treatment in both TG and WT mice (Fig.
4.8.2 b and d). This observation gives supporting evidence that NF-E2 does not
influence apoptosis during early hematopoiesis (both mouse and human) even when
its concentration reaches unphysiological values. Most likely SAHA treatment
106
induces cell cycle in the KSL population of the TG mice while not affecting the WT,
thus reversing the phenotype caused by hNF-E2 overexpression (compare Fig.4.8.1a
to Fig 4.8.2 a). The effect on the KL population was again an increase of the number
of cells entering the cell cycle, but the effect was equally present in both TG and WT
animals. More experimental data is necessary to confirm these observations (Fig.
4.8.2 a and c). SAHA treatment was previously connected to blockage in the G1
phase of the cell cycle by inducing p21 (cell cycle kinase inhibitor) in a study done
with a human bladder carcinoma cell line, but this effect was present only when low
dose of SAHA was used. Increasing SAHA concentrations resulted in an increase of
the number of cells in G2 phase
203
. Although the influence of SAHA on cell cycle is
dose and cell type specific it is possible that the induction of cell cycle in TG mouse
KSLs and WT and TG mice KL upon SAHA treatment is caused by the decrease of
NF-E2 amount which may influence the expression of p21.
5.6 Megakaryocytic differentiation is delayed in CD34+
cells overexpressing NF-E2, as in PV CD34+ cells
cultured in-vitro.
In this work the effect of NF-E2 overexpression in CD34+ cells on megakaryocytic
differentiation was tested in few cytokine conditions. In expansion medium the
number of cells expressing megakaryocytic markers was less when NF-E2 was
overexpressed. When using modified expansion medium containing 10 times more
TPO and 5 times less SCF the results were opposite: there were more
megakaryocytic cells than the Empty control (Fig.4.5.3 a).
Since TPO is the major cytokine influencing megakaryopoiesis and changes
in its concentration on the background of other cytokines (Flt3, IL-6 and SCF)
possibly promoted megakaryocytic differentiation of NF-E2 overexpressing CD34+
we have decided to test different concentrations of TPO. Moreover defects in TPO
signaling have been previously reported in PV patients
40, 41, 207
. When using culture
media containing only TPO and SCF the CD34+ cells become preferentially
megakaryocytic, unlike in expansion and modified expansion media that support
differentiation to all cell types of the myeloid lineage. Like this terminal
megakaryocytic differentiation can be studied in detail, while eliminating changes in
the amount of megakaryocytic cells due to shifted lineage commitment. For the
purpose media containing 50 and 200 ng/ml TPO and 25ng/ml SCF were used. A
107
combination of TPO and EPO was also tested where the cells differentiated either as
megarakaryocytic or erythroid. In all conditions NF-E2 overexpressing cells
demonstrated a delay in megakaryocytic differentiation (Fig.4.5.5 a) that recapitulated
the phenotype of CD34+ cells from PV patients (Fig.4.5.5 b). Silencing of NF-E2 in
PV cells did not have an effect on the amount of megakaryocytic cells generated,
except when 50ng/ml TPO was used where the strongest silencing was achieved
(Fig.4.5.5 c). However there was an effect on megakaryocytic ploidy in PV cells when
NF-E2 was silenced (Fig.4.5.6 b). PV cells had more megakaryocytes with 8N and
more than 8N ploidy than the healthy controls (Fig.4.5.6 a) and silencing of NF-E2 in
PV partially reversed the phenotype (Fig.4.5.6 b).
The data confirms that NF-E2 has an important role on different aspects of
megakaryocytic physiology and moreover its overexpression affects megakaryocytic
maturation in the same manner as in PV patients. Indeed megakyrocyte function is
impaired in PV patients. Bone marrow histology shows abnormally clustered and
large megakaryocytes in PV that have increased ploidy
proplatelet formation are also affected in PV patients
208
.The ultrastructure and
209, 210
. In-vitro cultured
megakaryocytes from both ET and PV patients formed more proplatelets that
generated more tips and shaft bifurcations compared to PMF an healthy control
megakaryocytes 210.
5.7 EPO enhances NF-E2 expression, while TPO has an
antagonistic effect.
To date not much is known about which cytokines regulate the expression of NFE2. While there are few publications reporting upregulation of NF-E2 after EPO
stimulation in cell lines (J2E, UT-7/GMT) and CD34+ cells
185, 186, 211
, not much is
known about how other cytokines influence NF-E2 expression. A publication from
1998 describes the expression levels of NF-E2 after TPO stimulation of the UT7/GMT cell line
211
. In this cell line signaling through the TPO receptor lead to an
increase in NF-E2 expression during the initial days of culture which was followed by
a slight decrease during later days of cell culture.
Since both TPO and EPO were previously reported to regulate NF-E2 expression
and moreover signaling through the EPO and TPO cascades is impaired in MPN
patients we have investigated the effect of these cytokines on NF-E2 expression in
differentiating CD34+ cells from healthy donors and PV patients. Stimulation with 2
108
different concentrations of TPO (50 ng/ml and 200ng/ml) increased NF-E2
expression 1 to 2 fold over the control. Increase of TPO concentration did not
influence NF-E2 expression. Stimulation with EPO as previously reported
185, 186
,
increased the amount of NF-E2 expression by 15 fold over the control in healthy
control in-vitro differentiated CD34+ cells (Fig.4.6.1). Interestingly, when the cells
were stimulated with both EPO and TPO the expression of NF-E2 went down to 2-3
fold over the control (Fig.4.6.1), which would mean that TPO can override EPO
signaling and downregulate NF-E2 expression in healthy CD34+ cells differentiated
in-vitro.
The disadvantage of the experimental approach used for these experiments is
that NF-E2 expression was scored only on day 12 of culture and not during earlier
time points of differentiation. On day 12 of culture the CD34+ cells have already
became terminally differentiated as erythroid when cultured with EPO or
megakaryocytic when cultured with TPO. Combination of EPO and TPO gave rise to
both erythoid and megakaryocytic cells. This would mean that the qRT-PCR data
represents
the
level
of
NF-E2
expression
in
erythroid
and
respectively
megakaryocytic cells. More frequent measurements of NF-E2 transcription in defined
populations would be necessary to determine the rate and differentiation stage on
which EPO and TPO signaling start differentially regulating NF-E2 expression.
The expression and the intensity of signaling of the EPO and TPO receptor is
variable during distinct stages of hematopoieis. There are few publications describing
the expression pattern of EPO and TPO receptors during hematopoiesis
80, 81, 187, 212
.
The expression of the EPO receptor starts from the CMP, is upregulated in MEPs
and terminally differentiating erythroid cells, while it gets downregulated in
megakaryocytic cells. On the other hand TPOR expression has been detected in
HSCs, CMPs, MEPs and committed erythroid and megakaryocytic cells. Its
expression gets elevated in MEPs, stays high in megakaryocytic cells while it gets
lower in erythroid cells (Fig.5.4.1). Putting together the pattern of EPO and TPO
receptor expression and NF-E2 expression in CD34+ cells terminally differentiated in
the presence of TPO, EPO or both TPO and EPO in combination leads to the
following conclusions:
-
Signaling through the EPO receptor increases NF-E2 expression and
promotes erythroid differentiation of otherwise undifferentiated CD34+ cells
109
-
Signaling through the TPO receptor maintains NF-E2 expression on basal
levels and most likely can modulate NF-E2 expression stimulated by EPO
signaling. TPO signaling promotes terminal megakaryocytic differentiation of
CD34+ cells cultured in-vitro
-
EPO receptor expression is higher in erythroid than in megakaryocytic cells,
while the opposite is true for the TPO receptor.
From these conclusions one can deduce that:
-
One possible mechanism by which EPO and TPO signaling guide erythroid
respectively megakaryocytic lineage commitment is by regulating the
concentration of NF-E2 (Fig.5.4.1).
5.8 The endogenous overexpression of NF-E2 in PV
CD34+ cells is not dependent on EPO and TPO
signaling cascades.
As discussed in the previous section signaling through EPO and TPO receptors
influences NF-E2 expression. Since NF-E2 expression is elevated in PV patients we
hypothesized that elevation of NF-E2 expression observed in PV patients may be
due to defective EPO and/or TPO signaling.
PV patients show several abnormalities regarding EPO and TPO signaling.
The main lesion affecting both EPO and TPO signaling cascades is the presence of
JAK2V617F mutated protein that renders the receptors constitutively active by
phosphorylating the receptors even in the absence of ligands. EPO signaling defects
in PV patients are demonstrated by the generation of EPO independent erythroid
colonies
8, 10
and lower serum levels of EPO. However, the expression, kinetics and
turnover of the EPO receptor in PV have not been studied in detail. TPO receptor
expression on PV platelets and megakaryocytes was reported to be low or
completely absent
40
. A micro RNA (miR-28) inhibiting TPOR translation was also
detected to be more in platelets from a subset of PV patients
213
. In peripheral blood
mononuclear cells TPOR (c-mpl) transcripts were present in all patients with CML
and only in 2 out of 5 PV patients
207
. On the other hand the amount of CD34+ cells
coexpressing the TPOR in bone marrow samples from healthy controls and PV
patients was equal - around 1% 207. Since TPO signaling in HSC is essential for their
development, self-renewal, proliferation and maintenance complete lack of its
expression wouldn’t be possible in HSCs of PV patients. Moreover, deficiency in
110
TPOR expression leads to congenital amegakaryocytic thrombocytopenia, a rare and
lethal hematological disorder which is demonstrated during early childhood
39, 214
which would mean that TPOR expression in PV is diminished during later stages of
megakaryocytic differentiation.
In our experimental model TPO stimulation of CD34+ cells from PV patients
resulted in generation of megakaryocytic cells overexpressing NF-E2 by 2 fold over
healthy controls. Changes in TPO concentration did not influence the expression of
NF-E2 in PV cells (Fig.4.6.1). Again NF-E2 overexpression was 2 fold higher in PV
cells cultured in the presence of EPO or EPO and TPO in combination. The same
difference was observed when a cytokine mix was (Flt3, TPO, IL-6, SCF) used
(Fig.4.6.1). This means that abnormal NF-E2 expression in PV cells is not influenced
by the cytokines investigated and cannot be modulated by controlling cytokine
concentrations or the intensity of the conveyed signal through the mentioned
signaling cascades.
111
6. Appendix
6.1 Abbreviations
aa
Ab
AML
APS
BCL-X
BCR-ABL
BFU
BM
C/EBP
CBFA2
CFU
CLP
CMEP
CML
CMP
CREB
Ct
CTD
DC
ddH2O
DMEM
DMSO
DNA
dNTPs
DTT
ECL
Edu
EEC
EKLF
EPOR
ET
EZH2
FACS
FCS
FLI-1
FOG-1
GATA-1
G-CSF
GFI1
GFP
GM-CSF
GMP
amino acid
antibody
Acute myeloid leukemia
Ammonium persulfat
Bcl-2–associated X protein
Breakpoint Cluster Region - Abelson Murine Leukemia fusion protein
Burst forming unit
bone marrow
CCAAT/Enhancer-Binding protein
Core-Binding Factor, RUNT domain Alpha subunit 2
Colony forming unit
common lymphoid progenitor
common myeloid-eyrthroid progenitors
Chronic Myelogenous Leukemia
common myeloid progenitor
cAMP-response elements binding protein
threshold cycle
COOH-terminal domain
dendritic cell
double destiled H2O
Dulbecco's Modified Eagle Medium
Dimethylsulfoxid
Deoxyribonucleic acid
Desoxyribonukleosidtriphosphate
Dithiothreitol
Enhanced chemiluminescence
5-ethynyl-2´-deoxyuridine
Endogenous erythroid colonies
Erythroid Kruppel-Like Factor
Erythropoietin receptor
Essential throbocythemia
Enhancer of zeste homolog 2
Fluorescent Activated Cell Sorting
foetal calf serum
Friend Leukemia Virus Integration 1
Friend of GATA 1
GATA-binding protein 1
granulocyte colony stimulating factor
Growth Factor independent 1
Green fluorescent protein
granulocyte-macrophage stimulating factor
granulocyte-monocyte progenitor
112
h
HBSS
HC
HDAC
HEPES
HPC
HRP
HS
HSA
HSC
IAA
IFN
IL
IMDM
IRES
ITCH
JAK2
JNK
LDL
LNK
M-CSF
mdr
MEP
MFI
MGG
min.
MNC
MPN
MPP
NaCl
NAPP
NEDD4
NK
NLS
NS
PB
PBS
PCR
PF4
PLT
PMF
POD
Pol II
PRV-1
PTEN
PV
PVDF
hour
Hank's Buffered Salt Solution
Healthy control
Histone deacetylase
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
hematopoietic progenitor cell
Horse raddish peroxidase
hypersensitive site
human serum albumin
hematopoietic stem cell
Iodoacetamide
Interferon
interleukine
Iscove's Modified Dulbecco's Medium
internal ribosome entry site
E3 ubiquitin-protein ligase Itchy homolog
Janus kinase 2
c-Jun N-terminal kinase
low density lipoprotein
lymphocyte adaptor protein
macrophage stimulating factor
multi drug resistance
megakaryocyte-erytroid progenitor
Mean fluorescent intensity
May-Grünwald Geimsa
minute
Mononuclear cells
Myeloproliferative Neoplasms
multipotent progenitor
natrium chloride
NF-E2 associated polypeptide
NPC-expressed, developmentally down-regulated 4
natural killer cell
nucleus locating sequence
statistically non-significant
Peripheral blood
Phosphate buffered saline
Polymerase Chain Reaction
Platelet Factor 4
platelets
Primary myelofibrosis
Promyelocytic oncogenic domain
RNA Polymerase II
Polycythemia rubra vera protein -1
Phosphatase and tensin homolog
Polycythemia vera
polyvinylidene difluoride
113
qRT-PCR
rhEPO
rhFlt3-Ligand
rhIL-6
rhSCF
rhTPO
RNA
ROS
rpm
RT
s
SAHA
SCL
scr
SDF-1
SDS
SFFV
shNF-E2
SOCS
SP
quantitative reverse transcribtion polymerase chain reaction
recombinant human erythropoietin
recombinant human FMS related tyrosine kinase 3 ligand
recombinant human interleukin 6
recombinant human stem cell factor
recombinant human thrombopoietin
Ribonucleic acid
reactive oxygen species
revolutions per minute
room temperature
second
suberoylanilide hydroxamic acid
Stem Cell Leukemia Hematopoietic Transcription Factor
scrambled shNF-E2 sequence
Stromal cell-Derived Factor 1
Sodium dodecyl sulfate
spleen focus-forming virus
short hairpin sequence against NF-E2
Suppressor of cytokine signaling
side population
TAF II 130
TBS
TEL
TET2
TF
TG
TPOR
USF
v/v
w/v
WHO
WT
YAP
TATA box-binding protein associated factor II 130
Tris buffered saline
Translocation ETS Leukemia
ten eleven translocation
transcription factor
Transgenic mice
Thrombopoietin receptor
Upstream Stimulatory Factor
volume per volume
weight per volume
World Health Organisation
Wild type mice
Yes associated protein
114
6.2 References
[1]
Fauser AA, Messner HA. Identification of megakaryocytes, macrophages, and
eosinophils in colonies of human bone marrow containing neurtophilic granulocytes
and erythroblasts. Blood. 1979; 53: 1023-7.
[2]
Iwasaki H, Akashi K. Myeloid lineage commitment from the hematopoietic
stem cell. Immunity. 2007; 26: 726-40.
[3]
Anger B, Janssen JW, Schrezenmeier H, Hehlmann R, Heimpel H, Bartram
CR. Clonal analysis of chronic myeloproliferative disorders using X-linked DNA
polymorphisms. Leukemia. 1990; 4: 258-61.
[4]
Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the
World Health Organization diagnostic criteria for polycythemia vera, essential
thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc
international expert panel. Blood. 2007; 110: 1092-7.
[5]
Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med.
2006; 355: 2452-66.
[6]
Adamson JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann L. Polycythemia
vera: stem-cell and probable clonal origin of the disease. N Engl J Med. 1976; 295:
913-6.
[7]
Prchal JF, Adamson JW, Steinmann L, Fialkow PJ. Human erythroid colony
formation in vitro: evidence for clonal origin. J Cell Physiol. 1976; 89: 489-92.
[8]
Gilliland DG, Blanchard KL, Levy J, Perrin S, Bunn HF. Clonality in
myeloproliferative disorders: analysis by means of the polymerase chain reaction.
Proc Natl Acad Sci U S A. 1991; 88: 6848-52.
[9]
Temerinac S, Klippel S, Strunck E, et al. Cloning of PRV-1, a novel member of
the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera.
Blood. 2000; 95: 2569-76.
[10] Adamson JW. Analysis of myeloproliferative disorders using cell markers in
culture. Haematol Blood Transfus. 1979; 23: 205-10.
[11] Pahl HL. Towards a molecular understanding of polycythemia rubra vera. Eur
J Biochem. 2000; 267: 3395-401.
[12] Hoffman R BEJ, Shattil SJ, et al. Hematology: Basic Principles and Practice.
3rd ed. New York: Churchill Livingstone, 2000; pp 1106-55, 72-205.
[13] Vaques LH. Sur une forme spéciale de cyanose s’accompagnant
d’hyperglobulie excessive et persistante. Paris: Comptes rendus de la Société de
biologie,, 1892; 384-8.
115
[14] Heuck G. Zwei Fälle von Leukämie mit eigenthümlichem Blut- resp.
Knochenmarksbefund. Virchows Archiv. 1879; 78: 475-96.
[15] Epstein E GA. Hemorrhagic thrombocythemia with a cascular, sclerotic
spleen. . Virchows Arch, 1934; 233-48.
[16] DAMESHEK W. Some speculations on the myeloproliferative syndromes.
Blood. 1951; 6: 372-5.
[17] Tefferi A. The history of myeloproliferative disorders: before and after
Dameshek. Leukemia. 2008; 22: 3-13.
[18] NOWELL PC, HUNGERFORD DA. Chromosome studies on normal and
leukemic human leukocytes. J Natl Cancer Inst. 1960; 25: 85-109.
[19] Rowley JD. Letter: A new consistent chromosomal abnormality in chronic
myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining.
Nature. 1973; 243: 290-3.
[20] Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr
genes in chronic myelogenous leukaemia. Nature. 1985; 315: 550-4.
[21]
8.
Levine RL, Gilliland DG. Myeloproliferative disorders. Blood. 2008; 112: 2190-
[22] Passamonti F, Maffioli M, Caramazza D, Cazzola M. Myeloproliferative
neoplasms: from JAK2 mutations discovery to JAK2 inhibitor therapies. Oncotarget.
2011; 2: 485-90.
[23] Passamonti F, Rumi E, Arcaini L, et al. Prognostic factors for thrombosis,
myelofibrosis, and leukemia in essential thrombocythemia: a study of 605 patients.
Haematologica. 2008; 93: 1645-51.
[24] Campbell PJ, Scott LM, Buck G, et al. Definition of subtypes of essential
thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F
mutation status: a prospective study. Lancet. 2005; 366: 1945-53.
[25] Passamonti F, Rumi E, Pungolino E, et al. Life expectancy and prognostic
factors for survival in patients with polycythemia vera and essential thrombocythemia.
Am J Med. 2004; 117: 755-61.
[26] Passamonti F, Rumi E, Caramella M, et al. A dynamic prognostic model to
predict survival in post-polycythemia vera myelofibrosis. Blood. 2008; 111: 3383-7.
[27] Beer PA, Delhommeau F, LeCouédic JP, et al. Two routes to leukemic
transformation after a JAK2 mutation-positive myeloproliferative neoplasm. Blood.
2010; 115: 2891-900.
116
[28] Tefferi A, Vardiman JW. Classification and diagnosis of myeloproliferative
neoplasms: the 2008 World Health Organization criteria and point-of-care diagnostic
algorithms. Leukemia. 2008; 22: 14-22.
[29] Vainchenker W, Delhommeau F, Constantinescu SN, Bernard OA. New
mutations and pathogenesis of myeloproliferative neoplasms. Blood. 2011; 118:
1723-35.
[30] Levine RL, Pardanani A, Tefferi A, Gilliland DG. Role of JAK2 in the
pathogenesis and therapy of myeloproliferative disorders. Nat Rev Cancer. 2007; 7:
673-83.
[31] Dawson MA, Bannister AJ, Göttgens B, et al. JAK2 phosphorylates histone
H3Y41 and excludes HP1alpha from chromatin. Nature. 2009; 461: 819-22.
[32] James C, Ugo V, Le Couédic JP, et al. A unique clonal JAK2 mutation leading
to constitutive signalling causes polycythaemia vera. Nature. 2005; 434: 1144-8.
[33] Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of
JAK2 in myeloproliferative disorders. N Engl J Med. 2005; 352: 1779-90.
[34] Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine
kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid
metaplasia with myelofibrosis. Cancer Cell. 2005; 7: 387-97.
[35] Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine
kinase JAK2 in human myeloproliferative disorders. Lancet. 2005; 365: 1054-61.
[36] McLornan D, Percy M, McMullin MF. JAK2 V617F: a single mutation in the
myeloproliferative group of disorders. Ulster Med J. 2006; 75: 112-9.
[37] Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia
vera and idiopathic erythrocytosis. N Engl J Med. 2007; 356: 459-68.
[38] Dusa A, Mouton C, Pecquet C, Herman M, Constantinescu SN. JAK2 V617F
constitutive activation requires JH2 residue F595: a pseudokinase domain target for
specific inhibitors. PLoS One. 2010; 5: e11157.
[39] Kaushansky K. Thrombopoietin and the hematopoietic stem cell. Blood. 1998;
92: 1-3.
[40] Moliterno AR, Hankins WD, Spivak JL. Impaired expression of the
thrombopoietin receptor by platelets from patients with polycythemia vera. N Engl J
Med. 1998; 338: 572-80.
117
[41] Pardanani AD, Levine RL, Lasho T, et al. MPL515 mutations in
myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood. 2006;
108: 3472-6.
[42] Vannucchi AM, Antonioli E, Guglielmelli P, et al. Characteristics and clinical
correlates of MPL 515W>L/K mutation in essential thrombocythemia. Blood. 2008;
112: 844-7.
[43] Tong W, Zhang J, Lodish HF. Lnk inhibits erythropoiesis and Epo-dependent
JAK2 activation and downstream signaling pathways. Blood. 2005; 105: 4604-12.
[44] Tong W, Lodish HF. Lnk inhibits Tpo-mpl signaling and Tpo-mediated
megakaryocytopoiesis. J Exp Med. 2004; 200: 569-80.
[45] Simon C, Dondi E, Chaix A, et al. Lnk adaptor protein down-regulates specific
Kit-induced signaling pathways in primary mast cells. Blood. 2008; 112: 4039-47.
[46] Oh ST, Simonds EF, Jones C, et al. Novel mutations in the inhibitory adaptor
protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms.
Blood. 2010; 116: 988-92.
[47] Pardanani A, Lasho T, Finke C, Oh ST, Gotlib J, Tefferi A. LNK mutation
studies in blast-phase myeloproliferative neoplasms, and in chronic-phase disease
with TET2, IDH, JAK2 or MPL mutations. Leukemia. 2010; 24: 1713-8.
[48] Krebs DL, Hilton DJ. SOCS: physiological suppressors of cytokine signaling. J
Cell Sci. 2000; 113 ( Pt 16): 2813-9.
[49] Suessmuth Y, Elliott J, Percy MJ, et al. A new polycythaemia vera-associated
SOCS3 SH2 mutant (SOCS3F136L) cannot regulate erythropoietin responses. Br J
Haematol. 2009; 147: 450-8.
[50] Jost E, do O N, Dahl E, et al. Epigenetic alterations complement mutation of
JAK2 tyrosine kinase in patients with BCR/ABL-negative myeloproliferative disorders.
Leukemia. 2007; 21: 505-10.
[51] Teofili L, Martini M, Cenci T, et al. Epigenetic alteration of SOCS family
members is a possible pathogenetic mechanism in JAK2 wild type myeloproliferative
diseases. Int J Cancer. 2008; 123: 1586-92.
[52] Quentmeier H, Geffers R, Jost E, et al. SOCS2: inhibitor of JAK2V617Fmediated signal transduction. Leukemia. 2008; 22: 2169-75.
[53] Haan S, Wüller S, Kaczor J, et al. SOCS-mediated downregulation of mutant
Jak2 (V617F, T875N and K539L) counteracts cytokine-independent signaling.
Oncogene. 2009; 28: 3069-80.
118
[54] Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;
324: 930-5.
[55] Ito S, Shen L, Dai Q, et al. Tet proteins can convert 5-methylcytosine to 5formylcytosine and 5-carboxylcytosine. Science. 2011; 333: 1300-3.
[56] Ko M, Huang Y, Jankowska AM, et al. Impaired hydroxylation of 5methylcytosine in myeloid cancers with mutant TET2. Nature. 2010; 468: 839-43.
[57] Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid
cancers. N Engl J Med. 2009; 360: 2289-301.
[58] Saint-Martin C, Leroy G, Delhommeau F, et al. Analysis of the ten-eleven
translocation 2 (TET2) gene in familial myeloproliferative neoplasms. Blood. 2009;
114: 1628-32.
[59] Tefferi A, Pardanani A, Lim KH, et al. TET2 mutations and their clinical
correlates in polycythemia vera, essential thrombocythemia and myelofibrosis.
Leukemia. 2009; 23: 905-11.
[60] Sauvageau M, Sauvageau G. Polycomb group proteins: multi-faceted
regulators of somatic stem cells and cancer. Cell Stem Cell. 2010; 7: 299-313.
[61] Cao R, Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine
27 in histone H3. Curr Opin Genet Dev. 2004; 14: 155-64.
[62] Ernst T, Chase AJ, Score J, et al. Inactivating mutations of the histone
methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010; 42: 722-6.
[63] Nikoloski G, Langemeijer SM, Kuiper RP, et al. Somatic mutations of the
histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet.
2010; 42: 665-7.
[64] Boise LH, González-García M, Postema CE, et al. bcl-x, a bcl-2-related gene
that functions as a dominant regulator of apoptotic cell death. Cell. 1993; 74: 597608.
[65] Dolznig H, Habermann B, Stangl K, et al. Apoptosis protection by the Epo
target Bcl-X(L) allows factor-independent differentiation of primary erythroblasts. Curr
Biol. 2002; 12: 1076-85.
[66] Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernández-Luna JL. Expression
of Bcl-x in erythroid precursors from patients with polycythemia vera. N Engl J Med.
1998; 338: 564-71.
119
[67] Silva M, Grillot D, Benito A, Richard C, Nuñez G, Fernández-Luna JL.
Erythropoietin can promote erythroid progenitor survival by repressing apoptosis
through Bcl-XL and Bcl-2. Blood. 1996; 88: 1576-82.
[68] Sachs UJ, Andrei-Selmer CL, Maniar A, et al. The neutrophil-specific antigen
CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31).
J Biol Chem. 2007; 282: 23603-12.
[69] Stroncek DF, Shankar R, Litz C, Clement L. The expression of the NB1
antigen on myeloid precursors and neutrophils from children and umbilical cords.
Transfus Med. 1998; 8: 119-23.
[70] Yaswen P, Smoll A, Peehl DM, Trask DK, Sager R, Stampfer MR. Downregulation of a calmodulin-related gene during transformation of human mammary
epithelial cells. Proc Natl Acad Sci U S A. 1990; 87: 7360-4.
[71] Klippel S, Strunck E, Busse CE, Behringer D, Pahl HL. Biochemical
characterization of PRV-1, a novel hematopoietic cell surface receptor, which is
overexpressed in polycythemia rubra vera. Blood. 2002; 100: 2441-8.
[72] Goerttler PS, Kreutz C, Donauer J, et al. Gene expression profiling in
polycythaemia vera: overexpression of transcription factor NF-E2. Br J Haematol.
2005; 129: 138-50.
[73] Pischedda C, Cocco S, Melis A, et al. Isolation of a differentially regulated
splicing isoform of human NF-E2. Proc Natl Acad Sci U S A. 1995; 92: 3511-5.
[74] Moroni E, Mastrangelo T, Razzini R, et al. Regulation of mouse p45 NF-E2
transcription by an erythroid-specific GATA-dependent intronic alternative promoter. J
Biol Chem. 2000; 275: 10567-76.
[75] Takayama M, Fujita R, Suzuki M, et al. Genetic analysis of hierarchical
regulation for Gata1 and NF-E2 p45 gene expression in megakaryopoiesis. Mol Cell
Biol. 2010; 30: 2668-80.
[76] Toki T, Arai K, Terui K, et al. Functional characterization of the two alternative
promoters of human p45 NF-E2 gene. Exp Hematol. 2000; 28: 1113-9.
[77] Chan JY, Han XL, Kan YW. Isolation of cDNA encoding the human NF-E2
protein. Proc Natl Acad Sci U S A. 1993; 90: 11366-70.
[78] Ney PA, Andrews NC, Jane SM, et al. Purification of the human NF-E2
complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an
associated partner. Mol Cell Biol. 1993; 13: 5604-12.
[79] Kaufmann KB, Gründer A, Hadlich T, et al. A novel murine model of
myeloproliferative disorders generated by overexpression of the transcription factor
NF-E2. J Exp Med. 2012; 209: 35-50.
120
[80] Edvardsson L, Dykes J, Olofsson T. Isolation and characterization of human
myeloid progenitor populations--TpoR as discriminator between common myeloid
and megakaryocyte/erythroid progenitors. Exp Hematol. 2006; 34: 599-609.
[81] Edvardsson L, Dykes J, Olsson ML, Olofsson T. Clonogenicity, gene
expression and phenotype during neutrophil versus erythroid differentiation of
cytokine-stimulated CD34+ human marrow cells in vitro. Br J Haematol. 2004; 127:
451-63.
[82] Akashi K, Traver D, Miyamoto T, Weissman IL. A clonogenic common myeloid
progenitor that gives rise to all myeloid lineages. Nature. 2000; 404: 193-7.
[83] Adolfsson J, Månsson R, Buza-Vidas N, et al. Identification of Flt3+ lymphomyeloid stem cells lacking erythro-megakaryocytic potential a revised road map for
adult blood lineage commitment. Cell. 2005; 121: 295-306.
[84] Andrews NC. The NF-E2 transcription factor. Int J Biochem Cell Biol. 1998; 30:
429-32.
[85] Moore A, Merad Boudia M, Lehalle D, Massrieh W, Derjuga A, Blank V.
Regulation of globin gene transcription by heme in erythroleukemia cells: analysis of
putative heme regulatory motifs in the p45 NF-E2 transcription factor. Antioxid Redox
Signal. 2006; 8: 68-75.
[86] Gavva NR, Gavva R, Ermekova K, Sudol M, Shen CJ. Interaction of WW
domains with hematopoietic transcription factor p45/NF-E2 and RNA polymerase II. J
Biol Chem. 1997; 272: 24105-8.
[87] Lee TL, Shyu YC, Hsu TY, Shen CK. Itch regulates p45/NF-E2 in vivo by
Lys63-linked ubiquitination. Biochem Biophys Res Commun. 2008; 375: 326-30.
[88] Chen X, Wen S, Fukuda MN, et al. Human ITCH is a coregulator of the
hematopoietic transcription factor NF-E2. Genomics. 2001; 73: 238-41.
[89] Gavva NR, Wen SC, Daftari P, et al. NAPP2, a peroxisomal membrane
protein, is also a transcriptional corepressor. Genomics. 2002; 79: 423-31.
[90] Lee TL, Shyu YC, Hsu PH, et al. JNK-mediated turnover and stabilization of
the transcription factor p45/NF-E2 during differentiation of murine erythroleukemia
cells. Proc Natl Acad Sci U S A. 2010; 107: 52-7.
[91] Hung HL, Kim AY, Hong W, Rakowski C, Blobel GA. Stimulation of NF-E2
DNA binding by CREB-binding protein (CBP)-mediated acetylation. J Biol Chem.
2001; 276: 10715-21.
[92] Amrolia PJ, Ramamurthy L, Saluja D, Tanese N, Jane SM, Cunningham JM.
The activation domain of the enhancer binding protein p45NF-E2 interacts with
121
TAFII130 and mediates long-range activation of the alpha- and beta-globin gene loci
in an erythroid cell line. Proc Natl Acad Sci U S A. 1997; 94: 10051-6.
[93] Wang X, Trotman LC, Koppie T, et al. NEDD4-1 is a proto-oncogenic ubiquitin
ligase for PTEN. Cell. 2007; 128: 129-39.
[94] Anindya R, Aygün O, Svejstrup JQ. Damage-induced ubiquitylation of human
RNA polymerase II by the ubiquitin ligase Nedd4, but not Cockayne syndrome
proteins or BRCA1. Mol Cell. 2007; 28: 386-97.
[95] Imhof MO, McDonnell DP. Yeast RSP5 and its human homolog hRPF1
potentiate hormone-dependent activation of transcription by human progesterone
and glucocorticoid receptors. Mol Cell Biol. 1996; 16: 2594-605.
[96] Shimohata T, Nakajima T, Yamada M, et al. Expanded polyglutamine
stretches interact with TAFII130, interfering with CREB-dependent transcription. Nat
Genet. 2000; 26: 29-36.
[97] Gonzalez GA, Montminy MR. Cyclic AMP stimulates somatostatin gene
transcription by phosphorylation of CREB at serine 133. Cell. 1989; 59: 675-80.
[98] Garingo AD, Suhasini M, Andrews NC, Pilz RB. cAMP-dependent protein
kinase is necessary for increased NF-E2.DNA complex formation during
erythroleukemia cell differentiation. J Biol Chem. 1995; 270: 9169-77.
[99] Casteel D, Suhasini M, Gudi T, Naima R, Pilz RB. Regulation of the erythroid
transcription factor NF-E2 by cyclic adenosine monophosphate-dependent protein
kinase. Blood. 1998; 91: 3193-201.
[100] Chrivia JC, Kwok RP, Lamb N, Hagiwara M, Montminy MR, Goodman RH.
Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature. 1993;
365: 855-9.
[101] Perdomo J, Fock EL, Kaur G, et al. A monopartite sequence is essential for
p45 NF-E2 nuclear translocation, transcriptional activity and platelet production. J
Thromb Haemost. 2010; 8: 2542-53.
[102] Shyu YC, Lee TL, Ting CY, et al. Sumoylation of p45/NF-E2: nuclear
positioning and transcriptional activation of the mammalian beta-like globin gene
locus. Mol Cell Biol. 2005; 25: 10365-78.
[103] Wickrema A, Crispino JD. Erythroid and megakaryocytic transformation.
Oncogene. 2007; 26: 6803-15.
[104] Pang L, Weiss MJ, Poncz M. Megakaryocyte biology and related disorders. J
Clin Invest. 2005; 115: 3332-8.
122
[105] Pruzina S, Hanscombe O, Whyatt D, Grosveld F, Philipsen S. Hypersensitive
site 4 of the human beta globin locus control region. Nucleic Acids Res. 1991; 19:
1413-9.
[106] Beaumont C, Seyhan A, Yachou AK, Grandchamp B, Jones R. Mouse ferritin
H subunit gene. Functional analysis of the promoter and identification of an upstream
regulatory element active in erythroid cells. J Biol Chem. 1994; 269: 20281-8.
[107] Magness ST, Tugores A, Diala ES, Brenner DA. Analysis of the human
ferrochelatase promoter in transgenic mice. Blood. 1998; 92: 320-8.
[108] Mignotte V, Eleouet JF, Raich N, Romeo PH. Cis- and trans-acting elements
involved in the regulation of the erythroid promoter of the human porphobilinogen
deaminase gene. Proc Natl Acad Sci U S A. 1989; 86: 6548-52.
[109] Taketani S, Inazawa J, Nakahashi Y, Abe T, Tokunaga R. Structure of the
human ferrochelatase gene. Exon/intron gene organization and location of the gene
to chromosome 18. Eur J Biochem. 1992; 205: 217-22.
[110] Motohashi H, Kimura M, Fujita R, et al. NF-E2 domination over Nrf2 promotes
ROS accumulation and megakaryocytic maturation. Blood. 2010; 115: 677-86.
[111] Demers C, Chaturvedi CP, Ranish JA, et al. Activator-mediated recruitment of
the MLL2 methyltransferase complex to the beta-globin locus. Mol Cell. 2007; 27:
573-84.
[112] Hosey AM, Chaturvedi C-P, Brand M. Crosstalk between histone modifications
maintains the developmental pattern of gene expression on a tissue-specific locus.
Epigenetics. 2010; 5: 273-81.
[113] Chaturvedi CP, Hosey AM, Palii C, et al. Dual role for the methyltransferase
G9a in the maintenance of beta-globin gene transcription in adult erythroid cells. Proc
Natl Acad Sci U S A. 2009; 106: 18303-8.
[114] Zhou Z, Li X, Deng C, Ney PA, Huang S, Bungert J. USF and NF-E2
cooperate to regulate the recruitment and activity of RNA polymerase II in the betaglobin gene locus. J Biol Chem. 2010; 285: 15894-905.
[115] Johnson KD, Grass JA, Boyer ME, et al. Cooperative activities of
hematopoietic regulators recruit RNA polymerase II to a tissue-specific chromatin
domain. Proc Natl Acad Sci U S A. 2002; 99: 11760-5.
[116] Xin L, Zhou GL, Song W, et al. Exploring cellular memory molecules marking
competent and active transcriptions. BMC Mol Biol. 2007; 8: 31.
[117] Sarge KD, Park-Sarge OK. Gene bookmarking: keeping the pages open.
Trends Biochem Sci. 2005; 30: 605-10.
123
[118] Shivdasani RA, Orkin SH. Erythropoiesis and globin gene expression in mice
lacking the transcription factor NF-E2. Proc Natl Acad Sci U S A. 1995; 92: 8690-4.
[119] Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, et al. Transcription factor
NF-E2 is required for platelet formation independent of the actions of
thrombopoietin/MGDF in megakaryocyte development. Cell. 1995; 81: 695-704.
[120] TILL JE, McCULLOCH EA. A direct measurement of the radiation sensitivity of
normal mouse bone marrow cells. Radiat Res. 1961; 14: 213-22.
[121] BECKER AJ, McCULLOCH EA, TILL JE. Cytological demonstration of the
clonal nature of spleen colonies derived from transplanted mouse marrow cells.
Nature. 1963; 197: 452-4.
[122] Wu AM, Till JE, Siminovitch L, McCulloch EA. Cytological evidence for a
relationship between normal hemotopoietic colony-forming cells and cells of the
lymphoid system. J Exp Med. 1968; 127: 455-64.
[123] SIMINOVITCH L, MCCULLOCH EA, TILL JE. THE DISTRIBUTION OF
COLONY-FORMING CELLS AMONG SPLEEN COLONIES. J Cell Physiol. 1963; 62:
327-36.
[124] Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of
mouse hematopoietic stem cells. Science. 1988; 241: 58-62.
[125] Doulatov S, Notta F, Eppert K, Nguyen LT, Ohashi PS, Dick JE. Revised map
of the human progenitor hierarchy shows the origin of macrophages and dendritic
cells in early lymphoid development. Nat Immunol. 2010; 11: 585-93.
[126] Kikushige Y, Yoshimoto G, Miyamoto T, et al. Human Flt3 is expressed at the
hematopoietic stem cell and the granulocyte/macrophage progenitor stages to
maintain cell survival. J Immunol. 2008; 180: 7358-67.
[127] Chao MP, Seita J, Weissman IL. Establishment of a normal hematopoietic and
leukemia stem cell hierarchy. Cold Spring Harb Symp Quant Biol. 2008; 73: 439-49.
[128] Morrison SJ, Weissman IL. The long-term repopulating subset of
hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity.
1994; 1: 661-73.
[129] Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell
differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U
S A. 2001; 98: 14541-6.
[130] Kondo M, Weissman IL, Akashi K. Identification of clonogenic common
lymphoid progenitors in mouse bone marrow. Cell. 1997; 91: 661-72.
124
[131] Wu L, Vandenabeele S, Georgopoulos K. Derivation of dendritic cells from
myeloid and lymphoid precursors. Int Rev Immunol. 2001; 20: 117-35.
[132] Seita J, Weissman IL. Hematopoietic stem cell: self-renewal versus
differentiation. Wiley Interdiscip Rev Syst Biol Med. 2010; 2: 640-53.
[133] Kawamoto H, Wada H, Katsura Y. A revised scheme for developmental
pathways of hematopoietic cells: the myeloid-based model. Int Immunol. 2010; 22:
65-70.
[134] Kawamoto H, Katsura Y. A new paradigm for hematopoietic cell lineages:
revision of the classical concept of the myeloid-lymphoid dichotomy. Trends Immunol.
2009; 30: 193-200.
[135] Taichman RS, Reilly MJ, Emerson SG. The Hematopoietic Microenvironment:
Osteoblasts and The Hematopoietic Microenvironment. Hematology. 2000; 4: 421-6.
[136] Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the
haematopoietic stem cell niche. Nature. 2003; 425: 841-6.
[137] Takano H, Ema H, Sudo K, Nakauchi H. Asymmetric division and lineage
commitment at the level of hematopoietic stem cells: inference from differentiation in
daughter cell and granddaughter cell pairs. J Exp Med. 2004; 199: 295-302.
[138] Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood.
1993; 81: 2844-53.
[139] Terskikh AV, Miyamoto T, Chang C, Diatchenko L, Weissman IL. Gene
expression analysis of purified hematopoietic stem cells and committed progenitors.
Blood. 2003; 102: 94-101.
[140] Hu M, Krause D, Greaves M, et al. Multilineage gene expression precedes
commitment in the hemopoietic system. Genes Dev. 1997; 11: 774-85.
[141] Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat
Rev Genet. 2000; 1: 57-64.
[142] Arinobu Y, Mizuno S, Chong Y, et al. Reciprocal activation of GATA-1 and
PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and
myelolymphoid lineages. Cell Stem Cell. 2007; 1: 416-27.
[143] Sherwood L, Klandorf, H, Yancey, P. Animal Physiology, Brooks/Cole,
Cengage Learning,. 2005.
[144] Spivak JL. The anaemia of cancer: death by a thousand cuts. Nat Rev Cancer.
2005; 5: 543-55.
125
[145] Hoffbrand AV. Essential haematology. In: Moss PAH, Pettit JE, eds. Malden,
Mass.: Blackwell Pub., 2006; 380.
[146] Chasis JA, Mohandas N. Erythroblastic islands: niches for erythropoiesis.
Blood. 2008; 112: 470-8.
[147] Perry C, Soreq H. Transcriptional regulation of erythropoiesis. Fine tuning of
combinatorial multi-domain elements. Eur J Biochem. 2002; 269: 3607-18.
[148] Debili N, Coulombel L, Croisille L, et al. Characterization of a bipotent erythromegakaryocytic progenitor in human bone marrow. Blood. 1996; 88: 1284-96.
[149] Nakorn TN, Miyamoto T, Weissman IL. Characterization of mouse clonogenic
megakaryocyte progenitors. Proc Natl Acad Sci U S A. 2003; 100: 205-10.
[150] Odell TT, Jackson CW, Gosslee DG. Maturation of rat megakaryocytes
studied by microspectrophotometric measurement of DNA. Proc Soc Exp Biol Med.
1965; 119: 1194-9.
[151] Lefebvre P, Winter JN, Meng Y, Cohen I. Ex vivo expansion of early and late
megakaryocyte progenitors. J Hematother Stem Cell Res. 2000; 9: 913-21.
[152] Starck J, Cohet N, Gonnet C, et al. Functional cross-antagonism between
transcription factors FLI-1 and EKLF. Mol Cell Biol. 2003; 23: 1390-402.
[153] Bouilloux F, Juban G, Cohet N, et al. EKLF restricts megakaryocytic
differentiation at the benefit of erythrocytic differentiation. Blood. 2008; 112: 576-84.
[154] Lehrer RI, Ganz T, Selsted ME, Babior BM, Curnutte JT. Neutrophils and host
defense. Ann Intern Med. 1988; 109: 127-42.
[155] Thomas EL, Lehrer RI, Rest RF. Human neutrophil antimicrobial activity. Rev
Infect Dis. 1988; 10 Suppl 2: S450-6.
[156] Bass DA, Szejda P. Eosinophils versus neutrophils in host defense. Killing of
newborn larvae of Trichinella spiralis by human granulocytes in vitro. J Clin Invest.
1979; 64: 1415-22.
[157] Bass DA, Szejda P. Mechanisms of killing of newborn larvae of Trichinella
spiralis by neutrophils and eosinophils. Killing by generators of hydrogen peroxide in
vitro. J Clin Invest. 1979; 64: 1558-64.
[158] Galli SJ. Mast cells and basophils. Curr Opin Hematol. 2000; 7: 32-9.
[159] Randolph GJ, Jakubzick C, Qu C. Antigen presentation by monocytes and
monocyte-derived cells. Curr Opin Immunol. 2008; 20: 52-60.
126
[160] Galli SJ, Gordon JR, Wershil BK. Cytokine production by mast cells and
basophils. Curr Opin Immunol. 1991; 3: 865-72.
[161] Cassatella MA. The production of cytokines by polymorphonuclear neutrophils.
Immunol Today. 1995; 16: 21-6.
[162] Traver D, Miyamoto T, Christensen J, Iwasaki-Arai J, Akashi K, Weissman IL.
Fetal liver myelopoiesis occurs through distinct, prospectively isolatable progenitor
subsets. Blood. 2001; 98: 627-35.
[163] Lieschke GJ. CSF-deficient mice--what have they taught us? Ciba Found
Symp. 1997; 204: 60-74; discussion -7.
[164] Liu F, Wu HY, Wesselschmidt R, Kornaga T, Link DC. Impaired production
and increased apoptosis of neutrophils in granulocyte colony-stimulating factor
receptor-deficient mice. Immunity. 1996; 5: 491-501.
[165] Okuda K, Foster R, Griffin JD. Signaling domains of the beta c chain of the
GM-CSF/IL-3/IL-5 receptor. Ann N Y Acad Sci. 1999; 872: 305-12; discussion 12-3.
[166] Ward AC, Loeb DM, Soede-Bobok AA, Touw IP, Friedman AD. Regulation of
granulopoiesis by transcription factors and cytokine signals. Leukemia. 2000; 14:
973-90.
[167] Friedman AD. Transcriptional control
development. Oncogene. 2007; 26: 6816-28.
of
granulocyte
and
monocyte
[168] Jamieson CH, Gotlib J, Durocher JA, et al. The JAK2 V617F mutation occurs
in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid
differentiation. Proc Natl Acad Sci U S A. 2006; 103: 6224-9.
[169] Anand S, Stedham F, Beer P, et al. Effects of the JAK2 mutation on the
hematopoietic stem and progenitor compartment in human myeloproliferative
neoplasms. Blood. 2011; 118: 177-81.
[170] Roelz R, Pilz IH, Mutschler M, Pahl HL. Of mice and men: human RNA
polymerase III promoter U6 is more efficient than its murine homologue for shRNA
expression from a lentiviral vector in both human and murine progenitor cells. Exp
Hematol. 2010; 38: 792-7.
[171] Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van
Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression
on B cells undergoing apoptosis. Blood. 1994; 84: 1415-20.
[172] Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and
functional properties of murine hematopoietic stem cells that are replicating in vivo. J
Exp Med. 1996; 183: 1797-806.
127
[173] Krishan A. Effect of drug efflux blockers on vital staining of cellular DNA with
Hoechst 33342. Cytometry. 1987; 8: 642-5.
[174] Ahmed F. Quantitative Real-time RT-PCR: Application to Carcinogenesis.
Cancer Genomics & Proteomics, 2005; 317-32.
[175] Randrianarison-Huetz V, Laurent B, Bardet V, Blobe GC, Huetz F, Duménil D.
Gfi-1B controls human erythroid and megakaryocytic differentiation by regulating
TGF-beta signaling at the bipotent erythro-megakaryocytic progenitor stage. Blood.
2010; 115: 2784-95.
[176] Chen L, Gao Z, Zhu J, Rodgers GP. Identification of CD13+CD36+ cells as a
common progenitor for erythroid and myeloid lineages in human bone marrow. Exp
Hematol. 2007; 35: 1047-55.
[177] Henney CS. Early events in lymphopoiesis: the role of interleukins 1 and 7. J
Autoimmun. 1989; 2 Suppl: 155-61.
[178] Henney CS. Interleukin 7: effects on early events in lymphopoiesis. Immunol
Today. 1989; 10: 170-3.
[179] Dai CH, Krantz SB, Dessypris EN, Means RT, Horn ST, Gilbert HS.
Polycythemia vera. II. Hypersensitivity of bone marrow erythroid, granulocytemacrophage, and megakaryocyte progenitor cells to interleukin-3 and granulocytemacrophage colony-stimulating factor. Blood. 1992; 80: 891-9.
[180] Dai CH, Krantz SB, Means RT, Horn ST, Gilbert HS. Polycythemia vera blood
burst-forming units-erythroid are hypersensitive to interleukin-3. J Clin Invest. 1991;
87: 391-6.
[181] Dai CH, Krantz SB, Green WF, Gilbert HS. Polycythaemia vera. III. Burstforming units-erythroid (BFU-E) response to stem cell factor and c-kit receptor
expression. Br J Haematol. 1994; 86: 12-21.
[182] Correa PN, Eskinazi D, Axelrad AA. Circulating erythroid progenitors in
polycythemia vera are hypersensitive to insulin-like growth factor-1 in vitro: studies in
an improved serum-free medium. Blood. 1994; 83: 99-112.
[183] Ney PA, Sorrentino BP, Lowrey CH, Nienhuis AW. Inducibility of the HS II
enhancer depends on binding of an erythroid specific nuclear protein. Nucleic Acids
Res. 1990; 18: 6011-7.
[184] Li Z, Cai X, Cai C, et al. Deletion of Tet2 in mice leads to dysregulated
hematopoietic stem cells and subsequent development of myeloid malignancies.
Blood. 2011.
128
[185] Sayer MS, Tilbrook PA, Spadaccini A, et al. Ectopic expression of transcription
factor NF-E2 alters the phenotype of erythroid and monoblastoid cells. J Biol Chem.
2000; 275: 25292-8.
[186] Mahajan MC, Karmakar S, Newburger PE, Krause DS, Weissman SM.
Dynamics of alpha-globin locus chromatin structure and gene expression during
erythroid differentiation of human CD34(+) cells in culture. Exp Hematol. 2009; 37:
1143-56.e3.
[187] Manz MG, Miyamoto T, Akashi K, Weissman IL. Prospective isolation of
human clonogenic common myeloid progenitors. Proc Natl Acad Sci U S A. 2002; 99:
11872-7.
[188] Lemoine F, Najman A, Baillou C, et al. A prospective study of the value of
bone marrow erythroid progenitor cultures in polycythemia. Blood. 1986; 68: 9961002.
[189] Eaves AC, Henkelman DH, Eaves CJ. Abnormal erythropoiesis in the
myeloproliferative disorders: an analysis of underlying cellular and humoral
mechanisms. Exp Hematol. 1980; 8 Suppl 8: 235-47.
[190] Prchal JF, Axelrad AA. Letter: Bone-marrow responses in polycythemia vera.
N Engl J Med. 1974; 290: 1382.
[191] Eaves CJ, Eaves AC. Erythropoietin (Ep) dose-response curves for three
classes of erythroid progenitors in normal human marrow and in patients with
polycythemia vera. Blood. 1978; 52: 1196-210.
[192] Shih LY, Lee CT, See LC, et al. In vitro culture growth of erythroid progenitors
and serum erythropoietin assay in the differential diagnosis of polycythaemia. Eur J
Clin Invest. 1998; 28: 569-76.
[193] Ugo V, Marzac C, Teyssandier I, et al. Multiple signaling pathways are
involved in erythropoietin-independent differentiation of erythroid progenitors in
polycythemia vera. Exp Hematol. 2004; 32: 179-87.
[194] Scharenberg CW, Harkey MA, Torok-Storb B. The ABCG2 transporter is an
efficient Hoechst 33342 efflux pump and is preferentially expressed by immature
human hematopoietic progenitors. Blood. 2002; 99: 507-12.
[195] Shapiro HM. Flow cytometric estimation of DNA and RNA content in intact
cells stained with Hoechst 33342 and pyronin Y. Cytometry. 1981; 2: 143-50.
[196] Kelly WK, O'Connor OA, Krug LM, et al. Phase I study of an oral histone
deacetylase inhibitor, suberoylanilide hydroxamic acid, in patients with advanced
cancer. J Clin Oncol. 2005; 23: 3923-31.
129
[197] Rubin EH, Agrawal NG, Friedman EJ, et al. A study to determine the effects of
food and multiple dosing on the pharmacokinetics of vorinostat given orally to
patients with advanced cancer. Clin Cancer Res. 2006; 12: 7039-45.
[198] Kelly WK, Richon VM, O'Connor O, et al. Phase I clinical trial of histone
deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously.
Clin Cancer Res. 2003; 9: 3578-88.
[199] Garcia-Manero G, Yang H, Bueso-Ramos C, et al. Phase 1 study of the
histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in
patients with advanced leukemias and myelodysplastic syndromes. Blood. 2008; 111:
1060-6.
[200] Rambaldi A, Dellacasa CM, Finazzi G, et al. A pilot study of the HistoneDeacetylase inhibitor Givinostat in patients with JAK2V617F positive chronic
myeloproliferative neoplasms. Br J Haematol. 2010; 150: 446-55.
[201] Shao Y, Gao Z, Marks PA, Jiang X. Apoptotic and autophagic cell death
induced by histone deacetylase inhibitors. Proc Natl Acad Sci U S A. 2004; 101:
18030-5.
[202] Rosato RR, Grant S. Histone deacetylase inhibitors: insights into mechanisms
of lethality. Expert Opin Ther Targets. 2005; 9: 809-24.
[203] Richon VM, Sandhoff TW, Rifkind RA, Marks PA. Histone deacetylase inhibitor
selectively induces p21WAF1 expression and gene-associated histone acetylation.
Proc Natl Acad Sci U S A. 2000; 97: 10014-9.
[204] Wardell SE, Ilkayeva OR, Wieman HL, et al. Glucose metabolism as a target
of histone deacetylase inhibitors. Mol Endocrinol. 2009; 23: 388-401.
[205] Vrana JA, Decker RH, Johnson CR, et al. Induction of apoptosis in U937
human leukemia cells by suberoylanilide hydroxamic acid (SAHA) proceeds through
pathways that are regulated by Bcl-2/Bcl-XL, c-Jun, and p21CIP1, but independent of
p53. Oncogene. 1999; 18: 7016-25.
[206] Ruefli AA, Ausserlechner MJ, Bernhard D, et al. The histone deacetylase
inhibitor and chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA)
induces a cell-death pathway characterized by cleavage of Bid and production of
reactive oxygen species. Proc Natl Acad Sci U S A. 2001; 98: 10833-8.
[207] Duensing S, Duensing A, Meran JG, et al. Molecular detection of c-mpl
thrombopoietin receptor gene expression in chronic myeloproliferative disorders. Mol
Pathol. 1999; 52: 146-50.
[208] Lagerlöf B. Cytophotometric study of megakaryocyte ploidy in polycythemia
vera and chronic granulocytic leukemia. Acta Cytol. 1972; 16: 240-4.
130
[209] Lagerlöf B, Franzén S. The ultrastructure of megakaryocytes in polycythaemia
vera and chronic granulocytic leukaemia. Acta Pathol Microbiol Scand A. 1972; 80:
71-83.
[210] Balduini A, Badalucco S, Pugliano MT, et al. In vitro megakaryocyte
differentiation and proplatelet formation in Ph-negative classical myeloproliferative
neoplasms: distinct patterns in the different clinical phenotypes. PLoS One. 2011; 6:
e21015.
[211] Yamada M, Komatsu N, Kirito K, et al. Thrombopoietin supports in vitro
erythroid differentiation via its specific receptor c-Mpl in a human leukemia cell line.
Cell Growth Differ. 1998; 9: 487-96.
[212] Klimchenko O, Mori M, Distefano A, et al. A common bipotent progenitor
generates the erythroid and megakaryocyte lineages in embryonic stem cell-derived
primitive hematopoiesis. Blood. 2009; 114: 1506-17.
[213] Girardot M, Pecquet C, Boukour S, et al. miR-28 is a thrombopoietin receptor
targeting microRNA detected in a fraction of myeloproliferative neoplasm patient
platelets. Blood. 2010; 116: 437-45.
[214] Kaushansky K. Mpl and the hematopoietic stem cell. Leukemia. 2002; 16: 7389.
131

Thesis fullx 1 - FreiDok plus

Transcription

Similar documents

How to Tie a Woggle

lnstallation - ConservationMart.com

Turkey - EOPOWER

Key - Scioly.org

RÉSoNANSS: a regional contribution to

Real Time Control of Hand Prosthesis Using Surface EMG

Kerntech Diagnose-System Loose Part Monitoring System

CoUNTerMEASURES CoUNTerMEASURES

PrimeFilm 1800 AFL Low-Cost 35mm Slide/Film Scanning Through

May 2013 - Caldwell Board of REALTORS