live and let die critical regulation of survival in - diva portal

Linköping University Medical Dissertations No. 1160

Live and Let Die

Critical regulation of survival in normal and malignant

hematopoietic stem and progenitor cells

Pernilla Eliasson

Experimental Hematology unit

Department of Clinical and Experimental Medicine

Faculty of Health Sciences

SE-581 85 Linköping, Sweden

Linköping 2009

Copyright © Pernilla Eliasson, 2009

Cover picture is an illustration made by the author of a hematopoietic stem cell in the

trabecular bone of the bone marrow




SE-581 85 Linköping, Sweden

Printed by LiU-tryck, Linköping, Sweden, 2009

Published articles have been reprinted with the permission from respective copyright

holder

During the course of the research underlying this thesis, Pernilla Eliasson was enrolled

in Forum Scientium, a multidisciplinary doctoral programme at Linköping University,

Sweden.

ISBN 978-91-7393-470-1

ISSN 0345-0082

“The universe is full of magical things patiently waiting for our wits to grow sharper.”

Eden Phillpotts

ABSTRACT

ABSTRACT

The hematopoietic stem cell (HSC) is characterized by its ability to self-renew and

produce all mature blood cells throughout the life of an organism. This is tightly

regulated to maintain a balance between survival, proliferation, and differentiation.

The HSCs are located in specialized niches in the bone marrow thought to be low in

oxygen, which is suggested to be involved in the regulation of HSC maintenance,

proliferation, and migration. However, the importance of hypoxia in the stem cell

niche and the molecular mechanisms involved remain fairly undefined. Another

important regulator of human HSCs maintenance is the tyrosine kinase receptor FLT3,

which triggers survival of HSCs and progenitor cells. Mutations in FLT3 cause

constitutively active signaling. This leads to uncontrolled survival and proliferation,

which can result in development of acute myeloid leukemia (AML). One of the

purposes with this thesis is to investigate how survival, proliferation and self-renewal

in normal HSCs are affected by hypoxia. To study this, we used both in vitro and in

vivo models with isolated Lineage-Sca-1+Kit+ (LSK) and CD34-Flt3-LSK cells from mouse

bone marrow. We found that hypoxia maintained an immature phenotype. In

addition, hypoxia decreased proliferation and induced cell cycle arrest, which is the

signature of HSCs with long term multipotential capacity. A dormant state of HSCs is

suggested to be critical for protecting and preventing depletion of the stem cell pool.

Furthermore, we observed that hypoxia rescues HSCs from oxidative stress-induced

cell death, implicating that hypoxia is important in the bone marrow niche to limit

reactive oxidative species (ROS) production and give life-long protection of HSCs.

Another focus in this thesis is to investigate downstream pathways involved in

tyrosine kinase inhibitor-induced cell death of primary AML cells and cell lines

expressing mutated FLT3. Our results demonstrate an important role of the PI3K/AKT

pathway to mediate survival signals from FLT3. We found FoxO3a and its target gene

Bim to be key players of apoptosis in cells carrying oncogenic FLT3 after treatment

with tyrosine kinase inhibitors. In conclusion, this thesis highlights hypoxic-mediated

regulation of normal HSCs maintenance and critical effectors of apoptosis in leukemic

cells expressing mutated FLT3.

SUPERVISOR

Jan-Ingvar Jönsson, Professor




Linköping University, Sweden

CO-SUPERVISOR

Mikael Sigvardsson, Professor





OPPONENT

Urban Gullberg, Professor

Division of Hematology and Transfusion Medicine

Faculty of Medicine

Lund University, Sweden

COMMITTEE BOARD

Marja Ekblom, Professor

Hematopoietic Stem Cell Laboratory

Department of Laboratory Medicine

Lund University, Sweden

Karin Öllinger, Professor

Division of Dermatology




Anders Rosén, Professor

Division of Cell Biology

Department of Experimental Medicine,



TABLE OF CONTENTS

TABLE OF CONTENTS

List of papers included in the thesis .............................................................................. 7

Abbreviations ................................................................................................................. 8

Hematopoiesis ................................................................................................................ 9

The hematopoietic stem cell ....................................................................................... 10

Identification of HSCs ................................................................................................ 11

In vitro and in vivo assays to detect HSCs ................................................................. 13

Clinical use of HSCs ................................................................................................... 15

Can HSCs be expanded in vitro? ............................................................................... 16

Regulation of the cell cycle in HSCs .......................................................................... 18

The hematopoietic stem cell niche ........................................................................... 21

The hypoxic stem cell niche ...................................................................................... 31

Hypoxia inducible factor-1 ........................................................................................... 27

HIF-1 and regulation of important niche molecules ................................................. 29

Glucose metabolism in hypoxic cells ........................................................................ 30

HIF-1 and Cell cycle ................................................................................................... 32

Apoptosis ...................................................................................................................... 33

Pro- and anti-apoptotic Bcl-2 members ................................................................... 33

Oxidative stress and aging of HSCs .............................................................................. 36

FoxO proteins are essential in the resistance to oxidative stress ............................ 37

The role of FLT3 in normal and malignant HSCs and progenitors .............................. 39

Tyrosine kinase receptors ......................................................................................... 39

The FLT3 receptor ..................................................................................................... 39

Stimulation of the FLT3 receptor .......................................................................... 40

Mutations in the FLT3 receptor ............................................................................ 41

FLT3 signaling in normal and mutated receptor................................................... 42

Other mutations implicated in AML ......................................................................... 44

Therapeutic strategies for AML ................................................................................... 46

FLT3 inhibitors ........................................................................................................... 46

Therapies that targets the leukemic stem cell niche ................................................ 47

BH3 mimicking drugs ................................................................................................ 48

Aims of the present investigation ............................................................................... 50

Methodological considerations ................................................................................... 51

Isolation of HSCs using FACS based cell sorting ........................................................ 51

RNA interference ...................................................................................................... 52

Real time PCR ............................................................................................................ 54

TABLE OF CONTENTS

Results and discussion of the papers in this thesis ..................................................... 57

Conclusions ................................................................................................................... 65

Future aspects .............................................................................................................. 67

En livsviktig balans mellan liv och död – ..................................................................... 69

en populärvetenskaplig sammanfattning .....................................................................

Acknowledgements ...................................................................................................... 72

References .................................................................................................................... 74

Paper I-IV ...................................................................................................................... 97

LIST OF PAPERS INCLUDED IN THE THESIS

I Hypoxia expands primitive hematopoietic progenitor cells from mouse

bone marrow during in vitro culture and preserves the colony-forming

ability.

P. Eliasson, R. Karlsson, and J-I. Jönsson.

Journal of Stem cells. 2006.1(4): 247-257

II Hypoxia, via hypoxia-inducible factor (HIF)-1α, mediates low cell cycle

activity and preserves the engraftment potential of mouse hematopoietic

stem cells

P. Eliasson, M.Rehn, P.Hammar, P. Larsson, O. Sirenko, L.A. Flippin, M. Arend,

J. Cammenga, and J-I. Jönsson

Manuscript revised submitted

III Hypoxia rescues hematopoietic stem cells from oxidative stress-induced cell

death and preserves the long-term repopulation ability.

P. Eliasson, E. Widegren, and J-I. Jönsson

Manuscript

IV BH3-only protein Bim more critical than Puma in tyrosine kinase inhibitor-

induced apoptosis of human leukemic cells and transduced hematopoietic

progenitors carrying oncogenic FLT3.

*A. Nordigården, *M. Kraft, *P. Eliasson, V. Labi, E. W-F. Lan, A. Villunger, and

J-I. Jönsson

Blood. 2009. 113 (10): 2302-2311

*Contributed equally to this work

ABBREVIATIONS

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ABBREVIATIONS

AML Acute myeloid leukemia

ATM ataxia telangiectasia mutated

Bcl-2 B cell lymphoma-2

Bim Bcl-2-interacting modulator

of cell death

BMT Bone marrow transplantation

BSO L-buthionine sulfoximine

CAFC Cobblestone-area-forming-

cells

CAT Catalase

CDK cyclin-dependent kinases

CFC Colony-forming cell

CLP Common lymphoid

progenitor

CMP Common myeloid progenitor

CXCR4 CX chemokine receptor 4

FACS Fluorescence-activating cell

sorting

FGF Fibroblast growth factor

FL FLT3 ligand

FLT3 human c-fms-like tyrosine

kinase 3 (Flt3 in mouse)

FoxO Forkhead box transcription

factor

GMP granulocyte/monocytes

progenitors

HIF-1 Hypoxia-inducible factor-1

HPP High proliferative potential

HSC Hematopoietic stem cell

HSCT hematopoietic stem cell

transplantation

IL Interleukin

IGF-2 insulin-like growth factor-2

ITD Internal tandem repeats

JAK Janus-activated kinases

JM Juxtamembrane

LSC Leukemic stem cells

LSK Lineage-Sca-1+c-Kit+

LT Long term

LTC-IC Long term cell-initiating cell

MAPK Mitogen-activated protein

kinase

MMP Matrix metalloproteinases

MPP Multi-potent progenitors

NPM1 Nucleophosmin-1

PDK1 Pyruvate dehydrogenase

kinase-1

PI3K Phosphatidylinositol-3 kinase

PIM Pimonidazole

Puma p53 upregulated modulator

of apoptosis

RNAi RNA interference

ROS Reactive oxygen species

RTK Receptor tyrosine kinases

Sca-1 Stem cell antigen-1

SCF Stem cell factor

SDF-1 Stromal derived factor-1

shRNA Short hairpin RNA

siRNA small interfering RNA

SOD superoxidedismutase

SP Side population

ST Short term

TKD Tyrosine kinase domain

TPO Thrombopoietin

VEGF Vascular endothelial factor

HEMATOPOIETIC STEM CELLS

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Background

HEMATOPOIESIS

In order to maintain a steady level of mature and functional cells in the blood system,

1 trillion (1012) new cells are produced every day in an adult man (Ogawa, 1993). The

hematopoietic cells have several distinct functions, such as delivering oxygen to all

tissue cells, protecting the organism against infectious agents, as well as regulating

the blood coagulation. Early in life, hematopoiesis takes place in the yolk sac, the

aorta-gonad-mesonephos (AGM)-region, spleen, and the fetal liver. The bone marrow

becomes the primary site for hematopoiesis from the time of birth (Muller et al.,

1994). Extramedullary hematopoiesis occurs if the bone marrow is damaged or

stressed due to irradiation or chemotherapy. A common extramedullary place for

hematopoiesis is the spleen. All blood cells, including erythrocytes, myeloid and

lymphoid leukocytes, and platelets (derives from megakaryocytes), arise from one

common stem cell, the hematopoietic stem cell (HSC). Once lineage commitment has

occurred, the choice is taken and cannot be reversed.

Figure 1. The classical hematopoietic tree. The HSCs are divided in long term (LT) HSCs, short term (ST) HSCs, and multipotent progentior cells (MPP) according to their self-renewal potential. They give rise to common myeloid progenitors (CMPs) and lymphoid progenitor cells (CLPs) which give rise to all myeloid and lymphoid cells, respectively. Mouse HSCs can be isolated by their distinct expression pattern of the surface markers c-Kit, Sca-1, Flt3 and CD34 (Modified from Reya et al., 2001).


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The differentiation to mature blood cells is a multistep process starting with the HSC

giving rise to multi-potent progenitors (MPP) followed by production and

commitment to lymphoid (common lymphoid progenitors, CLP) and myeloid

restricted progenitors (common myeloid progenitors, CMP) which divide frequently

and differentiate into distinct hematopoietic lineages (Figure 1) (Reviewed in (Reya et

al., 2001). Identification of the lymphoid primed multi-potent progenitors (LMPP),

which lack erythrocyte and megakaryocyte potential but possess the potential to

undergo lineage segregation into lymphoid and myeloid lineages, has questioned this

classical model of hematopoiesis (Adolfsson et al., 2005). How the hierarchy and the

lineage commitment for the hematopoietic three is ordered remains to be

investigated and the debate continues (Forsberg et al., 2006; Månsson et al., 2007).

Differentiation to specialized lineage cells is regulated by both intrinsic (transcription

factors) and extrinsic (cytokine receptor signaling) factors (Laiosa et al., 2006).

THE HEMATOPOIETIC STEM CELL

The true HSC is a rare cell type, constituting 1 per 105 bone marrow cells (Harrison

1988) and can maintain the turnover of new blood cells throughout a lifespan. The

first revolutionary experiments on, perhaps the best characterized stem cell today,

the HSC, was done nearly a half century ago by Till, McCulloch and Becker (Becker et

al., 1963; Till and Mc, 1961). They found a clone of murine marrow cells capable of

repopulating all lineages in an irradiated mouse host.

The definition of HSCs is that they are unspecialized cells capable of producing all

hematopoietic lineage cells as well as renewing themselves (Till and Mc, 1961). The

HSC is divided in long term (LT) stem cells, which have a lifelong reconstitution ability,

and short term (ST) stem cells, with a time-limit reconstitution of around 8 weeks

(Morrison and Weissman, 1994). Early studies in the stem cell research area

suggested that some HSCs remain outside the cell cycle in a dormant state (Kay,

1965). However, a more recent study has shown that 8% of the LT-HSCs are in the cell

cycle at any given time and that nearly all (99%) divide within a two month time

frame (Cheshier et al., 1999). The number of HSCs in young mice is relatively constant

(Harrison et al., 1988), and to be able to divide and differentiate into progenitor cells

they must have a self-renewal capacity in order not to decimate themselves. The HSC

must undergo asymmetric cell division to be able to self-renew itself and at the same

time produce progeny cells which divide further and mature into different cell fates


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(Knoblich, 2001). The determination of the HSC to self-renew or to differentiate is

thought to be regulated by intrinsic factors via a stochastic process (Abkowitz et al.,

1996; Mayani et al., 1993; Suda et al., 1984), but deterministic regulation by extrinsic

signals from the microenvironment is also of importance (Ho, 2005; Metcalf, 1998).

The regulation between asymmetric and symmetric cell division needs to be well

balanced in order to maintain homeostasis in the hematopoietic system.

Identification of HSCs HSCs are mainly located in the bone marrow, but can also be found in the spleen,

umbilical blood, and the placenta. The use of HSCs in the clinic requires an efficient

and highly purified isolation procedure. Two techniques which are frequently used to

isolate stem cells are enrichment with an immunomagnetic method and

fluorescence-activating cell sorting (FACS) selection, both based on labeling cell

surface markers with specific antibodies. Human HSCs and progenitors can be

isolated by a positive selection of CD34 expressing cells (Civin et al., 1984). However,

this population is very heterogeneous with only 1% being HSCs (Larochelle et al.,

1996). A way to achieve a purer stem cell population is to remove mature cells by

negative selection (Civin et al., 1984; Spangrude et al., 1988). As mentioned above,

the HSCs are unspecialized hematopoietic cells and do not express lineage specific

surface markers such as B220 (B cells), Mac-1 (myelomonocytic cells), Gr-1

(granulocytes), Ter-119 (erythrocytes), CD4 and CD8 (T cells). CD38 is another marker

that can be used to distinguish human progenitor cells from CD38- HSCs, as well as a

positive selection for CD133 (reviewed in (Wognum et al., 2003)).

The mouse HSC is better characterized than the human HSC and enables a purer

isolation of stem cells. The lineage negative (Lin-) selection method for mouse HSCs is

routinely used together with a positive selection for the stem cell antigen (Sca-1, also

referred to as Ly-6A/E) and c-KIT receptor (also called CD117) (Ikuta and Weissman,

1992; Li and Johnson, 1995), usually called the LSK compartment, which contains all

cells with repopulating activity (Spangrude et al., 1988; Uchida and Weissman, 1992).

Thy-1 can be used to further enrich the murine HSCs within the LSK population

because murine LT-HSCs, similar to human LT-HSCs, are found in the Thy-1low

population (Spangrude and Brooks, 1992). Mouse HSCs are CD34-/low, which are all

found in the LSK compartment (Osawa et al., 1996). Recently, expression of FMS-like

tyrosine kinase 3, Flt3 (also called Flk2 or CD135) has shown to be accompanied by a

loss of self-renewal capacity (Adolfsson et al., 2001; Christensen and Weissman,

2001). In addition, the LSKthy-1low compartment, containing the LT-HSC, completely


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overlaps the LSK phenotype lacking expression of Flt3. Staining the LSK phenotype

with antibodies against CD34 and Flt3, it is possible to distinguish ST-HSCs, expressing

CD34 but not Flt3, from LT-HSC negative for both markers (Yang et al., 2005).

However, FLT3 is ubiquitously expressed on hematopoietic cells in the entire human

bone marrow as well as in cord blood, including CLPs, granulocyte/monocytes

progenitors (GMPs), some CMP and HSCs with long term reconstitution capacity

(Figure 2) (Kikushige et al., 2008). This shows that the expression patterns for mouse

Flt3 and human FLT3 is different. Recently, Morrison and his group defined an

alternative way to identify and isolate mouse HSC by using the SLAM family markers,

in particular CD150 and CD48 (Kiel et al., 2005; Yilmaz et al., 2006). By isolating

CD150+CD48-Sca-1+Lineage-c-Kit+ bone marrow cells, they were able to nearly double

the fraction of cells capable of long-term reconstitution.

In addition to surface markers, isolation based on functional markers is also used to

identify HSCs. HSCs have the ability to efflux certain fluorescent dyes. This led to the

characterization of a Hoechst-low flow cytometric profile of HSCs, called the “side

population” (SP) because of its location in the lower left corner of a dot-plot for

Hoechst fluorescence (Goodell et al., 1996). The SP phenotype is associated with

long-term reconstitution potential and expression of high levels of a multidrug

resistant pump, the ABC transporter Bcrp1/ABCG2, which is responsible for mediating

the efflux of the Hoechst dye (Zhou et al., 2001). However, the use of SP to isolate

human HSCs has recently been questioned due to findings that the majority of cells in

the SP population were mature cells expressing lineage markers. Instead, another

characteristic for LT-HSCs, the high aldehyde dehydrogenase (ALDH) activity, is a

Figure 2. Proposed expression of FLT3 on human and mouse hematopoietic stem and progenitor cells. FLT3 in the human hematopoietic system is more widely expressed compared to a more restricted Flt3 expression in mouse cells. Human LT-HSCs express low levels of FLT3 and its expression increases during comittment to primitive lymphoid (CLP), myeloid (CMP) and granulocyte/monocytes (GMPs) progenitors. CMPs primed to develop megakaryocytes and erythrocytes do not express FLT3. In contrast, mouse Flt3 expression is limited to multipotent progenitor cells (MPP) and early lymphoid progenitors (CLP), although a fraction of CMPs express Flt3 at a low level (modified from (Kikushige et al., 2008)) .


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better marker for isolation of human HSCs (Jones et al., 1996). In contrast, the most

suitable functional method to isolate murine HSC is suggested to be the Hoeschst

exclusion and not the ALDH activity (Pearce and Bonnet, 2007). Although the purity of

HSC in the SP has been questioned, the most primitive quiescent human HSC is

suggested to be Lin-CD34+CD38-ALDHbrightSP+ (Pierre-Louis et al., 2009).

In vitro and in vivo assays to detect HSCs There are several in vitro assays for detection of hematopoietic stem and progenitor

cells (Figure 3). A common way to quantify lineage-committed progenitor cells is the

colony-forming cell (CFC) assay where cells are seeded in semi-viscous media dishes.

In this way, colonies from one single cell can be analyzed and scored in an inverse

light microscopy. To quantify more primitive human and mouse hematopoietic cells a

long-term culture (LTC) assay was established 20 years ago (Sutherland et al., 1989).

A layer of adherent feeder cells, primary bone marrow stromal cells or a stromal cell

line, are first established and irradiated. Then test cells, unseparated or purified

hematopoietic cells, are added and cultured for 4-5 weeks. During this time, more

committed progenitors differentiate, die and disappear and only very primitive cells

remain in the culture. The culture is then transferred to new dishes containing

methylcellulose, a semi-viscous media. After 12-14 days de novo CFCs, formed from a

LTC-initiating cell (LTC-IC) can be detected and scored. LTC-IC assays can detect some

but not all HSCs (Larochelle et al., 1996). Another variant to score primitive cells is to

analyze the test culture 28-45 days after seeding in situ on the feeder layer, where

they form very distinguished flat colonies of cells tightly adhered to the feeder cells

resembling cobblestones, hence called “cobblestone-area-forming-cells” (CAFC)

(Ploemacher et al., 1991). The CAFC assay can easily overestimate the number of

primitive hematopoietic cells and a validation of the method needs to be done to get

reliable results (Denning-Kendall et al., 2003).

Detection of colony forming cells with high proliferative potential (HPP-CFC) have

been frequently used for detecting, in particular human, hematopoietic stem cells.

HPP-CFCs have been defined as dark colonies greater than 0.5 mm and containing

more than approximately 50.000 cells (McNiece et al., 1990).


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Although in vitro assays are needed to further study and characterize stem cells, it is

important to state that the existence of a true HSC can only be proved by using in

vivo long-term reconstitution assays. A HSC has the capacity to recover the

endogenous hematopoietic system when transplanted into an irradiated, i.e.

myeloablated, mouse. A common in vivo assay used is the competitive repopulation

assay, which is a relative measurement of the repopulation ability of the test cells

compared to a reference standard of normal non-fractioned bone marrow cells. The

competitive bone marrow fraction is also necessary to support the hematopoietic

tissue initially before primitive stem and progenitor cells have produced mature and

functional hematopoietic cells. Myeloablated mice are transplanted with genetically

marked test cells from donor mice (Szilvassy et al., 1990). Using CD45 congenic

(genetically different in one locus) mouse strains whose leukocytes can be distinguish

by their expression of CD45.1 or CD45.2 forms of the alloantigen enables selective

tracking of test cells and competitor cells in congenic mice. If normal B6 (C57BL/6)

Figure 3. In vitro and in vivo stem and progenitor cell assays. To study HSCs and progenitors a variety of assays can be used. The colony-forming cell (CFC) assay can be used to detect committed progenitor cells. To quantify more primitive progenitors, long term cell initiating cell (LTC-IC), cobblestone-area forming cell (CAFC), and high proliferative progenitor (HPP)-CFC assays are commonly used. Transplantations of stem cells in mice enable detection of long term reconstitution cells.


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mice, expressing CD45.2, are used as recipient mice, then isolated HSCs from CD45.1

expressing B6.SJL mice can be used as donor cells. Competitor cells are syngenic to

the recipient strain (Figure 4).

To evaluate the self-renewal of human HSCs, immunedeficient xenogeneic nonobese

diabetic–scid/scid (NOD/SCID) mice are used as recipients (Conneally et al., 1997).

Engraftment potential for the test cells are evaluated for multilineage differentiation

in the peripheral blood of the recipient for various endpoints.

Clinical use of HSCs The pioneer for hematopoietic stem cell transplantation (HSCT) was E. Donnall

Thomas who developed bone marrow transplantation as a treatment for leukemia

more than 50 years ago (Thomas et al., 1957). Dr. Thomas, together with Dr. Joseph E

Murray, was awarded with the Nobel Prize in medicine in 1990. HSCT is used in the

treatment of multiple myeloma (Blade and Kyle, 1998) or severe leukemia (Michallet

et al., 1996), where the patient has become resistant to chemotherapy. For other

inherited blood diseases, such as severe combined immunodeficiency (SCID) and

sickle cell anemia, BMT is the only curative treatment (Pinto and Roberts, 2008). The

donor can either be the patient him-/herself (autologous bone marrow transplant) or

a genetically matched donor (allogenic bone marrow transplant). For the latter, graft

rejection is a major problem, which partly can be overcome with immunosuppressive

medicine. Most allogenic stem cell transplantations use mobilized peripheral blood as

a stem cell source instead of bone marrow. Granulocyte colony-stimulating factor (G-

Figure 4. Schematic representation of the competitive repopulation assay. Donor cells from C57B6/J mice (CD45.2) are, together with competitor bone marrow (BM) cells (CD45.1), injected in the lateral tail vein of lethally irradiated recipient B6.SJL mice (CD45.1). At different time points after transplantation, engraftment potential is analyzed using flow cytometry in samples taken from peripheral blood.


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CSF) is routinely used to mobilize bone marrow stem cells into the peripheral blood. A

recent study reported that usage of mobilized peripheral HSCs instead of using bone

marrow stem cells in BMT increases the risk of developing graft-versus-host disease

although survival rate is not affected (Gallardo et al., 2009). To establish engraftment

of hematopoiesis, a large amount of donor bone marrow cells are needed. A shortage

of donor cells is often a limiting factor. Ex vivo expansion of hematopoietic stem and

progenitor cells could increase both the usage of BMT in clinics and also enhance

hematopoietic recovery.

Can HSCs be expanded in vitro? Basically, although clearly oversimplified, the proliferation and maturation of mature

hematopoietic lineage cells are regulated by specific growth factors such as

erythropoietin (Epo) for erythrocytes, macrophage colony-stimulating factor (M-CSF)

for macrophages and granulocyte colony-stimulating factor (G-CSF) for granulocytes.

In contrast, proliferation and survival of multipotent progenitors are stimulated with

overlapping cytokines such as interleukin (IL)-3, granulocyte/macrophage colony-

stimulating factor (GM-CSF) and IL-4. Our knowledge about extrinsic factors

regulating self-renewal (asymmetric division) in long-term HSC, which natural state in

vivo is mainly dormant, is limited. A balanced hematopoiesis in vivo at steady-state is

dependent on the asymmetric cell division of HSCs, whereas expansion of stem cells

requires symmetric cell division. An interaction of early acting cytokines including IL-

6, IL-11, fibroblast growth factor (FGF), stem cell factor (SCF, or c-KIT ligand, or steel

factor), thrombopoietin (Tpo), and FLT3-ligand (FL) is thought to play a significant role

in survival and maintenance of HSCs (Borge et al., 1997; de Haan et al., 2003; Ogawa,

1993). However, a successful expansion of human HSCs in vitro by using cytokines has

shown to be difficult. Culture of stem cells with growth cytokines often results in

massive proliferation, which is thought to be coupled with the loss of self-renewal

(Ogawa, 1993; van der Loo and Ploemacher, 1995). Long-term culture of whole

mouse bone marrow was pioneered by Dexter and colleagues in 1984 (Dexter, 1984),

where bone marrow cells were cultured on stromal cells together with the addition

of growth factors.

Finding the optimal conditions to expand stem cells without affecting the “stemness”

would be of great importance for clinical use. Since the initial use of HSC in BMT

therapy in the late fifties, several improvements in the field have been made,

although clinical significance for in vitro expansion of human HSCs has not been

achieved today. Conflicting results for which combination of cytokines that is optimal


17

for maintenance and expansion of HSCs indicate that more reliable and reproducible

studies need to be done before expansion of human HSCs can be used in clinics.

Some early acting cytokines, such as FL, affect murine and human HSCs differently,

which makes the interpretation of data even harder.

Growth conditions for mouse HSCs are better established. The first known HSC self-

renewal division that occurred in in vitro cultures was reported in 1992 (Fraser et al.,

1992). Mouse marrow cells enriched for slow-dividing stem cells using 5-fluorouracil

(5-FU) were cultured for four weeks on an irradiated layer of marrow feeder cells. By

injecting cultured stem cells into myeloablated recipient mice, they demonstrated a

maintained ability for long-term repopulation. Several studies with similar results

followed showing maintenance of self-renewal but not expansion of stem cells. At

this time, attention focused mainly on finding soluble growth factors, cytokines, to

achieve a reproducible and controlled regulation of stem cell division. Five years

later, a combination of FL, SCF, and IL-11 in serum-free media showed to be a

successful combination for the expansion of HSCs with long-term self-renewal (Miller

and Eaves, 1997). Sca-1+Lin- marrow cells were cultured for 10 days and in vivo

studies showed a 3-fold net increase in multilineage repopulation ability.

FGF receptor (FGFR), involved in the maintenance and developing of a wide range of

tissues, is expressed on mouse LT-HSCs and stimulation with FGF-1 alone in serum-

free media is capable of their expansion (de Haan et al., 2003). It is, however,

possible that these results could be caused by indirect effects due to culture of

unfractionated cells instead of purified HSCs.

A combination of SCF and FL together with Tpo (Ramsfjell et al., 1996) or IL-6, FL, and

Tpo (Matsunaga et al., 1998) support survival of murine long-term HSCs. A body of

evidence implicates that Tpo is important for in vivo maintenance of HSC and capable

of generating de novo HSCs in culture (Borge et al., 1997; Kirito et al., 2003; Qian et

al., 2007; Yagi et al., 1999). While SCF and FL appear to be important for survival and

proliferation of HSCs, retention of stem cell activity needs stimulation with IL-11 or IL-

6 (Borge et al., 1997; Sauvageau et al., 2004). Recently, Dr Lodish and his group

developed a culture system for murine HSCs using SCF, Tpo, FGF-1 and the insulin-like

growth factor (IGF)-2 in serum-free media, and were able to expand repopulating

HSCs eight-fold compared to freshly isolated HSCs (Zhang and Lodish, 2005). A more

remarkable expansion of HSCs was achieved when any of the members of the

angiopoietin-like family of proteins were added to the culture showing a 30-fold

increase of LT-HSCs (Zhang et al., 2006a).


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It has also been shown that members of the Wnt growth factors are important for

regulation of HSCs. Overexpression of β-catenin, a downstream effector in the Wnt

pathway, results in an expansion of HSCs (Reya et al., 2003). Wnt induces expression

of HOXB4 and Notch1, which are both suggested to be important for self-renewal

(Reya et al., 2003; Stier et al., 2002; Varnum-Finney et al., 2000).

There is an increasing interest in culturing HSC on feeder layer of stromal cells or

other supporter cells, referred to as co-culture. The use of feeder cells for in vitro

culturing of stem cells would mimic the in vivo condition found in the bone marrow

(see section; The hematopoietic stem cell niche?). A wide range of soluble cytokines,

known and unknown, are secreted from the stromal feeder cells regulating self-

renewal, survival and expansion. In addition, the feeder cells provide important cell-

cell contact with the HSCs including ligand-receptor and interactions between

integrins and the extracellular matrix. Special interest in Notch signaling, because of

its role in supporting the HSCs, has lead to the finding that its ligands Delta1 and

Jagged1 had no or little effect on the increase of ST-HSCs expansion, but significantly

increased numbers of LT-HSCs (Kertesz et al., 2006; Suzuki et al., 2006).

Regulation of the cell cycle in HSCs The fact that all cells produced during hematopoiesis are derived from HSCs, which

are mostly dormant, implicates that a balanced regulation of proliferation and

maintenance of a quiescent state is fundamental to enable formation of new cells

without causing exhaustion of the HSC pool. Investigation of in vivo 5-bromo-2-

deoxyuridine (BrdU) labeling of dividing cells has shown that 50% of all LT-HSCs divide

every 6 days, whereas 99% of all LT-HSCs have entered the cell cycle after 57 days

(Cheshier et al., 1999). Furthermore, at any given time point, 75% of all LT-HSCs are in

a quiescent state. The cell cycle consists of four phases; G1, S, G2 and M with the

addition of G0, which is a quiescent state outside the cell cycle. Growth factors

stimulate cells to enter the G1 phase, where they grow and make preparations for

the DNA replication, which takes place in the S phase. The G2 phase is a second

growing phase needed to enable cell division, or mitosis, in the M phase. The cell

cycle is controlled by cyclins, cyclin-dependent kinases (CDK), and CDK-inhibitors. In

mammalian cells CDK4/6 and CDK2, which are involved in the activation of the cell

cycle, are inhibited by CDK-inhibitors of the INK4 family (p16INK4A, p15INK4B, p18INK4C

and p19 INK4D) and of the Cip/Kip family (p21Cip1/Waf1, p27Kip1, and p57Kip2), respectively

(Sherr and Roberts, 1999). I will highlight some of the CDK-inhibitors that have


19

received special attention in their role of regulating cell cycle activity in

hematopoietic stem cells.

A slow cell cycle and maintenance of quiescence is important to protect stem cells

and prevent exhaustion of HSC activity (Cheng et al., 2000b; Nygren and Bryder,

2008). Mice lacking the gene coding for p21Cip1/Waf1 (p21 hereafter) show increased

cycling of primitive HSCs with an impaired self-renewal capacity (Cheng et al., 2000b).

Cell cycle inhibition in the late G1 phase by p21 seems to be stem cell restricted, due

to the fact that progenitor cells from p21-/- mice show a decreased proliferation rate

(Braun et al., 1998; Mantel et al., 1996). In contrast, deletion of the early G1 phase

CDK inhibitor, p18INK4C (p18), increased the number of primitive HSCs with self-

renewal capacity (Yuan et al., 2004). Together this shows that different CDK inhibitors

can have distinct effects on the cell cycle control of HSCs. Furthermore, deletion of

p18 counteracted the exhaustion of stem cells caused by p21 deficiency (Yu et al.,

2006).

The CDK inhibitor p27Kip1 (p27) is, in contrast to p21, not regulated by the tumor

suppressor p53 (Polyak et al., 1994) and is controlled both by translational and post-

translational mechanisms (Hengst and Reed, 1996; Pagano et al., 1995). Deletion of

p27 does not affect the number of stem cells and they show a normal cell cycle,

whereas the number of progenitors in the hematopoietic system is increased (Cheng

Figure 5. Maintained quiescence is critical for self-renewal in HSCs. Several CDK inhibitors are important in the control of the cell cycle. For HSCs, upregulation of p21 and p57 are suggested to retain HSCs in a quiescent state. In contrast, p16 and p19 are thought to induce cell cycling. Bmi-1, causes accumulation of HSCs in G0 by inhibition p16 and p19. Abrogation of the transcription factor Mef causes quiescence, whereas deletion of either PTEN or FoxO proteins results in increased proliferation and exhaustion of HSCs (modified from (Orford and Scadden, 2008).


20

et al., 2000a). The finding that there is dominance of p21 in stem cell kinetic

regulation and of p27 in progenitor cells implicates a divergent function of different

CDK inhibitors in distinct cell stages depending on intrinsic mechanisms. This

difference in the CDK inhibitor function might contribute to the explanation why stem

cells have a slower cell cycle compared to highly proliferative progenitor cells.

Bmi-1 is necessary for the maintenance of HSCs and is involved in the regulation of

cell cycle by repressing the expression of p16INK4A (p16) and p19 INK4D (p19) (Park et

al., 2003), which are both involved in cell aging.

Until recently, the significance for p57Kip2 (p57) in HSC kinetic was unknown.

Expression analysis of different CDK inhibitors in the quiescent bone marrow SP cells

and non-SP cells revealed novel data showing p57 specific expression in SP cells

(Umemoto et al., 2005). This suggests a critical role of p57 in maintaining HSCs in a

quiescent state. Similarly, data from Nakauchi and his group showed abundant

expression of p57 in freshly isolated primitive CD34-LSK cells, whereas p57 was

downregulated in CD34+LSK cells, HSCs with less self-renewal capacity (Yamazaki et

al., 2006). The predominance of p57 in HSC, in contrast to p21 and p27, might imply a

major role of p57 as a specific CDK inhibitor within the HSC cell cycle. It has also been

shown that quiescence of HSCs by two growth factors, transforming growth factor

(TGF)-β (Scandura et al., 2004) and Tpo (Qian et al., 2007), is mediated by p57

upregulation.

Another regulator of retaining HSC in the G0 phase is the phosphatase and tensin

analog (PTEN). Abrogation of PTEN increases the cycling of HSCs and causes

exhaustion of the stem cell pool (Zhang et al., 2006b). PTEN controls the cell cycle

through inhibition of the phosphatidylinositol-3 kinase/AKT pathway and is

commonly mutated in malignant tumor cells (Vivanco and Sawyers, 2002). Similar to

the p21-/- mouse, depletion of PTEN, as well as the growth factor independent 1

(Gfi1) or Forkhead box transcription factors (FoxO) 1,3 and 4, show a HSC phenotype

with increased cycling leading to HSCs exhaustion (Orford and Scadden, 2008).

The transcription factor MEF/ELF4 regulates both self-renewal and quiescence of

HSCs (Lacorazza et al., 2006). Mef null mice have increased number of HSCs, which

are more quiescent than wild type HSCs. Recently, it was found that this increase in

HSCs quiescence in Mef null mice was abrogated in the absence of p53, suggesting an

important role of p53 in the regulation of stem cell quiescence (Liu et al., 2009).

Furthermore, maintenance of quiescence by p53 is thought to be mediated by the


21

p53 target genes Gfi1 and Necdin. Gfi-1 has an essential role in restricting the

proliferation of HSCs and retaining their self-renewal capacity (Hock et al., 2004).

Contradictory data for the role that p53 plays in HSCs engraftment have been

reported (Akala et al., 2008; Chen et al., 2008; Hock et al., 2004; TeKippe et al., 2003).

Therefore, p53 seems to be important for quiescence of HSCs, whereas its

significance for increasing functional HSCs is controversial (Figure 5).

The interaction of the tyrosine kinase receptor Tie-2, expressed on HSCs, and its

ligand angiopoietin-1 (Ang-1) is another important way to maintain the HSC

quiescence (Arai et al., 2004). This is one, of many, receptor-ligand pairs involved in

the maintenance of HSCs in the stem cell niche, which will be explained in more detail

in the next chapter.

The hematopoietic stem cell niche Although the HSC is the best characterized adult stem cell today, its precise location

in the bone marrow where it self-renews and differentiates is not fully defined. In

contrast, the place for stem cells in other tissues, such as the skin and brain, is well

identified and described. The microenvironment in the bone marrow provides the

HSCs with important signals for their maintenance (self-renewal and survival),

migration and differentiation. These signals are mediated by secreted- and

membrane-bound factors from bone marrow cells. The concept of a

microenvironment with a special architecture housing the stem cells was first

proposed by Schofield more than 30 years ago when he introduced the term “stem

cell niche” (Schofield, 1978). The criteria for a stem cell niche are: first, the number of

stem cells is well regulated; second, an interaction with a heterogeneous population

of other cells is necessary for the stem cell maintenance; third, a balanced regulation

of stimulatory and inhibitory signals from membrane-bound and secreted molecules;

and fourth, non-stem cells can acquire stem cells-like properties when located in the

niche (Adams and Scadden, 2006). Although the bone marrow is the common place

for HSCs, they undergo regular trafficking into the peripheral blood where they reside

for shorter periods and then return to the bone marrow (Wright et al., 2001). The

function for the stem cells in the peripheral blood is unknown, but their mobilization

into the peripheral blood following various stresses such as chemotherapy or

administration of G-CSF has been utilized in BMT therapy. In the niche, HSCs reside in

fragments of spongious bone, called the trabecular bone, in close contact to the

osteoblasts (Figure 6). This part of the bone contains a variety of cells such as


22

osteoblasts, adipocytes, reticular stromal cells, vascular endothelial cells, and small

blood vessels called sinusoids.

The crucial role of the niche has been recognized for a long time, starting with the

finding that mutation in the SCF gene, expressed on niche cells, in Sl/Sld mice, had a

dramatic effect on the bone marrow HSCs (McCulloch et al., 1965). The first evidence

that osteoblasts were important for the HSC niche came when a conditional (tissue

specific) inactivation of the bone morphogenic protein (BMP)-1 receptor showed a

bone defect causing an increase of the number of osteoblasts coupled to an increase

of HSCs (Zhang et al., 2003). They found that LT-HSCs appeared to be attached to

early spindle-shaped N-cadherin expressing osteoblastic (SNO) cells. Another study

showed that the deletion of the oncogene c-Myc in HSCs resulted in severe cytopenia

and an accumulation of HSCs in the bone marrow, whereas overexpression of c-Myc

was shown to decrease the expression of N-cadherin on HSCs with lost self-renewal

(Wilson et al., 2004). This indicates that c-Myc controls the balance between self-

Figure 6. The hematopoietic stem cell niche. In the bone marrow, HSCs reside in the spongious bone called trabecular bone. HSCs receive important survival and maintenace signals from other cells in the niche (e.g. osteoblasts, reticulocytes, and endothelial cells). The nutrient and blood supply in the niche is rather limited and carried out via small blood vessels called sinusoids.


23

renewal and differentiation. Lately, the role of N-cadherin in the HSC niche has been

questioned. Two recent studies have shown that conditionally deletion of N-cadherin

in HSCs did not affect the bone marrow cellularity or the maintenance of the HSCs

(Kiel et al., 2009; Kiel et al., 2007). Another evidence for the importance of

osteoblasts in the niche was reported in a study showing that constitutively activation

of parathyroid hormone receptors, expressed on osteoblasts, led to an increase of

trabecular osteoblasts supporting the expansion of HSCs (Calvi et al., 2003).

Furthermore, the expansion of HSCs caused by increased number of osteoblastic cells

showed to be dependent on Notch signaling, although the role of Notch in the niche

has been challenged (Mancini et al., 2005). The mineral environment in the bone

marrow with a high quantity of calcium has shown to play an important role in the

niche. Ca2+ ion concentration is recognized by the seven-transmembrane calcium-

sensing receptor (CaR) which is expressed on HSCs (Adams et al., 2006). Mice

deficient for CaR show a distinguished decrease in HSCs in the trabecular bone

whereas the numbers of more mature progenitors were intact. Further analysis

revealed that HSCs had entered the circulation and the spleen, however, no

differences in the cell cycle profile were detected. It is likely that the calcium gradient

in the niche has a major impact on homing and retaining HSCs in the bone marrow

niche. Quiescence is a signature for HSCs located in the stem cell niche. The

interaction of angiopoietin (Ang)-1 expressed on osteoblasts with the tyrosine kinase

receptor Tie-2 on HSCs plays a critical role in maintaining the quiescent HSC state and

strengthening of the adhesion to the endosteal surface (Figure 7) (Arai et al., 2004).

Lately, the role of the osteoblasts as the single niche cell important for HSC

maintenance has been questioned in a study reporting that depletion of osteoblasts

in the trabecular zone did not affect the frequency of HSCs (Kiel et al., 2009; Kiel et

al., 2007). It is likely that there is interplay between several cell types in the niche,

and that not only direct signaling with cell-cell contact is importance. One possibility

is that soluble factors secreted by endosteal, perivascular or other cells create a

gradient of secreted factors which contribute to the regulation of HSCs in the niche.

Ang-1, Tpo, as well as the CXC chemokine ligand (CXCL) 12, also known as stromal

derived factor (SDF)-1, are secreted by niche cells and known to regulate HSC

maintenance (Arai et al., 2004; Sacchetti et al., 2007; Yoshihara et al., 2007). Several

findings reveal that quiescent HSCs with long-term potential are associated with

osteoblasts in the “osteoblastic stem cell niche” (Arai et al., 2004; Calvi et al., 2003;

Zhang et al., 2003), while other findings point towards the importance of vascular

cells to maintain HSCs in the “vascular stem cell niche” (Kiel et al., 2005).


24

It has been suggested that the vascular niche resides more mitotically active HSCs

and play a role in the mobilization, while quiescent HSCs are located in the

osteoblastic niche (Avecilla et al., 2004; Heissig et al., 2002). This indicates that the

osteoblastic niche is the primary niche for maintenance of LT-HSC and the vascular

niche is a secondary niche which function is to produce progenitor cells and maintain

the homeostasis in the hematopoietic system (Wilson and Trumpp, 2006). A body of

evidence shows the importance of osteoblasts, but it remains to be investigated if

they are actually required for HSC maintenance (reviewed in (Kiel and Morrison,

2008)). Recent evidence support the earlier findings that quiescent HSCs are closer

located to the osteoblasts, whereas more mitotically active HSCs are found more

distant to osteoblasts (Lo Celso et al., 2009). However, sinusoids are also present in

the endosteal niche (Kubota et al., 2008; Lo Celso et al., 2009; Xie et al., 2009) and it

is therefore likely that the HSC niche is created through a combined influence of

several specialized niche cells. Like adipocytes and osteoblasts, endothelial cells

Figure 7. A simplified picture of some of the signal transduction pathways in the HSC niche. Both soluble and membrane-bound interactions between osteoblasts and HSCs in the niche are thought to regulate the balance between quiescence, self-renewal, migration, and adhesion (Rizo et al., 2006).


25

surrounding sinusoids, are derived from mesenchymal stem cells. Recent data has

revealed that endothelial cells express the HSC regulators CXCL12 and Ang-1

(Sacchetti et al., 2007). Experiments have shown that deletion of the receptor for

CXCL12, CXCR4, had a severe effect on the number of HSCs, indicating that CXCL12-

CXCR4 signaling plays an essential role in maintaining quiescent HSCs. In addition,

bone marrow HSCs seem to be co-localized to special reticular cells expressing high

amounts of CXCL12, called CXCL12-abundent reticular (CAR) cells, which are found

near endothelial cells or the endosteum (Sugiyama et al., 2006). However, another

study has reported that CXCL12 and CXCR4 have a key role in the G-CSF-induced

mobilization of HSCs (Petit et al., 2002). Endothelial cells are thought to regulate HSC

functions in the niche and maintaining stem cells properties in vitro (Kiel et al., 2007;

Kiel et al., 2005; Li et al., 2004; Ohneda et al., 1998). These data together raise the

possibility that HSCs can reside at different locations in the bone marrow and that

several different cells are involved in the regulation of HSCs. So far, it is known that

the maintenance of HSCs in the niche is dependent on a variety of membrane-bound

and soluble factors secreted from osteoblasts, endothelial cells and other known or

unknown cells. The anatomic microenvironment in the bone marrow is dynamic and

the question of whether there is one or more niches remains.

The hypoxic stem cell niche To better understand the stem cell niche in the bone marrow and how the regulation

of HSCs is controlled, many studies have focused on the interplay between niche cells

and HSCs. The oxygen level in the bone marrow microenvironment is another factor

that lately has been given special attention in the hematopoietic stem cell research. It

is suggested that the bone marrow is low in oxygen (hypoxic) (Ceradini et al., 2004;

Harrison et al., 2002). The hypoxic region is thought to be caused by lower blood

perfusion. Due to difficulties in measuring the partial pressure of oxygen (pO2) in the

bone marrow, the exact physiological concentration of oxygen in the endosteal area

has not been specified. A provocative, but sensational, study used a mathematical

model to estimate the oxygen concentration in bone marrow and speculated that

HSCs were located in areas of low oxygen, whereas more proliferative blood cells

were located in areas of higher oxygen concentration (Chow et al., 2001).

Physiological measurements with a blood gas syringe in the bone marrow of a

healthy human adult revealed a pO2 average of 54.9 mmHg (Harrison et al., 2002).

This could be compared to a pO2 in capillaries of 95 mmHg and 40 mmHg in the

interstitial fluids that surround tissue cells (Guyton and Hall, 1996), whereas in

ischemic tissues the O2 tension can decrease to 4 mmHg (Ceradini et al., 2004). A


26

recent study by Parmar et al tested the hypothesis that HSCs were located in areas of

low oxygen by utilizing that hypoxic regions have a lower blood perfusion which was

measured by injecting mice with the fluorescent dye Hoechst 33342 (Ho). Bone

marrow cells were isolated and divided according to a Ho dye diffusion gradient

which simulates the in vivo level of oxygen. To investigate the stem cell character of

cells with low and high Hoechst fluorescence, transplantations into lethally irradiated

mice were done. The results showed that the bone marrow fraction with the lowest

Ho uptake, inferred to be hypoxic, had the highest amount of long-term repopulating

cells. Furthermore, HSCs in the SP showed high staining for the hypoxic probe

pimonidazole (PIM), which covalently binds to protein thiol groups when pO2 is below

10 mmHg (corresponding to less than 1.3% oxygen) (Parmar et al., 2007). Moreover,

another study shows that PIM stains hypoxic areas in vivo in the bone marrow of

mice. PIM staining was intense in endosteal areas and decreased rapidly within a

short distance of 50 µm (Levesque et al., 2007), postulating that the osteoblastic

niche is hypoxic. Recently it was shown that quiescent cells, label-retaining cells

stained with BrdU, are found in hypoxic sinusoids containing areas distant from the

“vascular niche” (Kubota et al., 2008). This finding, together with Lo Celsos data that

dormant stem cells are closer to osteoblasts compared to cells with a more active cell

cycle (Lo Celso et al., 2009), indicates that areas containing sinusoids, close to the

endosteum, distant from capillaries and likely hypoxic, maintain HSCs in a quiescent

state.

Consistently, it has been shown that cultivation of bone marrow cells in hypoxia in

vitro increases the number of primitive colony forming cells, and sustains their

repopulating ability better compared to normoxic conditions and inhibited

differentiation (Cipolleschi et al., 1993; Eliasson, 2006; Ivanovic et al., 2000; Ivanovic

et al., 2002; Ivanovic et al., 2004). Moreover, cultivation of purified human HSCs (Lin-

CD34+CD38-) for 4 days in hypoxia revealed a 6-fold increase of repopulating cells

compared to stem cells cultured in normoxia and also a nearly 4-fold increase

compared to freshly isolated HSCs (Danet et al., 2003). This is consistent with findings

in a recent study on human HSCs showing that hypoxia supports the maintenance of

HSCs but reduces proliferation (Shima et al., 2008). Similarly, culture of CD34+ cord

blood cells in very low (0.1%) oxygen levels maintains survival and favors return of

cycling stem cells to G0 (Hermitte et al., 2006).

HYPOXIA INDUCIBLE FACTOR-1α

27

HYPOXIA INDUCIBLE FACTOR-1

The main regulator in hypoxic cells is the transcription factor hypoxia-inducible factor

(HIF)-1. HIF-1 regulates multiple genes involved in glucose metabolism, as well as

stem cell mobilization, proliferation, survival, and differentiation. HIF-1 was

discovered in 1988 when a hypoxia-response element (HRE) was found in the

enhancer for erythropoietin, a protein involved in erythropoiesis (Goldberg et al.,

1988; Semenza et al., 1991). HIF-1 is a heterodimeric transcription factor consisting of

a 120 kDa subunit HIF-1α and a constitutively expressed β subunit, which is also

called aryl hydrocarbon receptor nuclear translocator (ARNT). Three different HIF-α

subunits have been characterized, HIF-1α, HIF-2α and HIF-3α. HIF-2α is

predominately expressed in lung and endothelial tissues, whereas HIF-3α is more

selectively expressed in neuronal cells and corneal epithelium (reviewed in (Ke and

Costa, 2006)). The two subunits of HIFα and HIFβ are members of the basic helix-

loop-helix Per-ARNT-Sim (bHLH-PAS) protein family, which are responsible for the

heterodimerisation of the protein. In the domain structure of HIFα, two

transactivation domains (TAD) have been identified; one N-terminal (N-TAD) and one

C-terminal (C-TAD) (Figure 8). Regulation of HIF-1 activity is mediated by

posttranslational modification of the oxygen-dependent degradation domain (ODD)

on the HIF-1α subunit.

At oxygen levels above 5%, hydroxylation of two conserved proline residues, Pro 402

and Pro 564 in the ODD, enables binding of the ubiquitin ligase von Hippel-Lindau

(VHL) tumor suppressor protein, which leads to polyubiquitination and rapid

degradation of HIF1-α by the proteosome (Maxwell et al., 1999). During hypoxic

conditions, hydroxylation is inhibited, leading to the stabilization of HIF-1α and

translocation to the nucleus where it dimerizes with HIF-1β and becomes

transcriptionally active (Figure 9). The oxygen sensors in the oxygen dependent

regulation of HIF-1α are prolyl hydroxylases (PHDs) which requires both oxygen and

ferrous ions (Fe2+) to be able to hydroxylate residues of the ODD. Diminished levels of

Fe2+ caused by iron chelators or metal ions such as cobalt (Co2+) are able to stabilize

Figure 8. Domain structure of HIF-1α. HIF-1α belongs to the bHLH and PAS protein family. The oxygen dependent degradation domain (ODDD) is important for oxygen regulated stability of HIF-1α. Oxygen levels higher than 5% cause hydroxylation of P402 and P564 and acetylation of K532. HIF-1α contains two transaction domains (TAD) domains involved in activation of gene transcription.


28

HIF-1α (reviewed in (Ke and Costa, 2006)). Once HIF-1α is stabilized, a further

decrease in oxygen inhibits the activity of factor inhibiting HIF (FIH), which modulates

C-TAD of HIF-1α and enables recruitment of the transcriptional coactivator p300 and

CREB binding protein (p300/CBP) (Mahon 2001).

HIF-1α activity is in addition to hydroxylation, regulated be several post-translational

modifications such as ubiquitination, acetylation, phosphorylation, and SUMOylation

(Agbor and Taylor, 2008; Ke and Costa, 2006). Phosphorylation of HIF-1α by the

mitogen-activated protein kinase (MAPK) p42/44 has shown to enhance the

transcription activity but not the stability of the protein (Richard et al., 1999). Lately,

it has become evident that HIF activity can be induced in normal oxygen levels by

specific cytokines, such as PDGF, FGF, IGF, SCF, and Tpo (Conte et al., 2008; Pedersen

et al., 2008; Yoshida et al., 2008). The biological significance for induction of HIF-1 by

cytokines, which is less intense, compared to hypoxic-induced expression, is under

discussion. It is thought that the induction of HIF-1 by growth factors is a result of

increased transcription of HIF-1, although stabilization of the protein is also

suggested (Pedersen et al., 2008). The stabilization could be mediated by reactive

oxidative species (ROS). The question whether ROS generated from the mitochondria

in the electron transport chain is sufficient to stabilize HIF-1α under normoxic

conditions is controversial (Brunelle et al., 2005; Gorlach et al., 2001; Yoshida et al.,

Figure 9. Oxygen-dependent regulation of HIF-1α stabilization. In normal oxygen concentration, prolyl hydroxylases (PDHs) mediate hydroxylation of proline residues of HIF-1α. This enables binding of the ubiquitin ligase von Hippel Lindau (VHL) following ubiquitination and degradation by the proteosome. In low oxygen, however, HIF-1α proteins accumulate and form dimers with the partner HIF-1β. Cofactors such as p300/CBP bind to the HIF-1 dimer, which results in gene transcription of HIF-1 target genes.


29

2008). Together, this shows that the regulation of HIF activity is a fine balance

between HIF stabilization, promoted mainly by hypoxia, and PHD dependent

degradation. It is possible that this regulation of HIF is cell type specific.

HIF-1 activates expression of several genes involved in angiogenesis and

erythropoiesis. Abrogation of HIF via deletion of Hif-1α or Arnt genes have shown to

defect the numbers of hematopoietic progenitors in the yolk sac (Adelman et al.,

1999) and cause embryonic death before birth (reviewed in (Ke and Costa, 2006)),

which makes it difficult to fully study the involvement of HIF-1 in adult hematopoietic

progenitor and stem cells. HIF is known to activate 70 genes via binding to the HRE

site, located in the promoter and enhancer of the target genes. More than 200 target

genes are thought to exist, although not all are regulated by direct binding to a HRE

region (Wenger et al., 2005). Furthermore, whether HIF-1 and HIF-2 have the same

target genes is under discussion, but it is proposed that it is the cooperation with

other transcription factors that distinguishes the expression pattern of specific genes.

As mentioned earlier, HIF-1 regulates the transcription of erythropoietin, which is

required for the formation of erythrocytes. Another well known and important target

gene for angiogenesis is the vascular endothelial factor (VEGF), which has the

function to recruit and increase proliferation of endothelial cells (Josko et al., 2000;

Neufeld et al., 1999). In addition, HIF-1 induces transcription of matrix

metalloproteinases (MMP), which are involved in matrix metabolism and vessel

formation (Ben-Yosef et al., 2002).

HIF-1 and regulation of important niche molecules As discussed above, it is suggested that hematopoietic stem and progenitor cells are

distributed along an oxygen gradient in the bone marrow, where the HSCs are sited in

hypoxic areas and more proliferative progenitors are located in more oxygen-rich

areas closer to large blood vessels (Levesque et al., 2007; Parmar et al., 2007). This

implies that a hypoxic niche plays a fundamental role in the maintenance of HSCs.

Many of the genes induced by HIF-1 are expressed in HSCs and the HSC niche (Figure

10). The HIF-1 target gene VEGF has been shown to have an important role for

survival and in vivo repopulation of HSCs (Gerber et al., 2002). Moreover,

perichondrial cells and chondrocytes, present in the osteoblastic niche, are known to

express high levels of VEGF. A recent study showed that suppressing VEGF in mice

inhibits niche formation (Chan et al., 2009), which indicates that VEGF has an

essential role in the niche, both for maintenance of HSCs and non-hematopoietic

niche cells. Hypoxia is known to increase growth factor signaling by inducing c-KIT,


30

Notch-1 (Jogi et al., 2002), IGF-2 (Feldser et al., 1999), and FGF (Conte et al., 2008),

which are suggested to be regulated in a HIF-1 dependent way. However, it is not

clear whether these regulatory mechanisms are transcriptional and if they are HIF-1

targets in HSCs. During hypoxia, Notch-1 is known to interact with HIF-1, which leads

to expression of Notch-1 downstream target genes and a block of differentiation of

neuronal and myogenic progenitor cells (Gustafsson et al., 2005). CXCL12, which

binds to the chemokine receptor CXCR4 expressed on hematopoietic cells are

important for homing to the bone marrow (Hattori et al., 2001), as well as

maintaining quiescence of HSCs (Sugiyama et al., 2006). HIF-1 regulated CXCL12

expression in hypoxic regions in the bone marrow recruits CXCR4-expressing stem

and progenitor cells, suggesting a fundamental role of HIF-1 in homing of HSCs

(Ceradini et al., 2004).

HIF-1 has also been proposed to be involved in the mobilization of hematopoietic

progenitor cells (Levesque et al., 2007). G-CSF, used in the clinic to elicit mobilization

of transplantable HSCs, increases the number of granulocytes, which deplete the

storage of oxygen in the bone marrow, leading to stabilization of HIF-1. This is

accompanied by increased levels of VEGF that increase the permeability of the blood

vessels. HIF-1 induced expression of MMP (Ben-Yosef et al., 2002), needed for

Figure 10. A possible niche for the HSC. It is suggested that HSCs reside in close contact with osteoblasts in distance from large blood vessels. This creates an micoenvironment low in oxygen, which might play a significat role in regulation of HSC maintenance. Several known niche molecules important for quiescence, self-renewal and survial are found to be regulated by hypoxia (marked in bold) and in some cases in a HIF-1 dependent way. HSCs in more vascularized niches are thought to be more proliferative with higher intracellular ROS levels causing decreased self-renewal.


31

penetration of the endothelium, causes degradation and inactivation of CXCR4,

CXCL12 and c-KIT, which are necessary to retain stem and progenitor cells in the bone

marrow (Levesque et al., 2007). Although paradoxically, this implicates that HIF-1 can

be involved both in mobilization and homing as well as maintenance of HSCs in the

bone marrow niche.

Another target gene for HIF-1 and is the Bcrp1/ABCG2 gene (Krishnamurthy et al.,

2004b), which is highly expressed on LT-HSCs. BCRP-1/ABCG2 is important for both

the survival as well as protection of HSCs from toxic agents. This indicates that HIF-1

might protect primitive HSCs from severe damage caused by the accumulation of

toxic compounds. HIF-1 has also been reported to control self-renewal by inducing

the expression of telomerase (though hTERT) (Nishi et al., 2004), Oct4 (Covello et al.,

2006) and Notch-1 (Gustafsson et al., 2005), although it remains to elucidate if these

genes are increased by HIF-1 in hematopoietic cells.

Glucose metabolism in hypoxic cells Under low oxygen supply, cells switch their glucose metabolism from the oxygen-

dependent tricarboxylic acid cycle (TCA) to the oxygen-independent glycolysis, a

process thought to be regulated by HIF-1 (Seagroves et al., 2001; Semenza et al.,

1996). Glycolysis is a less energy effective pathway and hypoxic cells compensate this

by increasing glucose uptake. This is achieved by upregulation of glucose transporters

on the cell surface, which are transcriptionally regulated by HIF-1 (Wenger, 2002). In

addition, by increasing expression of pyruvate dehydrogenase kinase-1 (PDK1) (Kim

et al., 2006), lactate dehydrogenase A (LDH-A) (Semenza et al., 1996), and other

enzymes involved in glycolysis, HIF-1 enables a higher production of adenosine

triphosphate (ATP) through an anaerobic metabolism. PDK1 attenuates the pyruvate

metabolism in TCA by inactivation of pyruvate dehydrogenase (PDH), the enzyme

that converts pyruvate to acetyl-coenzyme A (Kim et al., 2006; Papandreou et al.,

2006; Semenza et al., 1996). Instead, LDH-A favors the cytosolic lactate production

from pyruvate (Figure 11). Recently, it was found that mice lacking prolyl hydroxylase

1 (Phd1), a negative regulator of HIF-1 and HIF-2 stabilization, showed signatures of

oxygen-independent glucose metabolism (Aragones et al., 2008), indicating that HIF

proteins play a key role in hypoxic metabolism. The ability of cells to adapt to

conditions of limited oxygen supply and maintain ATP production is of particular

importance in tumor development and stem cell maintenance.


32

HIF-1 and Cell cycle Despite the crucial role of hypoxia in the regulation proliferation of normal and tumor

cells, the mechanism behind this is poorly understood. Hypoxic conditions can cause

cell cycle arrest in the G1 phase of primary cells and also in immortalized cell lines

(Gardner et al., 2003). HIF-1α is suggested to induce cell cycle arrest by activating p21

(Goda et al., 2003; Koshiji et al., 2004) and p27 expression (Goda et al., 2003). Until

recently, the HIF-1α activated p21 expression has been a mystery due to the fact that

p21 lack a HRE sequence in the promoter. Koshiji and colleagues showed that neither

the DNA binding domain, nor the transcriptional activity was needed for HIF-1α-

induced p21 expression. Instead, HIF-1α binds to c-Myc and counteracts its

transcriptional repression of p21 (Koshiji et al., 2004). Lack of HIF-1α increases

proliferation in hypoxia and leads to decreased levels of p21, whereas p27 is

unchanged (Carmeliet et al., 1998). However, another study claimed that p21 was not

required for HIF-1α mediated cell cycle arrest, and that p27 was the key regulator of

reduced proliferation in hypoxia independently of HIF-1 (Gardner et al., 2001).

Whether HIF-1α regulates expression of p27 or not is under discussion and remains

to be investigated. Low levels of oxygen decreases proliferation of human cord blood

HSC and increases the number of quiescent cells without disturbing engraftment

potential (Hermitte et al., 2006; Shima et al., 2008). This was associated with

increased levels of p21 and p57 (Shima et al., 2008).

Figure 11. In hypoxia, cells switch their glucose metabolism to glycolysis. Under low oxygen supply, HIF-1α is stabilized and increases expression of glycolytic enzymes (e.g. lactate dehydrogenase A, LDH-A). This promotes anaerobic metabolisation of pyruvate to lactate.

APOPTOSIS

33

APOPTOSIS

Apoptosis is important in maintaining a balance between the formation of new blood

cells and the depletion of aged and damaged cells in the hematopoietic system with a

high turnover rate. Deregulation of apoptosis is involved in many diseases such as

cancer, where tumor cells through mutations have evolved mechanisms to avoid

apoptosis, whereas several neurodegenerative diseases, such as Alzheimer´s and

Parkinson´s disease, are associated with an increase of apoptosis of neurons.

Apoptosis is mediated by the activation of a cascade of asparatate specific cystein

proteases (caspases), which through proteolytic activity cause degradation of

proteins or activate DNAses which in turn cause degradation of DNA. The activation

of caspases leading to apoptosis can be triggered by two pathways, the intrinsic and

the extrinsic pathway. The intrinsic pathway is characterized by increased

permeability of the outer mitochondrial membrane, which leads to the release of

several pro-apoptotic mediators such as cytochrome c. The mechanism for

permeabilization of the outer membrane is not fully understood, but it is likely that

the Bcl-2 family members play a significant role (Green and Reed, 1998). Once

released, cytochrome c forms, together with the adaptor protein Apaf-1 and

procaspase-9, a complex called the apoptosome (Zou et al., 1997; Zou et al., 1999).

The apoptosome activates procaspase-9, which in turn cleaves and activates the

executioner caspases-3 leading to cell death. The extrinsic pathway is initiated by the

activation of special cell surface death receptors, the tumor necrosis factor receptor 1

(TNFR1) and the Fas receptor, which are activated by the binding of TNF or Fas ligand

(Locksley et al., 2001; Screaton and Xu, 2000). Both receptors contain death domains

(DDs), which interact with specific adaptor proteins with trigger an intracellular

cascade of cleavage and activation of executer caspases.

Pro- and anti-apoptotic Bcl-2 members The first Bcl-2 (B cell lymphoma-2) member, provided the name to the family

containing both pro- and anti-apoptotic members, was identified in 1985 for its

involvement in human follicular B cell lymphoma (Tsujimura et al., 1999). The

following years, studies in D. melanogaster and C. elegans led to much of the

knowledge we know today concerning the Bcl-2 proteins as critical regulators in

apoptosis. The different members of the Bcl-2 family are classified according to their

function and the presence of one to four conserved Bcl-2 homology domains (BH1-4).

The anti-apoptotic members, namely Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1/BFL-1, share

three or four BH domains, which are necessary for their ability to bind and sequester

APOPTOSIS

34

pro-apoptotic members. The pro-apoptotic members are divided into two subgroups

according to if they contain one or multiple BH domains. Bax, Bak, Bok, among

others, belong to the later group, whereas Bad, Bid, Bik, Bim, and Puma are examples

of members in the subgroup sharing the BH3 domain, and are therefore named BH3-

only members. The BH3 domain is responsible for binding and inactivating the anti-

apoptotic members. The release of cytochrome c is thought to be regulated by a

complex interaction between pro- and anti-apoptotic Bcl-2 members forming homo-

and heterodimers in the outer mitochondrial membrane. It is suggested that both

Bax and Bak can form homodimers in the mitochondria upon an apoptotic stimuli,

leading to increased permeabilization, possible through the formation of pores

inducing mitochondrial protein release (Eskes et al., 2000; Wei et al., 2001). The pore

formation is proposed to be inhibited by the anti-apoptotic members, Bcl-2 and Bcl-XL

(Cheng et al., 2001; Zong et al., 2001), whereas Bim and other BH3-only members

facilitate the activity of Bax and Bak by binding and sequester Bcl-2 and Bcl-XL (Figure

12) (Desagher et al., 1999). Precisely how the Bcl-2 proteins form monomers,

homodimers or heterodimers which activate or inhibit their activity to regulate the

permeability in the mitochondria is unclear and controversial. Regulation of Bcl-2

proteins is complex and involves both transcriptional and post-transcriptional

mechanisms. Upon stress signals such as DNA damage or metabolic stress, p53 and

FoxO3a, can induce expression of the Puma or Bim gene, respectively (Dijkers et al.,

2000; Nakano and Vousden, 2001). Lately, it has become evident that p53 and FoxO

signaling pathways interact to determine the choice between cell death and survival

(You and Mak, 2005). Both transcription factors are regulated by posttranslational

modification such as phosphorylation and acetylation, but the outcome might be

different. Post-transcriptional modification, such as phosphorylation, regulates

several pro-apoptotic proteins. For instance, growth factor stimulation of the

PI3K/AKT pathway leads to the phosphorylation of Bad, which attract 14-3-3 proteins

resulting in the sequestration of Bad due to transportation out of the nucleus (Datta

et al., 1997; del Peso et al., 1997). Bid is instead regulated by caspases-8 cleavage into

an active truncated form, resulting in Bax-dependent protein leakage from the

mitochondria. In this way, the extrinsic and intrinsic pathways are integrated

together to activate apoptosis (Li et al., 1998).

To study the individual role of Bcl-2 members in hematopoiesis and development,

knockout technology in mice has been performed. Bcl-2-/- mice have an

immuodeficient phenotype due to apoptosis in peripheral lymphocytes (Veis et al.,

1993). Studies have shown that HSCs ablated of Bcl-2 can perform long-term

reconstitution of myeloid but not lymphoid cells, suggesting an important role of Bcl-

APOPTOSIS

35

2 in lymphopoiesis (Matsuzaki et al., 1997). In contrast, Bim-deficient mice have

increased numbers of lymphoid and myeloid cells, which are resistant to growth

factor starvation but vulnerable to apoptosis induced by DNA damages (Bouillet et

al., 1999). This is likely explained by different pathways for specific apoptotic stimuli

mediated via the Forkhead and p53 pathways described above. Mcl-1-/- mice show

peri-implantation lethality, which complicates the investigation regarding its role in

adult hematopoiesis. However, a recent study that performed conditional deletion of

Mcl-1 in mice, which were anemic, showed ablation of bone marrow cells (Opferman

et al., 2005). The requirement of Mcl-1 for survival of hematopoietic cells was

particular pronounced in HSCs and progenitor cells. Furthermore, Mcl-1 was highly

expressed in HSCs and induced by early-acting cytokines, such as SCF.

Figure 12. Regulation of apoptosis by Bcl-2 family members. In the absence of growth factors, apoptosis can be triggered through the intrinsic pathway. This inhibits important survival signaling pathways such as PI3K/AKT. A possible effect can be transcription of the BH3 only protein Bim. In the mitochondria, anti-apoptotic Bcl-2 proteins bind and sequester Bax proteins. However, in the presence of Bim, this survival promoting effect of Bcl-2 will be inhibited. Instead Bax proteins form pores in the mitochondria membrane resulting in cytochrome c release. Cytochrome c forms a complex with Apaf-1 and procaspase 9, called the apoptosome, which results in activation of caspases 9. Caspase 9 activates the executioner caspases-3 that starts a cascade leading to cell death. Apoptosis can also be induced by death receptors via the extrinsic pathway, which activate caspases without involving the mitochondria.

OXIDATIVE STRESS AND HEMATOPOIETIC STEM CELLS

36

OXIDATIVE STRESS AND AGING OF HSCs

Several thousand DNA damages occur every day in every cell and the major part of

these are caused by ROS (Conger and Fairchild, 1952). During production of energy,

ATP, in the mitochondrial respiratory chain, water is formed when oxygen molecules

accept electrons. Some O2 molecules are, however, only partially reduced and form

superoxide O-2. The superoxide undergoes rapid dismutation by superoxide

dismutase (SOD) to form hydrogen peroxide, H2O2, and O2. H2O2 is more stable but

can be extinguished by three different processes: 1) it is removed by catalase and

glutathione (GSH) peroxidase; 2) it is spontaneously transformed into the highly

reactive hydroxyl radical, OH, in the fenton reaction catalyzed by Fe2+; or in

neutrophils 3), H2O2 is converted by myeloperoxidase to hypochlorous acid, a toxic

product used in the defense against bacteria (reviewed in (Ghaffari, 2008)).

HSCs seem to be particular sensitive for oxidative stress and the accumulation of ROS

causes defects in the regulation of hematopoiesis (Ito et al., 2004; Ito et al., 2006;

Miyamoto et al., 2007; Tothova et al., 2007). It is therefore not surprising that HSCs

are mostly in a dormant quiescent state with low metabolic activity, which spares

them from DNA and lipid damages caused by ROS formed in the respiratory chain.

The ataxia telangiectasia mutated (ATM) gene is essential for the regulation of

genomic stability and studies by Suda and colleagues have showed that deletion of

the Atm gene results in bone marrow failure associated with increased levels of ROS

in HSCs, leading to a decrease in stem cell numbers and defects in their reconstitution

ability. The accumulation of ROS in HSCs from Atm-/- mice results in upregulation of

the tumor suppressor genes p16 and p19 caused by the activation of the p38

mitogen-activated protein kinase (MAPK) (Ito et al., 2004). The inhibition of p38

MAPK or treatment with antioxidant reagent N-acetylcysteine (NAC) allowed HSCs

from Atm-/- mice to maintain a quiescent state and improved the self renewal of LT-

HSCs (Ito et al., 2004; Ito et al., 2006). In addition to the dysfunction of cell cycle

regulation caused by p38 MAPK, it is thought that adhesion molecules in the niche,

such as N-cadherin, are affected by ROS, leading to the exit of HSCs from the niche

(Hosokawa K, 2007). Increased levels of ROS and upregulation of p16 and p19 leads

to a reduced repopulation ability and is associated with the aging of stem cells (Ito et

al., 2004; Janzen et al., 2006; Krishnamurthy et al., 2004a). Primitive bone marrow

cells from young mice deficient of p16 showed no difference in cell cycle or apoptosis

compared to cells from wild type mice, whereas cells from older p16-/-mice showed

higher cycling activity and more dead cells compared to their wild type counterparts


37

(Janzen et al., 2006). Lately, the significance of p16 as a role in HSCs aging has been

questioned (Attema et al., 2009). Another suggested signature for aging of HSCs is a

myeloid skewing of differentiation (Janzen et al., 2006; Liang et al., 2005; Morrison et

al., 1996), which has been suggested to depend on the increased levels of ROS in

aging of HSCs (Janzen et al., 2006). The capacity to generate lymphoid differentiation

is dramatically reduced in aging HSCs, whereas the myeloid potential is maintained

and these changes are accompanied by upregulation of myeloid specific genes in LT-

HSCs from older mice, indicating that aging of HSCs is regulated by intrinsic

mechanisms (Rossi et al., 2007).

FoxO proteins are essential in the resistance to oxidative stress During the last decade, several studies have underscored the importance of the FoxO

family in the regulation of cell cycle arrest, stress resistance, apoptosis, glucose

metabolism, and DNA repair. Particular in stem cell homeostasis, FoxO has attracted

a lot of attention due to its role in the regulation of maintenance, survival, and

quiescence. In mammals, the FoxO family consists of four members: FoxO1, FoxO3,

FoxO4 and FoxO6. In hematopoietic cells the first three mentioned play a critical role.

The FoxO protein was first characterized in C.elegans as the ortholog DAF-16 involved

in the extension of lifespan (Kenyon et al., 1993). The FoxO family has diverse

physiological functions and is well conserved from yeast to humans, which proclaims

their importance in development. It seems like a paradox that FoxO proteins can

regulate both cell death and resistance to oxidative stress. However, this is explained

by a tight regulation of post-translational modifications controlling the diversity of

functions achieved by FoxO factors. These include phosphorylation, acetylation and

ubiquitylation (reviewed in (van der Horst and Burgering, 2007)). Insulin, insulin-like

growth factors (IGF), as well as other growth factors including IL-3, SCF, FL, and Tpo

activate the PI3K/AKT signaling pathway which leads to the inactivation of FoxO by

phosphorylation of three specific serine and threonine residues (Thr32, Ser253 and

Ser315 for FoxO3), subsequently leading to the interaction with the 14-3-3 and

translocation out of the nucleus (Brunet et al., 1999; Engström et al., 2003; Jönsson

et al., 2004; Tanaka et al., 2001). Instead, inactivation of the PI3K/AKT pathway or

stress stimuli leads to activation of FoxOs (Greer and Brunet, 2005). In contrast to the

phosphorylation of AKT, phosphorylation of FoxO caused by another kinase, JNK,

results in nuclear translocation and activation (Essers et al., 2004). Oxidative stress

activates FoxO proteins by mammalian sterile 20 like kinase 1 (MST-1)

phosphorylation, monoubiquitination, as well as interaction with β-catenin, and SIRT1

deacetylase (reviewed in (Ghaffari, 2008)). Today, it is not known if JNK regulates


38

FoxO proteins in hematopoietic cells. The function of FoxO proteins to protect cells

from oxidative stress by detoxification of ROS is thought to be mediated by

transcriptional regulation of the scavenger enzymes SOD2, catalase (CAT),

glutathione, and peroxidase-1 (Kops et al., 2002). Recently, studies have shown that

FoxO proteins, in particular FoxO3, protect the maintenance of HSCs by regulating

oxidative stress (Figure 13) (Miyamoto et al., 2007; Tothova et al., 2007). Although

proliferation and differentiation of hematopoietic progenitor cells in Foxo3-/- mice

were normal, serial transplantations showed an obvious failure of HSCs to maintain

long term repopulation (Miyamoto et al., 2007). Due to suggested functional

redundant mechanism for FoxO proteins in the hematopoietic system, mice with

simultaneous conditional deletion of Foxo1, Foxo3 and Foxo4 were engineered. A

marked decrease in HSCs as well as increased cell cycle and defects in long-term

repopulation together with increased levels of apoptosis, which was not detected in

mice deficient for a single FoxO member (Miyamoto et al., 2007; Tothova et al.,

2007). This demonstrates that functional loss of one member can, at least partially,

be compensated by other FoxO members.

Figure 13. FoxO proteins are critical mediators of HSC stress resistance. FoxO activity is regulated by several posttranslational modifications. Stimuli can regulate FoxO proteins differently. Activation of the PI3K/AKT pathway causes inhibition of FoxO, whereas oxidative stress activates FoxO by phosphorylation, monoubiquitination, and through interaction with β-catenin and the SIRT1 deacetylase. This leads to transcription of FoxO target genes involved in cell cycle arrest and oxidative stress resistance. FoxO proteins are thought to be important for the decrease of ROS levels in HSCs and maintenance of long term repopulation ability.

FLT3 SIGNALING IN HSCS AND PROGENITORS

39

THE ROLE OF FLT3 IN NORMAL AND MALIGNANT HSCs AND

PROGENITORS

As mentioned earlier, there are a number of cytokines important for stem cells and

progenitors in the hematopoietic system for regulating survival, proliferation,

differentiation, and self-renewal. While the knowledge which extrinsic factors are

necessary for controlling self-renewal are fairly limited, much has been learned

regarding cytokines important for survival and proliferation of HSCs. One cytokine

with well-known capabilities to mediate survival and proliferation of HSCs is FL (Veiby

et al., 1996). Although there are other cytokines important for survival-mediated

pathways in HSCs, the focus in this thesis will be FL and its receptor FLT3.

Tyrosine kinase receptors The majority of growth factors bind to receptors with intrinsic tyrosine kinase activity.

Ligand binding causes the receptors to assemble into receptor dimers enabling cross-

phosphorylation of the cytoplasmic part of the receptors. This initiates intracellular

signaling cascades involved in controlling cellular processes such as growth,

differentiation, migration, chemotaxis, and angiogenesis. There are 58 different

human receptor tyrosine kinases (RTK); TIE2, FGFR, IGF, FLT3 and c-KIT just to name a

few, which all have the ability to self-interact as stable dimers within the membrane

(Finger 2009). FLT3 belongs to the subset of class III RTK family, which also includes

FMS and the platelet-derived growth factor receptor (PDGFR). The RTK class III family

is characterized by an extracellular domain compromised of five immunoglobulin-like

domains, a transmembrane domain, a juxtamembrane (JM) domain and a

cytoplasmic tyrosine kinase domain that are split into two motifs (reviewed in

(Gilliland and Griffin, 2002)).

The FLT3 receptor The murine Flt3 receptor was cloned independently by two groups in 1991. One

group applied a hybridization technique using a probe from the FMS receptor to

clone the novel receptor which was named FMS-like tyrosine kinase 3 (Flt3) (Rosnet

et al., 1991). The other group cloned Flt3 from the fetal liver and named the receptor

fetal liver kinase 2 (FLK-2) (Matthews et al., 1991). Today both names are used but

the term FLT3 will be used hereafter in this text. Shortly after, the human FLT3 gene,

also called the stem cell tyrosine kinase 1 (STK1), was cloned (Rosnet et al., 1993;

Small et al., 1994), showing 85% similarity in the amino acid sequence with the

murine Flt3. FLT3 ligand (FL), cloned two years later (Lyman et al., 1993), can activate


40

the tyrosine kinase receptor both in a soluble homodimeric form as well as a cell

surface bound ligand. FLT3 is mainly expressed on human HSCs and other immature

hematopoietic cells such as early lymphoid and myeloid progenitors, but not on more

committed cells (Rasko et al., 1995; Rosnet et al., 1996). Bone marrow

transplantations with HSCs lacking FLT3 has shown a reduction in both T cells and

myeloid cells, whereas targeted disruption of FLT3 results in a decrease of primitive B

cell progenitors, natural killer (NK) cells, and dendritic cells (reviewed in (Gilliland and

Griffin, 2002; Small, 2006). Together this shows that FLT3 is essential for a multi-

lineage differentiation. While the FLT3 expression is restricted to primitive

hematopoietic stem and progenitor cells, FL is ubiquitously expressed on several cell

types, including hematopoietic B-, T-, and myeloid cells, fibroblasts in the bone

marrow microenvironment and also by non-hematopoietic tissues, including

prostate, ovary, testis, heart, and placenta (Brasel et al., 1995; Lisovsky et al., 1996;

Stirewalt and Radich, 2003). FLT3 signaling is important for the survival and

proliferation in HSCs and progenitor cells. FL is by itself not a strong growth factor,

but in synergizes with other cytokines to stimulate proliferation ((Gilliland and Griffin,

2002)).

Stimulation of the FLT3 receptor

Stimulation with FL results in receptor homodimerization and the activation of

intracellular pathways involved in survival and proliferation. Inactive FLT3 receptors

are found as monomeric proteins in the plasma membrane. Due to steric inhibition,

the inactive state is unable to form receptor dimers. The JM domain stabilizes the

inactive kinase conformation through an autoinhibitory mechanism. The JM domain

is divided in three distinct topological components: the JM binding motif (JM-B), the

JM-switch motif (JM-S) and the zipper segment (JM-Z). The JM-B stabilizes a closed

conformation and buries the activation loop in the cleft between the N and C lobes of

the FLT3 kinase, thereby blocking access to peptide substrate and ATP-binding sites.

The JM-S is positioned next to the C-lobe, which enables the binding of the JM-B. The

role of the JM-Z is simply to correctly align and maintain the JM-S in the right position

during the inactive state of the kinase protein (Griffith et al., 2004). Activation of the

FLT3 by FL leads to rapid conformational changes promoting dimerization of the

receptor. This leads to transphosphorylation of specific tyrosine residues in the JM

domain which allows it to move away from the active center of the kinase and enable

the autophosphorylation of Tyr842 in the activation loop, which is the final step for

activation of the kinase (Choudhary et al., 2005).


41

Mutations in the FLT3 receptor

In addition to the normal expression of FLT3 on primitive immature hematopoietic

cells, FLT3 is also expressed in a wide range of hematopoietic malignancies, including

B-lineage acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). High

expression of FLT3 has been reported on 70-100% of the cases of AML (Birg et al.,

1992; Carow et al., 1996; Nakao et al., 1996; Stacchini et al., 1996). This implicates a

significant role of FLT3 in leukemic malignancies. An aberrant form of the FLT3 has

been found in about one third of the patients with AML. Two types of mutations

leading to constitutively active receptor signaling have been found; internal tandem

duplications (ITDs) in the JM domain and point mutations in the activation loop in the

tyrosine kinase domain.

The ITD mutation was first reported by Nakao and collegues in 1996 (Nakao et al.,

1996). They investigated FLT3 expression in 30 patients with AML and in 50 patients

with ALL and found 73% and 78% expression, in the respective groups. Interestingly,

5 of the AML patients had unexpectedly longer FLT3 transcripts. Sequence analyses

revealed that this was caused by tandem repeats of certain regions in the JM domain,

which were all in-frame enabling formation of non-truncated proteins. The length of

the ITD varies from 3 to more than 400 base pairs (Stirewalt and Radich, 2003). The

FLT3-ITD mutation, the second most common mutation in AML after mutations in the

nucleophosmin (NPM1) gene (Falini et al., 2005), has been associated with increased

relapse risks and poor overall survival rates (Kottaridis et al., 2001; Thiede et al.,

2002). FLT3-ITD expression causes factor-independent growth and resistance to

radio-induced apoptosis in factor-dependent cells lines (Mizuki et al., 2000). Mice

injected with these cells developed leukemia-like syndromes. The frequency of FLT3-

ITD mutation in adult AML ranges between 23-35%, whereas the incidence of FLT3-

ITD in pediatric AML is strikingly lower ranging from 5-16%. However, pediatric AML

with FLT3-ITD is associated with an inferior prognosis compared to adult AML with

ITD (reviewed in (Parcells et al., 2006)). This implicates that age-related mechanisms

are involved in the pathogenesis of AML. In addition, the incidence of AML increases

with age (Appelbaum et al., 2006). The aging marker p16 increases in normal human

CD34+ cells, which is suggested to be important in order to induce apoptosis to

protect against accumulation of DNA damages that might lead to cancer. A low

expression of p16 in older AML patients have been reported to be associated with

decreased overall survival rates (de Jonge et al., 2009). Surprisingly, this pattern of

p16 as a prognostic marker could only be seen in AML without FLT3-ITD.


42

The second most common mutation in the FLT3 receptor involved in AML is the point

mutation of aspartate 835 to tyrosine in the activation loop in the tyrosine kinase

domain (TKD) (Abu-Duhier et al., 2001). It is suggested that Asp835 provides a crucial

stabilization of the closed loop configuration although this has been questioned

(Griffith et al., 2004). This could explain how the TKD mutation causes the loop to

transform into an open configuration allowing access to the kinase. Like the FLT3-ITD

mutation, the FLT3-TKD mutation is associated with the constitutively activation of

the receptor (Yamamoto et al., 2001). The prognostic impact of FLT3-TKD mutations

in AML has been uncertain. A recent study reported that the 10-year overall survival

rates for AML patients with FLT3-TKD was 51% compared to 23% for patients with ITD

mutations, indicating that the former has a favorable prognostic outcome (Mead et

al., 2007). The prevalence of AML patients with FLT3-TKD has been reported to be 7%

(Thiede et al., 2002).

FLT3 signaling in normal and mutated receptor

The downstream signaling of FLT3 is today not fully elucidated. Several studies have

demonstrated the involvement of different survival pathways in FLT3 expressing cells,

both in primary cells and cell lines. Restriction of the interpretation has to be

considering due to the fact that FLT3 signaling between cell types and organisms

might differ. Although both FLT3-TKD and FLT3-ITD, like normal FLT3 stimulated with

FL, leads to activation of the FLT3 receptor, the downstream signaling may differ

(Choudhary et al., 2005). The ITD insertion occurs mainly in the JM-Z which offsets

the position of JM-S and thereby disrupts the insertion of JM-B into its autoinhibitory

binding site, which results in a configuration of the receptor that is continuously

active (Griffith et al., 2004). Both normal and mutated FLT3 activate several

downstream signaling pathways, including STAT5, PI3K/AKT and RAS/MAPK pathways

(Figure 14) (Mizuki et al., 2000; Zhang and Broxmeyer, 1999; Zhang et al., 2000).

The RAS/MAPK pathway

Stimulation of FLT3 activates the RAS/MAPK pathway. FL induced phosphorylation of

son of sevenless (SOS), which associates with the adaptor protein GRB2, which in

turn, binds to the phosphorylated tyrosine residues in the activated receptor via its

SH2 domain. This binding to the phosphorylated receptor causes translocation of SOS

to membrane-bound RAS, which becomes activated through GTP-binding. RAS-GTP

then activates RAF-1, which in turn, activates the two MAPK proteins, ERK-1 and ERK-

2 (Figure 14). ERK-1 and ERK-2 are kinases primarily involved in regulation of cell

growth and survival, whereas two other kinases, p38 and JNK are involved in


43

apoptosis caused by stress (Wada and Penninger, 2004). Activation of p38 MAPK is

associated with dysfunction of hematopoietic stem cells (Ito et al., 2006).

Similar to FLT3-ITD, the FLT3-TKD mutation leads to the constitutively activation of

ERK1 and ERK2 (Mizuki et al., 2000).

The PI3K/AKT pathway

Similar to Ras-MAPK signaling, the activation of the PI3K and its downstream target

protein kinase B (PKB or AKT) is important for cell proliferation, growth, survival, and

metabolism in many cell types (reviewed in (Vanhaesebroeck and Alessi, 2000)). Type

IA PI3K is a heterodimer consisting of a catalytic subunit (p110α) and a regulatory

subunit (p85α). PI3K is a lipid kinase that catalyzes the phosphorylation of inositol

phospholipids at the 3´position generating lipids called phosphatidylinositol-3,4-

bisphosphate, PI(3,4)P2, or phosphatidylinositol-3,4,5-triphosphate, PI(3,4,5)P3. The

lipids products, PI(3,4)P2 and PI(3,4,5)P3, recruit phosphatidylinositol-dependent

kinase 1 and the serine/threonine kinase AKT to the plasma membrane, leading to

phosphorylation and activation of AKT. The activation of murine Flt3 associates

directly with p85, whereas human FLT3 lacks a binding site for p85. However, adaptor

proteins, such as GRB2 binding protein (GAB) -1, GAB-2 and SHP-2, become

phosphorylated and activated upon FL stimulation and associate to p85 (Zhang and

Broxmeyer, 1999). Activation of AKT by FL stimulation leads to inactivation of the

transcription factor FoxO3a through phosphorylation (Jönsson et al., 2004), which

enables docking of the transporter protein 14-3-3 and nuclear export (Brunet et al.,

2002). This prevents transcription of Bim and p27 promoting survival and cell cycle.

Another downstream target of AKT is the pro-apoptotic protein Bad. AKT

phosphorylates Bad, which results in sequestration and blocked apoptosis (Figure 14).

In AML blasts and in cell lines with FLT3-ITD mutations increased activation of AKT

has been observed, which negatively regulates FoxO3a and thereby promotes cell

survival and proliferation (Brandts et al., 2005; Scheijen et al., 2004).

The JAK/STAT pathway

The Janus-activated kinases (JAKs) are cytoplasmic tyrosine kinases that mediate

survival and proliferation signaling via activation of signal transducer and activator of

transcription (STAT) proteins. Comparative analyses of normal FLT3 with FLT3-TKD

and FLT3-ITD signaling has revealed differences in the activation of STAT5 (Choudhary

et al., 2005; Mizuki et al., 2000). Preferential activation of STAT5 by FLT3-ITD, weak

activation of FLT3-TKD and absence of STAT5-signaling by ligand-activated normal

FLT3 have been described. Although the majority of STAT5 activation in AML is

associated with autophosphorylation of FLT3 (Knapper et al., 2006), some cases of

constitutively STAT5 phosphorylation have shown no activation of FLT3 (Birkenkamp


44

et al., 2001). This suggests that there might be a FLT3-independent mechanism for

the activation of STAT5 in AML. An activation of the STAT5 leads to expression of its

target genes PIM1 and PIM2. Increased expression of both PIM1 and PIM2 has been

observed in AML patients with FLT3-ITD mutation (Kim et al., 2005; Mizuki et al.,

2003). This upregulation of PIM is thought to contribute to proliferation and survival

following FLT3-signaling.

Other mutations implicated in AML FLT3 mutations block myeloid differentiation and ligand-dependent proliferation.

However, this is not sufficient to induce acute leukemia. It has been suggested that

two classes of mutations are required to develop AML. Another mutation often found

expressed together with FLT3-ITD is the t(15:17). This is a balanced reciprocal

translocation of the promyelocytic leukemia (PML) gene at chromosome 15 and the

retinoic acid receptor-α (RARA) gene at chromosome 17 causing impaired

differentiation of hematopoietic cells. The t(8;21) gene arrangement, resulting in

Figure 14. The FLT3 signaling pathways. The figure shows signaling cascades that are thought to be triggered through FLT3 activation. Binding of FL causes activation of the PI3K/AKT pathway involved in proliferation and survival. Association of GRB2 with the activated receptor results in activation of RAS, which in turn activates the RAF/MEK/ERK cascade. Another mediator of survival and proliferation activated by FLT3 is the STAT5 protein, although this activation likely is restricted to mutated FLT3 signaling (Stirewalt and Radich, 2003).


45

expression of the fusion gene AML1/ETO, is also listed in this class of mutations that

frequently occur in conjugation with FLT3 mutations. This cooperation between

mutations leading to inhibited differentiation with mutations associated with

proliferation and survival is thought to be sufficient to cause leukemic malignancy.

This “two-hit” model of leukemia proposes that only one mutation in each class is

necessary for AML to develop. In support of this, mutation in the RAS gene, leading to

increased proliferation and survival, never or very seldom coexist with FLT3

mutations (Gilliland and Griffin, 2002; Stirewalt and Radich, 2003). Ras mutations

occur in 15-20% of AML patients, but have less prognostic significance compared to

mutations in FLT3 (reviewed in (Levis and Small, 2005)). Another mutations

associated with poor prognosis are c-KIT mutations, although the frequency is

relatively rare (1-5%) in AML cases (Gilliland and Griffin, 2002).

Recently, a mutation in the NPM1 gene was detected and described in 35% of AML

cases (Falini et al., 2005). A high frequency of co-expression of NPM1 mutations and

FLT3-ITD mutations in AML has been reported. Interestingly, NPM1 mutation alone is

associated with a significantly better prognosis than absence of this mutation (Thiede

et al., 2006). NPM1 protects cells from stress-induced cell death by inhibition of p53

(Li et al., 2005). Mutation and the loss of functional NPM1 are suggested to be

explained by increased sensitivity to chemotherapy.

TREATMENT OF AML

46

THERAPEUTIC STRATEGIES FOR AML

AML is an aggressive malignancy with short survival rates if not treated. AML is

thought to arise in leukemic stem cells (LSCs) and is characterized by aberrant

proliferation of myeloid progenitor cells coupled to a partial block in differentiation.

This in turn causes bleeding, anemia and immune suppression due to the decreased

number of mature functional leukocytes, erythrocytes, and thrombocytes. The overall

incidence is 3.4 cases per 100 000 inhabitants, but increases significantly with age

(Tallman et al., 2005). Poor prognosis is often associated to AML due to therapy-

induced mortality or resistance to chemotherapy (reviewed in (Weisberg et al.,

2009)). As described above, deregulation of tyrosine kinases in particular FLT3, has

for the last decade been implicated in the molecular pathology of AML.

The current treatment of AML is chemotherapy and BMT. Growing insight in specific

pathogenic molecular mechanism has led to the development of novel therapeutic

drugs, such as FLT3 inhibitors, inhibitors of bone marrow niche targets and BH3

mimetic drugs.

FLT3 inhibitors The high frequency of FLT3 mutations associated with a poor prognosis has made

FLT3 an interesting molecular target for treatment of AML. To date, many tyrosine

kinase inhibitors have been developed where some are in clinical trials. Although they

all interact with and inhibit FLT3 signaling, no FLT3 kinase-specific inhibitor has been

developed. FLT3-inhibitors often recognize other RTKs such as PDGFR, VEGFR, and c-

KIT. After the success of the tyrosine inhibitor imatinib (Glivec; Novartis) used in

treatment of chronic myeloid leukemia (CML) by inhibiting the kinase activity in the

fusion protein BCR/ABL, there has been a massive struggle to find an inhibitor as

effective for treatment of AML. It must however be noticed that BCR/ABL might be

the only molecular abnormality leading to the development of CML in contrast to

AML, which is a multi-mutational leukemia, where constitutively active FLT3 is only

one of many genetic alterations. The molecular mechanism for these small molecule

kinase inhibitors is to mimic the adenosine structure and compete with ATP for

binding to the ATP-binding pocket in the kinase domain. The first report that FLT3 is a

potential therapeutic target for AML used the FLT3 inhibitor AG1295. AG1295 inhibits

FLT3-ITD and wild type FLT3 and noticeably reduced the number of AML blasts in

vitro (Levis et al., 2001). Because of its low bioavailability and low potency, AG1295

TREATMENT OF AML

47

cannot be used clinically but is used in research to study cellular and molecular

mechanisms influenced by FLT3-inhibition. Since then, several FLT3 inhibitors, such as

SU11248, CEP701, PKC412, and MLN-518, have been discovered and investigated.

PKC412 was originally developed as an inhibitor against protein kinase C, but was

shown to be a more powerful inhibitor of FLT3 kinase activity. Through its inhibition

of FLT3-ITD and FLT3-TKD it caused cell cycle arrest and apoptosis in leukemic cells

(Weisberg et al., 2002). In addition, administration of PKC412 to mice with FLT3-ITD-

induced leukemia showed prolonged survival. In a phase 2 study, PKC412 reduced

peripheral blast count to 50% or less in 14 of 20 patients, whereas bone marrow blast

counts were reduced to 50% in only 6 patients (Stone et al., 2005). This low response

in reduction of bone marrow blasts is generally described for all FLT3 inhibitors used

in clinical trials so far. In addition, other clinical problems, such as maintaining plasma

concentrations of the drug and avoiding drug toxicity, need to be resolved before

tyrosine inhibitors can be used in treatment of AML. The acquisition of additional

mutations in the kinase domain inhibiting drug binding, increased expression of FLT3,

and the activation of compensatory signaling pathways for survival are potential

mechanisms by with the drug response can be reduced. The rapid developed

resistance to tyrosine inhibitors as sole therapeutic agents has lead to clinical trials of

FLT3 inhibitors in combination with conventional cytotoxic therapy. Hopefully, this

would target the pathologic mechanism of AML while avoiding some toxicity from

chemotherapy, as well as resistance to kinase inhibitors (reviewed in (Knapper,

2007)).

Therapies that targets the leukemic stem cell niche The high incidence of relapse in AML is thought to be explained by leukemic stem

cells (LSCs) escaping conventional chemotherapy. LSCs have self-renewal capacities

and the ability to give rise to more mature hematopoietic cells (Somervaille and

Cleary, 2006). A high percentage of leukemic stem cells at the time of diagnosis often

reflect a number of remaining chemotherapy-resistant cells associated with poor

survival (van Rhenen et al., 2005). If FLT3-ITD mutations occur in leukemic stem cells

remains to be investigated. Data regarding the FLT3-ITD expression on AML blasts at

the time for diagnosis and relapse provide useful information for this question. In

addition, the mutant-to-wild type ratio might provide meaningful information to

when the FLT3-ITD mutation occurs. Some studies have shown that patients

diagnosed with FLT3-ITD all showed FLT3-ITD expressing blast cells at relapse,

whereas others have shown the mutations were lost at relapse (reviewed in (Levis

and Small, 2003)). A loss of FLT3-ITD indicates that this is a “late mutation” not

TREATMENT OF AML

48

present in cancer stem cells. If instead, the mutation occurs early in stem cells with

self-renewal capacity, the mutant-to-wild type ratio would be 1 or greater.

Leukemic cells can escape chemotherapy by “hiding” in special niches in the bone

marrow referred to as “the leukemic stem cell niche”, which provides them with

survival signals activating expression of anti-apoptotic proteins (Konopleva et al.,

2002). Bone marrow stromal cells protect leukemic cells from chemotherapy-induced

death (Konopleva et al., 2002; Panayiotidis et al., 1996; Tabe et al., 2004). CXCR4 has

been shown to be important for migration and homing of AML cells to the bone

marrow niche (Tavor et al., 2004). Interestingly, high expression of CXCR4 in AML cells

with FLT-ITD has been reported to be associated with poor prognosis and an

increased risk for relapse (Rombouts et al., 2004), indicating that FLT3-ITD expressing

AML cells are favored by signals from the leukemic stem cell niche. One explanation

to why peripheral blood blasts are more responsive to treatment than bone marrow

blast might be that the bone marrow protects leukemic cells from toxicity. Therefore,

disruption of protective niche-signals could be used to increasing responses to

current therapies and reducing resistance to conventional chemotherapy and

tyrosine kinase inhibitors. However, consideration is required before niche targeted

therapies can be used.

BH3 mimicking drugs Aberrant overexpression of Bcl-2 proteins in AML patients is associated with

increased resistance to chemotherapy and FLT3 inhibitors. A deregulation of

apoptosis caused by increased expression of anti-apoptotic proteins is common in the

majority of malignancies which make them ideal targets for developing anti-cancer

drugs. Overexpression of anti-apoptotic members inhibits Bax and Bak from forming

homodimers in the mitochondria membrane and causing release of cytochrome c.

Tumor cells with overexpression of Bcl-2 are therefore less sensitive to drug-induced

apoptosis. In a normal cell, the Bcl-2 family proteins are controlled by BH3 only pro-

apoptotic members. The docking of the BH3 domain to Bcl-2 annuls their protective

effects against increased mitochondrial permeability. An attractive approach has

been to mimic the BH3 domains and thereby reducing the anti-apoptotic ability for

Bcl-2 protein family members. Several BH3 mimic peptides have been described

where some are in clinical trials (reviewed in (Zhang et al., 2007)). The most

promising Bcl-2/Bcl-XL inhibitor in the treatment of cancer is ABT-737. It was recently

reported that treatment of AML cells, expressing high levels of Bcl-2, with ABT-737

neutralized achieved resistance to FLT3 inhibitors (Kohl et al., 2007; Konopleva et al.,

TREATMENT OF AML

49

2006). ABT-737 induced apoptosis in primary AML blast cells but showed no effect in

normal lymphocytes. However, AML cells with a high expression of Mcl-1 showed

significantly lower sensitivity to ABT-737. Together, this demonstrates that normal

hematopoietic cells are tolerant to treatment with BH3 mimetics which might lower

the dose of conventional chemotherapy required for response in combination with

specific BH3 mimetics. Furthermore, the expression pattern of different Bcl-2 family

proteins might indicate which treatment is the most proper to choose. For instance,

treatment with ABT-737 should only be used in malignancies with low expressions of

Mcl-1. Hopefully, new BH3 mimetics with specificity for Mcl-1 will be developed in

the near future.

AIMS

50

AIMS OF THE PRESENT INVESTIGATION

The overall goal of the present study was to gain an increased understanding in how

maintenance is regulated in normal and leukemic hematopoietic stem and progenitor

cells. Towards this the main objects were;

To investigate the role hypoxia and HIF-1α play in survival and self-renewal of

hematopoietic stem and progenitor cells.

To study pro- and apoptotic signaling pathways activated by the tyrosine

kinase receptor FLT3 in primitive AML cells.

The specific aims in the papers included in the thesis were the following;

Paper I: To evaluate the affect hypoxia has on survival and expansion of in vitro

culturing of primitive hematopoietic progenitor cells from mouse bone

marrow.

Paper II: To investigate the role of hypoxia in HSCs maintenance and expansion in

vitro and in vivo, focusing on the importance of HIF-1α-dependent

regulation of proliferation and cell cycle.

Paper III: To study the involvement of hypoxia and HIF-1α in HSCs exposed to

oxidative stress.

Paper IV: To determine which apoptotic pathways are involved in FLT3 inhibitor-

induced cell death of AML cells.

METHODOLOGICAL CONSIDERATIONS

51


Detailed description of methods used is written in the material and methods section

in each paper. Still, there are some issues that need to be clarified and explained

more thoroughly.

Isolation of HSCs using FACS based cell sorting Bone marrow is normally taken from femur and tibia from sacrificed mice. After

crushing the bones in a mortar a single cell suspension is prepared by washing and

filtering. Before sorting of HSCs using fluorescent activating cell sorting (FACS),

primitive multipotent cells are enriched by depletion of mature lineage positive cells

(express lineage specific markers e.g. Ter119 for erythrocytes) or concentrated for

stem- and progenitor cells (express CD117/KIT ligand). Both ways generate a

population of cells containing a heterogeneous population of stem- and progenitor

cells. In brief, bone marrow cells are labeled with immunomagnetic beads conjugated

with either antibodies specific for lineage surface antigens (lineage depletion) or

antibodies specific for CD117 (CD117 positive selection). Labeled cells are then

passed through a magnetic field, which enable separation of labeled and unlabeled

cells. By using this pre-separation method, the time needed for sorting is reduced

with a factor of 50 to 100 times, which is of advantage for the operator as for the

viability of the cells.

The two most frequent populations used in this thesis are the lineage-Sca-1+c-Kit+

(LSK) cells and the CD34-Flt3-LSK cells, containing MPP, ST-HSCs, and LT-HSCs or LT-

HSCs, respectively. The enriched stem- and progenitor cell sample is labeled with

fluorochromes-conjugated antibodies specific for lineage markers, Sca-1, c-Kit, Flt3

and CD34 and further sorted on a FACS cell sorter. The cell sorter used in this study is

the FACSAria from BD Bioscience. It is a high-speed cell sorter (approximately 20 000

cells/second) connected to a FACS analyzing unit. The sample runs from the sample

tube to the sample injection chamber to the cuvette flow cell and finally to the

collection device. Sheet fluid is pumped from the fluid reservoirs into the cuvette

flow cell where hydrodynamic forces focusing the sample into a single-file stream

through the cuvette. Within the cuvette single cells are focused in the interrogation

point by the laser beam and enter thereafter the nozzle (Figure 15).


52

Fluorochrome-conjugated antibodies are excited by different lasers. The FACSAria

used here has four lasers; 488nm (blue), 633nm (red), 407nm (violet), and 355nm

(ultra violet). The lasers can detect 2-6 different fluorochromes, which give the

possibility to distinguish 14 colors in one sample at the same time. When the cells

pass through the laser beam the fluorochromes absorb photons and become excited.

Returning to their ground state results in release of energy as emitted light also

referred to as fluorescence, which is transferred to specific detectors. The stream

containing the cells is accelerated in the nozzle where the stream is broken into

droplets. When a cell is detected that meets the criteria for the sorting, an electric

charge is applied to the droplet at the time it leaves the nozzle tip. The charged

droplets passes between two charged deflection plates and is attracted or repulsed

towards the plates depending on its charge. This causes the droplet containing the

cell to move to the right or the left collection tube. Uncharged droplets pass straight

down in the waste container. By calibrating the instrument before use and select

specific criteria for sorting, high purity of sorted population can be achieved.

RNA interference In year 2006 Andrew Fire and Craig C. Mello were rewarded with the Nobel Prize in

medicine for their work on RNA interference in C. elegans. They showed that anti-

sense RNA is important for regulation of gene expression (Fire et al., 1991). The

Figure 15. Sorting of specific cells using FACS selection. In the nozzle, the laser beam is focused on the sample core stream. Signals from flourescent molecules bound to specific cells are detected and analyzed. Charge is applied to droplets containg a cell with the desired criteria. Deflection plates attract or repel the charged droplet and the cells are sorted into collection tubes.


53

discovery of RNA interference (RNAi) is one of the most important breakthroughs in

modern time both for basic and for applied research. Exploring the mechanism for

the RNAi machinery has resulted in a valuable research tool that can be utilized in

therapeutic applications. RNAi is triggered by double-stranded (ds) short RNA

exogenously transferred as small interfering RNAs (siRNAs) or endogenously

produced as microRNAs (miRNAs). These short dsRNAs are around 21-23 nucleotides

and is today commonly used in research to induce sequence-specific gene silencing.

Exogenously supplied siRNAs induce gene-silencing by triggering RNA cleavage after

binding to their target mRNA. The endogenous miRNAs are important regulators of

genes involved in development, proliferation, hematopoiesis and apoptosis (reviewed

in (Rana, 2007)). One important difference between miRNAs and siRNAs is the way of

inhibiting the production of functional mRNA. Unlike siRNAs, miRNAs do not usually

cause cleavage of RNA, but instead repress translation of mRNA. However, synthetic

siRNAs can suppress mRNA translation by binding partially complementary to target

sequences and simulate functions similar to endogenous miRNAs (Doench et al.,

2003; Zeng et al., 2003). This less stringent mechanism to control RNA silencing can

explain nonspecific effects sometimes observed in experiments using siRNAs.

RNA interference using siRNAs is transient and to be able to achieve a stable gene-

silencing DNA vectors expressing short hairpin (sh) RNAs have been developed. Long

dsRNAs, hairpin RNAs and siRNAs can all initiate the RNAi machinery. In the

cytoplasm, both long dsRNAs and hairpin RNAs are first processed by the RNase III-

enzyme Dicer to shorter siRNAs with an overhang at the 3’ end and a phosphate

group at the 5´end. The anti-sense is known as the guide strand because it is

responsible for complementary binding to the target RNA sequence. The siRNA oligo

assemble into a complex with proteins called the RNA-induced silencing complex

(RISC). This results in unwinding of the helix and the sense strand is rapidly degraded.

The single stranded RNA-RISC complex then recognizes and binds the target RNA

resulting in cleavage between nucleotide 10 and 11 upstream of the 5’ end of the

guide strand (Figure 16) (reviewed in (Rana, 2007)).


54

In the present investigation, both siRNA (paper IV) and shRNA (paper II and III) are

used as molecular tools to silencing specific genes. The choice to use shRNA was

taken considering several aspects. First, a sustained long-term silencing of our target

gene was desired. Second, our target cells are non-dividing primary stem cells that

require lentiviral delivery of RNAi oligos to introduce gene-silencing. Third, using

lentiviral vectors containing shRNA oligos encoding the selection marker enhanced

green fluorescent protein (EGFP) enables sorting of cells expressing shRNA shortly

after introduction of the viral vectors. The lentiviral vectors used in this study carry

the human U6 polymerase III promoter for expression of the GFP reporter and the

shRNA oligos. In addition to the lentiviral shRNA vector, vectors containing viral

encoding gag, pol, and env, which are structural and enzymatic genes necessary for

formation of viral particles and replication.

Real time PCR To measure the expression of a gene of interest, quantification of transcribed mRNA

molecules can be used. One common method to quantitate mRNA expression is real

time polymerase chain reaction (PCR). Real time PCR is a sensitive method that

Figure 16. Schematic process over the RNAi machinery. Long double-stranded RNA (dsRNA) and short hairpin RNA (shRNA) are cleaved by Dicer into shorter siRNA. These small double-stranded oligos are recognized by the multicomponent enzyme-complex, RISC. Assembling with RISC results in unwinding of the helix and the sense strand is removed. The antisense strand, instead, binds to its complementary mRNA sequence causing cleavage by the RISC complex. Cleaved mRNA products are degraded and the RISC complex is reused to cleave additional mRNA targets.


55

requires minimal amounts of starting material which makes it useful for determine

difference in gene expression when only a few number of primary cells are available.

Before running a real time PCR, mRNA is converted by reverse transcriptase to

complementary DNA (cDNA). The initial step for real time PCR is denaturation of the

two DNA strands which enables amplification of the target gene sequence using

specific primers. The PCR product accumulates for every cycle in an exponential way

and in contrast to traditional PCR, the amplification product is measured before the

final plateau phase, which increases the sensitivity and precision for quantification.

Detection of the PCR product in real time PCR can be monitored in two ways, non-

specific detection using DNA binding dyes or specific detection using target specific

probes. The latter includes a fluorescent labeled probe for detection, whereas the

former, used in this study, utilize double-strand DNA-specific dyes. The most widely

used fluorescent dye that bind double-stranded DNA is SYBR® green. Unbound SYBR®

green exhibits a very weak fluorescent signal, which increases extensively upon

binding to the minor groove in the DNA helix. Due to the fact that binding of SYBR®

green to DNA is non-specific it is important to optimize the settings to generate only

one gene product. Evaluation of the melting curve that monitors the kinetics of the

melting temperature for the DNA helix which depends on the products length and

the nucleotides composition is important to make sure the true target gene is

detected and not unspecific gene products or primer-dimer artifacts. To normalize

the total amount of RNA between the different samples, detection of an endogenous

constitutively expressed housekeeping gene is used. Mouse β-actin and human β-

glucuronidase (GusB) are used in paper II and III, and paper IV, respectively. The

increase in SYBR® green fluorescence will follow the amplification of the gene

product. Measurement of the cycle number is where the increase of fluorescence

(and therefore amplification) is exponential. This measuring point is called CT (cycle

threshold) value. The CT value is inversely proportional to the amount of DNA

product, meaning the lower CT value the higher amount of starting DNA copies in the

sample. In this way comprising in expression of a target gene between different

samples can be achieved. A common method to analyze the CT values generated from

different samples is the relative quantitative method 2-∆∆CT (Livak and Schmittgen,

2001), which have been used in the present investigation. First, the CT value for the

target gene (CT,X) is normalized to the value for the endogenous housekeeping gene

(CT,R), also referred to as reference gene. The equation that describes this is;

CT,X - CT,R = ∆CT

The next step is to relate the ∆CT for the sample (∆CT,q) to the untreated sample

(∆CT,cb), referred to as calibrator, given by;


56

∆CT,q - ∆CT,cb = ∆∆CT

A simplified calculation of the fold change of target gene normalized to the

endogenous reference gene and calibrated to the untreated sample is given by;

Fold change of target = 2-∆∆CT

Calibrators used in this study are sample cultured in normoxia (paper II and III) and

sample treated with 0 nM PKC412 (paper IV).

RESULTS AND DISCUSSION

57

RESULTS AND DISCUSSION OF THE PAPERS IN THIS THESIS

Hypoxia expands quiescent primitive progenitor in vitro and maintains

functional HSCs in vivo (Paper I and II)

It has been suggested by several investigators that HSCs are located in a

microenvironment in the bone marrow low in oxygen (Ceradini et al., 2004; Chow et

al., 2001; Kubota et al., 2008; Levesque et al., 2007; Parmar et al., 2007). To simulate

the oxygen conditions in the HSC niche, we cultured primary bone marrow cells in

low oxygen levels (1% O2). The key findings in paper I were an expansion of primitive

hematopoietic multipotential colony forming cells and maintenance of a primitive

stem cell phenotype in hypoxic in vitro cultures compared to cultures in ambient

oxygen levels. The functional in vitro colony assays used to detect primitive

progenitors were pre-CFCmulti, HPP-CFC, and CAFC. These colony forming cells

resemble the true HSC with long term potential (Cheng et al., 2000b; Iscove and Yan,

1990), which make them suitable in vitro systems to detect stem cell activity. After

hypoxic cultivation we could detect a preserved LSK phenotype, whereas more

committed progenitors generating CFU-GM were decreased in number. Together,

these observations indicate that hypoxia promotes expansion of cells with

multipotent ability and inhibits differentiation of hematopoietic cells, which is in

agreement with two previous studies using unfractionated murine bone marrow cells

(Cipolleschi et al., 1993; Ivanovic et al., 2000). The significance of hypoxia in

regulating differentiation of stem- and progenitor cells has been stated earlier by

several groups and in various tissues, including neural crest stem cells and adipocytes

(Morrison et al., 2000; Yun et al., 2002). Hypoxia-induced maintenance of an

undifferentiated state is dependent on Notch-signaling (Gustafsson et al., 2005). A

model where HIF-1α interacts with and stabilizes the cleaved Notch intracellular

domain, promoting transcription of Notch downstream targets important for stem

cell maintenance, has been proposed (Gustafsson et al., 2005). Similar to our

observation of HSCs cultured in hypoxia, constitutively active Notch-signaling in HSCs

generates a primitive phenotype and a morphology resembling immature blasts

(Varnum-Finney et al., 2000). Together with our results from paper I it is possible that

maintenance of HSCs in response to hypoxia could be elicited by activation of the

Notch-pathway.

This study was followed by paper II where functional HSCs were detected using a

competitive repopulation in vivo assay. In this study, a purer population of stem cells


58

was isolated allowing a more direct evaluation of the stem cell response to hypoxia.

In a heterogeneous cell population, the influence of non-stem cells on HSCs cannot

be ignored. In paper II we focused on the role HIF-1α has in hypoxic culture of HSCs.

One of the major findings from paper II was that hypoxia maintained quiescence of

HSCs and reduced the proliferation rate which resembles the steady-state of HSCs.

Furthermore, similar findings on proliferation and cell cycle arrest were observed by

overexpression of HIF-1α in a multipotent progenitor cell line. Interestingly, despite a

lower number of cells generated in the hypoxic culture, transplantation of these cells

generated equal long-term reconstitution capacity compared to transplanted

normoxic cell cultures, containing several fold higher total cell numbers. This

indicates that hypoxia can maintain LT-HSCs whereas proliferation of more

committed progenitor cells with lost long-term potential is reduced. In contrast,

normoxia increased the proliferation of more mature progenitors in addition to

decreased preservation of a primitive progenitor morphology and phenotype

observed in paper I. An increase in cell cycle can cause an exhaustion of stem cells

with long-term engraftment potential. It is therefore possible that the effects of

hypoxia in maintaining stem cells capable of long-term engraftment can be explained

by a reduced proliferation rate in addition to reduced differentiation. However, it

cannot be excluded that hypoxia in vitro could regulate maintenance of functional

stem cells by other mechanisms. Several niche molecules important for self-renewal

of HSCs are regulated by hypoxia; VEGF, Notch-1, Bcrp1/ABCG2 just to name a few,

demonstrating the importance of hypoxia in vivo. It has been shown that slowly

cycling HSCs are most abundant in hypoxic regions of the bone marrow (Kubota et al.,

2008; Parmar et al., 2007), which goes hand in hand with our observation of HSCs

response to hypoxia in culture.

In paper II, we showed that hypoxia increased the expression of the CDK inhibitor p21

in CD34-FLT3-LSK cells, enriched for LT-HSCs. Two other CDK inhibitors, p27 and p57,

were not affected by hypoxia in LT-HSCs. To our cultures we add the survival factor

Tpo, which is suggested to increase the expression of p57 (Qian et al., 2007). Tpo-

induced p57 expression and a predominance of p57 in CD34-FLT3-LSK cells (Yamazaki

et al., 2006) might conceal possible increased levels caused by hypoxia. Earlier studies

have shown that p21 is selective for regulating proliferation of HSCs, whereas p27 is

more important for regulation of cell cycle in progenitors (Cheng et al., 2000a; Cheng

et al., 2000b). Today, the interaction between hypoxia, p21, p27, and p57 is not fully

understood. It has to be taken into consideration that p27 also can be regulated

posttranscriptionally (Hengst and Reed, 1996; Pagano et al., 1995). In most cases

mRNA expression is direct related to protein abundance (Greenbaum et al., 2002),


59

but some proteins are regulated at the protein levels (e.g. HIF-1α through

degradation by the ubiquitin-pathway). This might explain the conflicting results from

earlier studies in how hypoxia regulates p27. It is known that the regulation of CDK

inhibitors is multiple and dependent on the microenvironment and cell type. Our

results showed that stable expression of HIF-1α in normoxic conditions could, similar

to hypoxia, reduce proliferation and induce cell cycle arrest. Therefore, it is tempting

to speculate that the effect hypoxia has on stem cell proliferation is HIF-1α mediated.

However, cultivation of primitive progenitors in normoxic conditions in the presence

of FG-4497, an oxygen-independent stabilizer of HIF-1α, reduced the initial

engraftment potential, whereas the long-term engraftment was unaffected.

Interestingly, we observed that HIF-1α induces expression of p27 and p57 in LSK cells,

whereas p21 levels were stable. Those findings indicate that HIF-1α mainly decreases

proliferation of progenitor cells through increased levels of p27, whereas p21

expression is stable and thereby maintain the number of stem cells with long-term

potential. This could explain the decrease in initial engraftment, which is dependent

on the activity and the number of progenitor cells, after treatment with the HIF-1α-

stabilizer. In agreement with this, primary bone marrow cells with stable expression

of constitutively active HIF-1α showed an initially reduction of engraftment potential.

However, this decline in engraftment was maintained over time. A possible

explanation could be that HIF-1α must be transiently expressed in order to enable

proliferation and differentiation to more committed progenitors and further

development of the hematopoietic lineages. According to our results, stable

expression of HIF-1α would induce cell cycle arrest of all hematopoietic cells tested.

Due to the fact that HIF-1α regulates a variety of signaling pathways involved in

apoptosis, differentiation, glucose metabolism, in addition to cell cycle regulation, it

is likely that overexpression of HIF-1α can cause a disturbance in regulation of other

cell fates. Another explanation for the reduced repopulation ability could therefore

be HIF-1α inducing cell death through transcriptional induction of the pro-apoptotic

gene BNIP3 (reviewed in (Wenger et al., 2005)). However, overexpression of the anti-

apoptotic protein Bcl-2 failed to rescue reconstitution of donor cells expressing

constitutively active HIF-1α, indicating that the negative effects we see on

repopulation cannot be explained by HIF-1α induced apoptosis. A third factor that

can affect reconstitution is homing, which has not been investigated in the present

study.

To determine how loss of HIF-1α affects HSCs we have used HIF-1α specific shRNA.

We were able to knock down endogenous HIF-1α mRNA levels in primary HSCs by

75% in normoxia and 68% in hypoxia. We cannot rule out that remaining HIF-1α


60

mRNA transcripts can be translated into proteins which can activate HIF-1α target

genes. Gene-silencing using shRNA does not induce a total knock-out of a gene, in

contrast to genes in cells from knock-out mice. Studying the biological role of HIF-1a

in bone marrow hematopoietic cells has been hampered due to the fact that HIF-1α-/-

mice die before birth in the uterus (Adelman et al., 1999). A HIF-1α-conditional

knockout mouse would be the optimal way to directly study the effects of HIF-1α, but

until then, partial silencing with HIF-1α specific shRNAs is a comparatively good

substitute.

Hypoxia protects HSCs from oxidative stress (paper III) One essential role for the hypoxic stem cell niche could be to protect HSCs from

exhaustion caused by oxidative stress. To investigate this, we induced oxidative stress

in primary HSCs by adding L-buthionine sulfoximine (BSO) that disturbs the synthesis

of the important ROS scavenger, glutathione peroxidase. The most important

discovery in paper III was the ability of hypoxia to maintain survival and

reconstitution capacity of primary HSCs even at high concentration of BSO. In

contrast, HSCs exposed to oxidative stress in normal oxygen conditions showed a

pronounced increase of cell death. The significance of hypoxia in HSC maintenance is

further supported by the finding that HSCs with high self-renewal capacity and low

ROS levels are associated with high expressions of niche molecules known to be

induced by hypoxia (Jang and Sharkis, 2007).

As demonstrated in paper II, hypoxia decreases cell cycle and favors HSCs in a

quiescent state. A slow cell cycle of HSCs is thought to prevent depletion of the stem

cell pool (Cheng et al., 2000b; Nygren and Bryder, 2008). Likewise, a low metabolic

activity can also spare HSCs from damages caused by ROS generated as by-products

from the mitochondrial respiratory chain during aerobic glucose metabolism. Another

possible mechanism of how hypoxia could save HSCs from oxidative stress is by lower

the accumulation of ROS through HIF-1α-induced PDK1 expression. In normal oxygen

conditions, energy is formed during mitochondrial respiration, which is the major

source of ROS. However, in hypoxic cells, increased levels of PDK1 increase glycolysis

and maintain a sufficient ATP production necessary for survival and growth at the

same time as the ROS generation from the mitochondria is attenuated (Semenza,

2007). In line with this, our results showed a pronounced increase in intracellular ROS

levels (H2O2) in cells cultured in normoxia, whereas cells cultured in hypoxia after

addition of BSO showed continuous low levels of ROS. It has been shown by others

that overexpression of PDK1 in cells deficient of HIF-1α, cultured in hypoxia, can

rescue cells from cell death and decrease oxidative stress (Kim et al., 2006). Similar to


61

this, we observed increased cell death when HSCs deficient for HIF-1α or PDK1 were

cultured in hypoxia, which indicate that PDK1 as well as other HIF-1α targets are

necessary for survival of hypoxic cells. Furthermore, we observed that bone marrow

transplantations with HSCs silenced for PDK1 or HIF-1α revealed a significant

decrease in engraftment capacity. A likely explanation could be that the transplanted

cells with insufficient levels of HIF-1α and its target gene PDK1 die in a hypoxic in vivo

microenvironment in the bone marrow. These findings might reflect the presence of

a hypoxic stem cell niche.

We observed that hypoxia can depress accumulation of ROS and that HIF-1α and

PDK1 are important for survival of cells in hypoxia. With these observations and the

theoretical knowledge that PDK1 is important to reduce mitochondrial ROS

production, it was therefore surprising that silencing of either HIF-1α or PDK1 did not

abolish the resistance to oxidative stress generated by hypoxia. When we challenged

the system by increasing the amounts of ROS through disturbance of the scavenger

system in attenuating ROS, hypoxia was able to reduce apoptosis despite decreased

levels of HIF-1α or PDK1. In this model, it is possible that increase of ROS might be

generated in a mitochondrial independent way, which could explain why PDK1

expression is not important for hypoxic mediated resistance to oxidative stress.

Noteworthy, mitochondria are not the only major source of ROS, they are also very

sensitive to damages caused by ROS. Damages on mitochondrial DNA coding for

proteins involved in the respiratory chain might result in cell death due to lack of ATP

(reviewed in (Gogvadze et al., 2008)). The protection from oxidative stress-induced

cell death we observed in hypoxic treatment might be explained by a decrease in

metabolism. HSCs in hypoxia only consume a minimal amount of ATP, and therefore,

lack of energy production caused by dysfunctional mitochondria or decrease in

glycolysis due to decreased expression of HIF-1α-regulated glycolytic enzymes does

not cause lethal effects in hypoxic conditions. Conversely, damaged mitochondrial

ATP production caused by ROS would have detrimental effects on survival of

normoxic cells with high metabolic activity. This might explain why HSCs survive

better in hypoxia when exposed to oxidative stress.

Another possible explanation to hypoxia-mediated rescue of HSCs from ROS-induced

cell death might be increased levels of FoxO proteins. As mentioned earlier, FoxO

proteins, in particular FoxO3, are important for maintenance of HSCs by

counteracting oxidative stress (Miyamoto et al., 2007; Tothova et al., 2007). In our

study we observed an increase of FoxO3a followed by increased levels of SOD and

CAT in HSCs cultured in hypoxia. The two ROS-scavenger enzymes SOD and CAT are


62

responsible for dismutation of superoxide (O-2) to hydrogen peroxide (H2O2) and

converting H2O2 to water and oxygen, respectively. Hypoxia-induction of FoxO

expression could therefore be involved in the process by how hypoxia maintains

survival of HSCs exposed to oxidative stress. A cross-talk between FoxO3 and HIF-1

during hypoxia has been observed allowing fine-tuning of HIF-1 by a negative

feedback mechanism of FoxO on HIF-1 (reviewed in (Hoogeboom and Burgering,

2009)). Furthermore, oxidative stress can also trigger interaction of both FoxO and

HIF-1 with β-catenin leading to enhanced transcriptional activity (Essers et al., 2005).

In addition to activation of scavenger enzymes, other FoxO3 targets can also induce

cell cycle arrest (e.g. p27), which is important to allow more time for repair (e.g.

GADD45) of ROS-damaged DNA (reviewed in (van der Horst and Burgering, 2007)).

However, FoxO3 can also mediate apoptosis through increased levels of Bim and Fas

ligand (Brunet et al., 1999; Dijkers et al., 2000). How can one protein both protect

against cell death and induce apoptosis? One explanation is stimuli-specific post-

translational regulation of FoxO proteins. In response to oxidative stress, the

deacetylase SIRT1 associates with FoxO3 causing deacetylation of FoxO3 and evokes

cell cycle arrest and DNA repair, whereas FoxO3-mediated apoptosis is attenuated

(Brunet et al., 2004). In contrast, inhibition of many growth factor receptors,

including FLT3, prevents PI3K/AKT mediated phosphorylation and inactivation of

FoxO3, which then can translocate to the nucleus and activate transcription of Fas

ligand and Bim, resulting in apoptosis (See next section). In addition to hypoxia-

mediated survival benefits of oxidative-stressed cells, we observed maintained

reconstitution ability. The mechanism for how hypoxia restores HSC activity remains

to be elucidated. However, possible key components might be hypoxia-induced

quiescence and increased FoxO activity, strengthened by the fact that both a slow cell

cycle and presence of FoxO proteins are closely coupled to maintenance of self-

renewal capacity.

The PI3K/FoxO3/Bim pathway is critical in apoptosis induced by

inhibition of FLT3 (Paper IV) As mentioned earlier in the background, resistance to FLT3 inhibitors is a major

problem in the treatment of AML. Therefore, it is important to investigate

downstream signaling of FLT3 in order to find critical factors that could be possible

new targets for anti-leukemic drugs. In paper IV, we report our discovery that FoxO3a

and Bim play important roles in mediating apoptosis in AML cells after treatment

with the FLT3 inhibitors AG1295 and PKC412. We found that inhibition of PI3K was

essential to induce apoptosis of FLT3-ITD expressing leukemic cells through activation


63

of Bim due to inhibition of AKT and FoxO3a. In addition to Bim (Bcl-2-interacting

modulator of cell death), another BH3-only member, Puma (p53 upregulated

modulator of apoptosis), was increased following inhibition with tyrosine kinases and

was closely associated with cell death. Bim is a well-known target gene for FoxO3a,

whereas Puma is a classical p53-regulated mediator in response to genotoxic stress.

Recent data has revealed that Puma can also be induced by FoxO3a when the

PI3K/AKT pathway is inhibited (You et al., 2006). Although Puma was induced in a

similar manner as Bim, we showed, by gene silencing, that Puma was not critical for

mediating apoptosis upon tyrosine kinase inhibition. In contrast, FLT3-ITD expressing

AML cells, silenced for either Bim or FoxO, were resistant to apoptosis after RTK

inhibitor treatment. Both Puma and Bim are important effectors of apoptosis due to

the fact that they bind and inhibit all anti-apoptotic Bcl-2 like family members (Chen

et al., 2005). Recent evidence confers the importance of Bim in mediating apoptosis

in chemotherapy-treated leukemic cells (Kuroda et al., 2006). Bim can be upregulated

by other pathways in addition to PI3K/AKT signaling. A recent study has reported that

Bim is critical in triggering apoptosis in AML cells after treatment with the RTK

inhibitor Sorafenib (Zhang et al., 2008). Sorfenib induced Bim expression by inhibition

of the RAF/MEK/ERK pathway. However, our study shows that blocking of MAP

kinase activity with a more specific ERK inhibitor only had minor effect on FLT3-

induced survival, in contrast to blocking of PI3K activity which resulted in a markedly

increase in cell death through increased levels of Bim. This suggests that in AML cells,

signaling via PI3K plays a significant role for survival, although signaling differences

between patients do occur due to different molecular abnormalities. It is likely that

other pathways in addition to PI3K/AKT signaling are involved in FLT3-induced

transformation. This was demonstrated recently with microarray analysis of AML

samples with FLT3-ITD showing induction of genes involved in multiple signaling

pathways including PI3K/AKT, JAK/STAT, and RAS/MAPK, which likely together

contribute to cell cycle progression, and decreased apoptosis and differentiation (Kim

2007). Constitutively expression of STAT5 has been associated with FLT3-ITD (Levis

2002; Weisberg 2002; Knapper 2007). This is dependent on phosphorylation of

specific tyrosine residues in the juxtamembrane domain of FLT3-ITD, which is not

phosphorylated in normal FLT3 (Rocnic 2006). Activation of the PI3K/AKT and

RAS/MAPK are not always dependent on FLT3 signaling, which has been

demonstrated in AML cells resistant to RTK although FLT3 is inhibited (Piloto et al.,

2007). This justifies the development of new drugs targeting effector proteins (e.g.

Bcl-2 proteins) in the PI3K/AKT and RAS/MAPK pathways in order to increase the

sensitivity to RTK inhibitor treatment. This would result in a broader and more

effective treatment with decreased risk of developing drug resistance.


64

Overexpression of Bcl-2 proteins is common in several leukemic malignancies which

might lead to resistance to conventional chemotherapy and FLT3-inhibitors. Together

with our results, this implicates that increased activity of Bim-mediating effects could

trigger apoptosis in leukemic cells upon treatment with anti-cancer drugs and reduce

the risk of developing resistance. In fact, BH3-only mimicking drugs are in clinical

trials in combination with RTK inhibitors. Targeting downstream effectors can

overcome FLT3 inhibitory-resistance by inhibiting their pathway regardless of the

upstream protein activator. However, disturbing the balance between anti- and pro-

apoptotic Bcl-2 members can also have negative side effects on survival of normal

cells. Therefore, it is necessary to find a treatment that targets downstream signaling

effectors of survival at the same time as sensitivity of tumor cells to apoptosis is

increased. Using FLT3 inhibitors in a combination with drugs targeting effectors

molecules in the survival signaling pathways would allow an effective and tumor

specific treatment.

.

CONCLUSIONS

65

CONCLUSIONS

Maintenance of functional stem cells in the bone marrow is dependent on a balance

between survival, proliferation, differentiation, and self-renewal. We show here that

hypoxia regulates three of these; survival, proliferation, and differentiation.

Hypoxia maintains the primitive population of stem and progenitor cells and

decreases the differentiation of more committed progenitor cells. In addition,

hypoxia decreases cell cycle progression in progenitor cells possible through

upregulation of the CDK inhibitor p27, which partly is dependent on HIF-1α.

Furthermore, HSCs cultured in hypoxia maintain their slow cell cycle better compared

to HSCs in normal oxygen levels. This is important to preserve the long-term

engraftment capacity, which is coupled to expression of p21. In this study we show

that hypoxia increases p21 expression in LT-HSCs and maintains functional HSCs. We

suggest that HIF-1 activity declines the expansion of progenitor cells and decreases

initial repopulation of committed progenitor cells in vivo.

Based on findings in this thesis, we proclaim that hypoxia protects HSCs from

oxidative stress-induced cell death. The mechanism for this is not delineated, but

FoxO3 might be involved. HIF-1 and PDK1 do not seem to be involved in the rescue of

HSCs from oxidative stress. However, both are important for survival of HSCs in

hypoxia. Although PDK1 might be involved in protecting cells from uncontrolled

production of ROS from aerobic mitochondrial respiration, our findings implicate that

other mechanism for attenuating ROS are induced by hypoxia. In addition, oxidative

stress-exposed HSCs cultured in hypoxia maintain their engraftment potential,

indicating that hypoxia can protect HSCs from cell death as well as preserve

functional active HSCs.

To summarize, the major role of hypoxia in normal HSC biology is to protect them

from damages caused by persistent cell cycle as well as endogenous and externally

induced ROS accumulation. This enables HSCs to be maintained and functional

throughout an individual´s lifetime.

The PI3K/AKT pathway is critical in tyrosine kinase inhibitor-induced cell death of

AML cells expressing FLT3-ITD mutations. Inhibition of PI3K causes activation of

FoxO3 and its downstream target Bim. Loss of FoxO3 or Bim results in preserved

survival and resistance to RTK inhibitor, implicating that FoxO3 and Bim are essential

to mediate apoptosis in AML cells with constitutively active FLT3 receptors. This

CONCLUSIONS

66

findings provide increased knowledge about survival pathways critical in FLT3-ITD

signaling, which could have a clinical significance in the design of new effective drugs

in the treatment of AML patients.

FUTURE ASPECTS

67

FUTURE ASPECTS

This thesis adds novel knowledge on the importance of a balance between survival

and cell death in normal and malignant hematopoietic stem and progenitor cells.

During my work new questions have been raised just waiting to be answered. For

instance, we have found that hypoxia can protect HSCs from cell death caused by

oxidative stress, but the underlying molecular mechanisms are still unsolved.

However, we observed that hypoxia can induce expression of the transcription factor

FoxO3 that lately has caught a lot of attention due to its role in regulating

maintenance of HSCs and scavenging ROS (Kops et al., 2002; Miyamoto et al., 2007;

Tothova et al., 2007). We speculate that hypoxia might rescue HSCs from cell death

by attenuating oxidative stress in a FoxO-dependent way. In addition to scavenging

ROS, FoxO3, similar to HIF-1, regulates several cellular processes that include

apoptosis, cell cycle arrest, differentiation, and metabolism. In some pathways FoxO3

and HIF-1 synergize, but in others they counteract each other. Just to make things

more difficult to interpret, hypoxia can induce apoptosis in some cell types, whereas

others appear to be resistance. This could possibly be explained by differences in

intrinsic pathways. Maybe one of the roles of FoxO3 in HSCs is to fine-tune the effects

mediated by hypoxia and HIF-1. It would be highly interesting to investigate if cross-

talk between HIF-1 and FoxO3 exists and if so, is this of importance in stem cells? The

regulation of FoxO proteins activity is multiple and complex, and involves

transcriptional and post-transcriptional regulation as well as interaction with co-

factors. Therefore, to be able to understand if FoxO proteins are important in

hypoxic-mediated cell survival of HSCs exposed to oxidative stress a thorough

investigation of FoxO proteins activity is necessary. Investigating the effect gene

silencing of FoxO3 might have on hypoxic mediated survival would provide valuable

information. Furthermore, it would be interesting to compare FoxO3 activity in

purified primary stem cells and more mature progenitor cells exposed to oxidative

stress while cultured in normoxia or hypoxia in order to see if FoxO proteins act

differently depending on the cell stage. FoxO3 is known to activate different

transcriptional programs depending on the stimuli. Inactivation of the PI3K/AKT

pathway causes FoxO-mediated apoptosis, whereas stress-induced activation of FoxO

by the deacetylase SIRT1 causes cell cycle arrest and stress resistance. How hypoxia

interacts with these pathways in HSCs has not been investigated, but a favor of

quiescence and protection from oxidative stress is likely. Notable, in the present

study we show that hypoxia depresses accumulation of ROS and decreases the

proliferation of HSCs, which resembles the effect mediated by FoxO proteins.

FUTURE ASPECTS

68

Whether hypoxia mediates these effects via FoxO proteins or through other effectors

remains to be investigated. Increased knowledge of how FoxO proteins are regulated

both in normal and malignant stem cells could provide important potential

therapeutic applications.

In the present investigation we have also investigated the role of hypoxia on HSC

maintenance, postulating that hypoxia in the HSC niche provides critical regulation of

stem cell activity. However, as discussed earlier, signals from other niche cells are

necessary for proper regulation. It is possible that hypoxia upregulates molecules on

niche cells important for the interaction and maintenance of HSCs. Therefore,

investigation of how niche cells are affected, both functionally and genetically, by

cultivation in hypoxia would provide meaningful information to increase the

understanding of the interplay between different factors in the stem cell niche.

Moreover, co-culture of HSCs and niche cells in hypoxia could have a potential

influence on the HSC potential. To investigate whether this could increase the

expansion of functional HSCs would be intriguing. Increased knowledge of possible

hypoxic targets in the stem cell niche will help to understand how self-renewal and

survival of HSCs is regulated.

In this thesis, I discuss and highlight the importance of a hypoxic niche in maintaining

and protect normal HSCs. Can this also be applied to the maintenance of leukemic

stem cells (LSCs)? Dormant LSCs are located in the bone marrow where they are

supported by survival signaling from niche cells (Konopleva et al., 2002). Many

studies reveal evidence that this might retain LSCs and protect them from

chemotherapy, at least in part explaining the high occurrence of relapse in AML. A

possible way to get around this is by targeting niche molecules which would result in

disrupting of the interaction with LSCs. A recent study by Zheng and colleagues has

shown that inhibition of CXCR4 increases sensitivity of leukemic bone marrow blasts

to FLT3 inhibitors. Furthermore, decreased signaling of CXCR4 efficiently mobilizes

FLT3-ITD expressing human AML cells from murine bone marrow into the peripheral

blood, which causes enhanced sensitivity to anti-leukemic drugs (Zeng et al., 2009).

Since hypoxia and HIF-1 regulate many interaction molecules between niche cells and

HSCs (e.g. CXCR4/SDF-1 signaling and BCRP-1/ABCG2), which have proved to be

involved in survival and maintenance of both normal HSCs and LSCs, it is tempting to

speculate that hypoxia plays a key role in the leukemic stem cell niche. Maybe

hypoxia is an important factor in promoting the escape of leukemic stem cells from

chemotherapy.

POPULÄRVETENSKAPLIG SAMMANFATTNING

69

EN LIVSVIKTIG BALANS MELLAN LIV OCH DÖD –

en populärvetenskaplig sammanfattning

Blodstamceller, eller hematopoietiska stamceller som de också kallas, bildar alla

blodets celler. Blodceller lever kort och intensivt, vilket kräver en ständig

nyproduktion. Varje sekund bildas det mer än hundra tusen vita blodkroppar

(leukocyter) och flera miljoner röda blodkroppar (erytrocyter). Samtidigt dör lika

många. De vita blodkropparna är viktiga i försvaret mot bakterier och virus medan de

röda blodkropparna försörjer alla kroppens celler med syre. Utvecklingen mot

fungerande blodceller sker stegvis via omogna förstadieceller (progenitorer). Dessa

förstadieceller delar sig snabbt och bildar nya mer specifika förstadieceller som i sin

tur mognar ut till olika typer av blodceller. Blodstamcellen själv delar sig sällan och är

för det mest i ett vilande stadium, vilket tros vara förutsättningen för deras

överlevnad och funktionalitet. Förutom förmågan att bilda alla blodets celler kan den

även förnya sig själv, vilket är unikt för stamceller. På så vis bibehålls ett konstant

antal stamceller livet ut. Blodbildningen sker i benmärgen där blodstamcellen är

placerad i en speciell mikromiljö (nisch). Den här mikromiljön tros ha en låg

syrekoncentration, vilket antas vara till fördel för stamcellen. Celler som befinner sig i

en miljö av låg syrekoncentration blir mer vilande och mindre känsliga för skador.

Den här avhandlingen handlar om hur balansen mellan överlevnad och

programmerad celldöd regleras och vad som händer vid en störd balans. Jag har här

fokuserat på blodstamcellen och omogna förstadieceller. Valet mellan att självförnya

sig eller att producera andra blodceller regleras av speciella signalmolekyler som

kallas cytokiner vilka produceras av andra celler i omgivningen. Dessa binder till

specifika mottagarmolekyler (receptorer) på stamcellens cellyta och skickar budskap

som t.ex. ”Självförnya dig!” eller ”Bilda mer vita blodkroppar!”. Alla kroppens celler

kan även få budskap om att ”begå självmord”. Kroppen kan på så vis göra sig av med

gamla och skadade celler. Celler kan även få signaler från hormon och nerver samt

från andra fysiologiska faktorer.

I delarbete I-III har jag studerat hur en låg syrehalt (hypoxi) påverkar blodstamceller.

Andra forskargrupper har bl.a. visat att en låg syrehalt är viktigt för att bevara

blodstamceller, men vilka mekanismer som sätts igång är till stor del oklart. Vi har

studerat blodstamceller isolerade från benmärgen på möss. Mössens blodstamceller

är bättre karaktäriserade än människans och det är på så vis lättare att rena fram


70

stamceller från andra celler. Dessutom underlättar användandet av en djurmodell

genomförandet av transplantationsförsök, vilket krävs för att till fullo studera

stamcellens unika egenskaper att förnya sig och bilda alla blodets celler under en

organsims livstid. Här har vi undersökt hur odling av blodstamceller under 1-8 dagar i

en syrefattig miljö påverkar stamcellernas egenskaper i en cellkultur och

blodstamcellens förmåga att nybilda sig själv och dela sig efter att de transplanterats

till andra djur. Vidare har vi studerat om hypoxi skyddar blodstamceller mot fria

syreradikaler. Tidigare forskning har visat att en ökning av mängden fria syreradikaler

skadar stamcellens förnyelse förmåga. Vissa forskare anser även att fria syreradikaler

leder till åldrande, men mer forskning krävs för att förtydliga och befästa dessa fynd.

I arbete I fann vi att en låg syrehalt bibehåller stamcellens utseende (fenotyp), medan

utvecklingen mot mer mogna celler var hämmad. Detta visar på att hypoxi bevarar

blodstamcellen. Genom att sedan undersöka celldelning observerade vi i delarbete II

att hypoxi leder till långsam celldelning, vilket andra forskare har visat skyddar

stamcellens kapacitet. Trots att färre celler transplanterades var stamceller med en

hög förnyelsekapacitet väl bevarade till antalet. Dessa fynd tyder på att hypoxi

skyddar blodstamceller och deras unika funktionella egenskaper. Dessutom fann vi

att ett protein som är påslaget i låga syrekoncentrationer, som kallas HIF-1, är en

viktig medlare av vissa av de signaler som hypoxi skickar till stamcellen. Både hypoxi

och HIF-1 tonar ner ”Dela dig” signalen hos stamceller och förstadieceller. En annan

intressant upptäckt vi gjorde i delarbete III var att hypoxi skyddar stamceller mot att

dö av fria syreradikaler. Tillsammans visar fynden från delarbete I-III att hypoxi har en

viktig funktion att bevara och skydda blodstamcellen. Detta stämmer väl med

hypotesen att mikromiljön i benmärgen där vilande blodstamceller med hög

självförnyelseförmåga befinner sig har en låg syrekoncentration.

Om ett fel uppstår i regleringen mellan överlevnad och celldöd kan blodcancer,

leukemi, utvecklas. Akut myeloid leukemi (AML) är en speciell form av leukemi där en

viss typ av förstadieceller delar sig ohämmat och dessutom är okänsliga för signaler

att begå självmord. Ett vanligt fel som kan leda till AML är att det blir en DNA-skada,

en mutation, på en speciell receptor som kallas FLT3. Denna sitter på cellytan och kan

bli aktiverad att skicka signaler in i cellen som leder till överlevnad. En muterad FLT3

leder till en ständigt aktiv signal för överlevnad. AML tros ha sitt ursprung i

blodstamceller eller omogna förstadieceller. En ny typ av behandling av leukemi är

läkemedel som hämmar FLT3-signalen. Detta leder till att överlevnadssignalen i de

leukemiska cellerna stängs av, vilket resulterar i celldöd. Leukemiska celler uttrycker

ofta fler FLT3 mottagare och/eller muterade FLT3 mottagare, vilket gör att det främst


71

är de som drabbas och inte friska celler. Tyvärr har det visat sig att leukemiska

cellerna utvecklar resistens mot dessa FLT3-hämmare. Därför måste nya

behandlingsformer utvecklas. I arbete IV har vi studerad vilka signalmolekyler som är

av betydelse för att inducera celldöd av AML-celler efter behandling med FLT3-

hämmare.

Vi fann att Bim och FoxO3 är kritiska för att inducera celldöd med FLT3-hämmare.

Bim ett protein som är en potent aktivator av programmerad celldöd. FoxO3 är en

genregulator (transkriptionsfaktor) som sätter igång bildning av proteiner (bl.a. Bim),

vilka är involverade i bl.a. celldöd och celldelning. Med olika metoder för att stänga

av Bim och FoxO3 visade vi att de var kritiska för celldöd inducerad av FLT3-hämmare.

AML celler med avstängd produktion av Bim eller FoxO3 kunde inte slås ut av FLT3-

hämmare. Vi stängde även av andra vägar som tros vara viktiga reglerare av celldöd,

men effekten var inte densamma. Våra resultat möjliggör utvecklingen av nya

läkemedel för att behandla AML. En kombination av FLT3-hämmare och en drog som

förstärker eller efterliknar Bim-signalen kan kanske motverka uppkomsten av

läkemedelresistens.

Den här avhandlingen bidrar med nya forskningsfynd om hur överlevnad och celldöd

regleras i normala och leukemiska stam- och förstadieceller.

ACKNOWLEDGEMENTS

72

ACKNOWLEDGEMENTS

I would like to express my gratitude to everyone who has supported me and

contributed to the work resulting in this thesis. There are many I would like to thank,

however, there are some persons that I would like to thank in particular:

Jan-Ingvar Jönsson, my supervisor. Thank you for those years and guidance through

the world of hematopoietic stem cells and survival pathways. Your enthusiasm is

spread to those around you and your never ending optimism is admirable. Another

great thing I connect with you is your humor. I’m gonna miss it!

Mikael Sigvardsson, my co-supervisor. Thank you for your creativity and inputs to my

research.

Stefan Klintström and all the PhD students in Forum Scientium.

Pia, thank you for this time! You know me very well after all those years and I am

thankful for having you by my side both as a friend and a colleague. Always willing to

help or take a coffee. I will miss you and your way of telling stories!

Amanda, my roommate and friend, with the biggest heart ever! Thank you for all

small talks and support both in research and life. The title of this thesis I dedicate to

you! I already have a title for your thesis…

Emma, Petter, Camilla and Annika (yes, you are one of us!), the students that have

contributed to the studies in this thesis.

The Sigvardsson group: Hong, Sasan and Panos, always with a friendly smile and

willing to help. Jenny and Josefine for nice chats and sharing the frustration when a

device (no name given!) important for cell sorting brakes down.

Elin, Josefine, Gosia and Liza for interesting talks in the coffee room and cozy

evenings after work. I’m looking forward to more of those!

All old and new colleagues at the division of Cell biology floor 9 and “Labettan”floor

13. Work becomes so much nicer with friendly people around!

Calle, Jocke, Matilda (My HIF-1 soulmate!), Johan J, Johan O, Johan R, Patiyan, Saad,

Åsa (Push it!), Siri, Petter, Anna, Alex, Jonas, Anders and Git. My years as a PhD

student would have been really boring without you!

ACKNOWLEDGEMENTS

73

Ullis and Lisa, two dear friends. Much has been said and discussed about research,

love, and life through those years in Linköping. I hope our friendship will continue

although a common time together in Linköping is already over.

Maria, Pelle, Magda, Jason, Mattias and Jenny. Thank you for your friendship and

giving the world another perspective! Words can´t say how much I appreciate the

time we spent together and hopefully we can meet more often now and chitchat

about the real important things in life, such as “What to buy at MEC”, “How to get to

Lofoten as fast as possible” and “What exotic bird was observed close to Cloetta

center”.

The Schultz family, for introducing Schwarzwald, Sulkava and Spätzle into my life!

My family, for always being there for me and encouraging me when I needed it the

most. Mum and Dad, your love brought me where I am today. Mathias and Per-Emil,

my dear brothers with their lovely families, even though we don’t meet on a daily

basis I know that you always are there for me.

Finally, Sebastian, the love of my life. Your unwavering support, either at the

backcountry uphill slopes over difficult steep terrain or with the uphill struggles as a

researcher in the lab, has been tremendously valuable. And right now, the view from

the top has definitely been worth all the struggles. I’m looking forward to new

adventures with you by my side!

REFERENCES

74

REFERENCES

Abkowitz JL, Catlin SN, Guttorp P. 1996. Evidence that hematopoiesis may be a stochastic process in vivo. Nature medicine 2(2):190-197.

Abu-Duhier FM, Goodeve AC, Wilson GA, Care RS, Peake IR, Reilly JT. 2001. Identification of novel FLT-3 Asp835 mutations in adult acute myeloid leukaemia. British journal of haematology 113(4):983-988.

Adams GB, Chabner KT, Alley IR, Olson DP, Szczepiorkowski ZM, Poznansky MC, Kos CH, Pollak MR, Brown EM, Scadden DT. 2006. Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor. Nature 439(7076):599-603.

Adams GB, Scadden DT. 2006. The hematopoietic stem cell in its place. Nature immunology 7(4):333-337.

Adelman DM, Maltepe E, Simon MC. 1999. Multilineage embryonic hematopoiesis requires hypoxic ARNT activity. Genes & development 13(19):2478-2483.

Adolfsson J, Borge OJ, Bryder D, Theilgaard-Monch K, Astrand-Grundstrom I, Sitnicka E, Sasaki Y, Jacobsen SE. 2001. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15(4):659-669.

Adolfsson J, Mansson R, Buza-Vidas N, Hultquist A, Liuba K, Jensen CT, Bryder D, Yang L, Borge OJ, Thoren LA, Anderson K, Sitnicka E, Sasaki Y, Sigvardsson M, Jacobsen SE. 2005. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121(2):295-306.

Agbor TA, Taylor CT. 2008. SUMO, hypoxia and the regulation of metabolism. Biochemical Society transactions 36(Pt 3):445-448.

Akala OO, Park IK, Qian D, Pihalja M, Becker MW, Clarke MF. 2008. Long-term haematopoietic reconstitution by Trp53-/-p16Ink4a-/-p19Arf-/- multipotent progenitors. Nature 453(7192):228-232.

Appelbaum FR, Gundacker H, Head DR, Slovak ML, Willman CL, Godwin JE, Anderson JE, Petersdorf SH. 2006. Age and acute myeloid leukemia. Blood 107(9):3481-3485.

Aragones J, Schneider M, Van Geyte K, Fraisl P, Dresselaers T, Mazzone M, Dirkx R, Zacchigna S, Lemieux H, Jeoung NH, Lambrechts D, Bishop T, Lafuste P, Diez-Juan A, Harten SK, Van Noten P, De Bock K, Willam C, Tjwa M, Grosfeld A, Navet R, Moons L, Vandendriessche T, Deroose C, Wijeyekoon B, Nuyts J, Jordan B, Silasi-Mansat R, Lupu F, Dewerchin M, Pugh C, Salmon P, Mortelmans L, Gallez B, Gorus F, Buyse J, Sluse F, Harris RA, Gnaiger E, Hespel P, Van Hecke P, Schuit F, Van Veldhoven P, Ratcliffe P, Baes M, Maxwell P, Carmeliet P. 2008. Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nature genetics 40(2):170-180.

Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K, Ito K, Koh GY, Suda T. 2004. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 118(2):149-161.

Attema JL, Pronk CJ, Norddahl GL, Nygren JM, Bryder D. 2009. Hematopoietic stem cell ageing is uncoupled from p16 INK4A-mediated senescence. Oncogene 28(22):2238-2243.

REFERENCES

75

Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K, Jin DK, Dias S, Zhang F, Hartman TE, Hackett NR, Crystal RG, Witte L, Hicklin DJ, Bohlen P, Eaton D, Lyden D, de Sauvage F, Rafii S. 2004. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nature medicine 10(1):64-71.

Becker AJ, Mc CE, Till JE. 1963. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197:452-454.

Ben-Yosef Y, Lahat N, Shapiro S, Bitterman H, Miller A. 2002. Regulation of endothelial matrix metalloproteinase-2 by hypoxia/reoxygenation. Circulation research 90(7):784-791.

Birg F, Courcoul M, Rosnet O, Bardin F, Pebusque MJ, Marchetto S, Tabilio A, Mannoni P, Birnbaum D. 1992. Expression of the FMS/KIT-like gene FLT3 in human acute leukemias of the myeloid and lymphoid lineages. Blood 80(10):2584-2593.

Birkenkamp KU, Geugien M, Lemmink HH, Kruijer W, Vellenga E. 2001. Regulation of constitutive STAT5 phosphorylation in acute myeloid leukemia blasts. Leukemia 15(12):1923-1931.

Blade J, Kyle RA. 1998. Multiple myeloma in young patients: clinical presentation and treatment approach. Leukemia & lymphoma 30(5-6):493-501.

Borge OJ, Ramsfjell V, Cui L, Jacobsen SE. 1997. Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38- bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin. Blood 90(6):2282-2292.

Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM, Strasser A. 1999. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science (New York, NY 286(5445):1735-1738.

Brandts CH, Sargin B, Rode M, Biermann C, Lindtner B, Schwable J, Buerger H, Muller-Tidow C, Choudhary C, McMahon M, Berdel WE, Serve H. 2005. Constitutive activation of Akt by Flt3 internal tandem duplications is necessary for increased survival, proliferation, and myeloid transformation. Cancer research 65(21):9643-9650.

Brasel K, Escobar S, Anderberg R, de Vries P, Gruss HJ, Lyman SD. 1995. Expression of the flt3 receptor and its ligand on hematopoietic cells. Leukemia 9(7):1212-1218.

Braun SE, Mantel C, Rosenthal M, Cooper S, Liu L, Robertson KA, Hromas R, Broxmeyer HE. 1998. A positive effect of p21cip1/waf1 in the colony formation from murine myeloid progenitor cells as assessed by retroviral-mediated gene transfer. Blood cells, molecules & diseases 24(2):138-148.

Brunelle JK, Bell EL, Quesada NM, Vercauteren K, Tiranti V, Zeviani M, Scarpulla RC, Chandel NS. 2005. Oxygen sensing requires mitochondrial ROS but not oxidative phosphorylation. Cell metabolism 1(6):409-414.

Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME. 1999. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96(6):857-868.

Brunet A, Kanai F, Stehn J, Xu J, Sarbassova D, Frangioni JV, Dalal SN, DeCaprio JA, Greenberg ME, Yaffe MB. 2002. 14-3-3 transits to the nucleus and participates in dynamic nucleocytoplasmic transport. The Journal of cell biology 156(5):817-828.

Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R, Cohen HY, Hu LS, Cheng HL, Jedrychowski MP, Gygi SP, Sinclair DA, Alt FW, Greenberg

REFERENCES

76

ME. 2004. Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science (New York, NY 303(5666):2011-2015.

Calvi LM, Adams GB, Weibrecht KW, Weber JM, Olson DP, Knight MC, Martin RP, Schipani E, Divieti P, Bringhurst FR, Milner LA, Kronenberg HM, Scadden DT. 2003. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature 425(6960):841-846.

Carmeliet P, Dor Y, Herbert JM, Fukumura D, Brusselmans K, Dewerchin M, Neeman M, Bono F, Abramovitch R, Maxwell P, Koch CJ, Ratcliffe P, Moons L, Jain RK, Collen D, Keshert E. 1998. Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394(6692):485-490.

Carow CE, Levenstein M, Kaufmann SH, Chen J, Amin S, Rockwell P, Witte L, Borowitz MJ, Civin CI, Small D. 1996. Expression of the hematopoietic growth factor receptor FLT3 (STK-1/Flk2) in human leukemias. Blood 87(3):1089-1096.

Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. 2004. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nature medicine 10(8):858-864.

Chan CK, Chen CC, Luppen CA, Kim JB, DeBoer AT, Wei K, Helms JA, Kuo CJ, Kraft DL, Weissman IL. 2009. Endochondral ossification is required for haematopoietic stem-cell niche formation. Nature 457(7228):490-494.

Chen J, Ellison FM, Keyvanfar K, Omokaro SO, Desierto MJ, Eckhaus MA, Young NS. 2008. Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice. Experimental hematology 36(10):1236-1243.

Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL, Adams JM, Huang DC. 2005. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17(3):393-403.

Cheng EH, Wei MC, Weiler S, Flavell RA, Mak TW, Lindsten T, Korsmeyer SJ. 2001. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8(3):705-711.

Cheng T, Rodrigues N, Dombkowski D, Stier S, Scadden DT. 2000a. Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nature medicine 6(11):1235-1240.

Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT. 2000b. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science (New York, NY 287(5459):1804-1808.

Cheshier SH, Morrison SJ, Liao X, Weissman IL. 1999. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proceedings of the National Academy of Sciences of the United States of America 96(6):3120-3125.

Choudhary C, Schwable J, Brandts C, Tickenbrock L, Sargin B, Kindler T, Fischer T, Berdel WE, Muller-Tidow C, Serve H. 2005. AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations. Blood 106(1):265-273.

Chow DC, Wenning LA, Miller WM, Papoutsakis ET. 2001. Modeling pO2 Distributions in the Bone Marrow Hematopoietic Compartment. II. Modified Kroghian Models. Biophysical Journal 81(2):685-696.

Christensen JL, Weissman IL. 2001. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proceedings of the National Academy of Sciences of the United States of America 98(25):14541-14546.

REFERENCES

77

Cipolleschi MG, Dello Sbarba P, Olivotto M. 1993. The role of hypoxia in the maintenance of hematopoietic stem cells. Blood 82(7):2031-2037.

Civin CI, Strauss LC, Brovall C, Fackler MJ, Schwartz JF, Shaper JH. 1984. Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 133(1):157-165.

Conger AD, Fairchild LM. 1952. Breakage of Chromosomes by Oxygen. Proceedings of the National Academy of Sciences of the United States of America 38(4):289-299.

Conneally E, Cashman J, Petzer A, Eaves C. 1997. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proceedings of the National Academy of Sciences of the United States of America 94(18):9836-9841.

Conte C, Riant E, Toutain C, Pujol F, Arnal JF, Lenfant F, Prats AC. 2008. FGF2 translationally induced by hypoxia is involved in negative and positive feedback loops with HIF-1alpha. PloS one 3(8):e3078.

Covello KL, Kehler J, Yu H, Gordan JD, Arsham AM, Hu CJ, Labosky PA, Simon MC, Keith B. 2006. HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes & development 20(5):557-570.

Danet GH, Pan Y, Luongo JL, Bonnet DA, Simon MC. 2003. Expansion of human SCID-repopulating cells under hypoxic conditions. J Clin Invest 112(1):126-135.

Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg ME. 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91(2):231-241.

de Haan G, Weersing E, Dontje B, van Os R, Bystrykh LV, Vellenga E, Miller G. 2003. In vitro generation of long-term repopulating hematopoietic stem cells by fibroblast growth factor-1. Developmental cell 4(2):241-251.

de Jonge HJ, de Bont ES, Valk PJ, Schuringa JJ, Kies M, Woolthuis CM, Delwel R, Veeger NJ, Vellenga E, Lowenberg B, Huls G. 2009. AML at older age: age-related gene expression profiles reveal a paradoxical down-regulation of p16INK4A mRNA with prognostic significance. Blood 114(14):2869-2877.

del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. 1997. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (New York, NY 278(5338):687-689.

Denning-Kendall P, Singha S, Bradley B, Hows J. 2003. Cobblestone area-forming cells in human cord blood are heterogeneous and differ from long-term culture-initiating cells. Stem cells (Dayton, Ohio) 21(6):694-701.

Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC. 1999. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. The Journal of cell biology 144(5):891-901.

Dexter TM. 1984. Blood cell development. The message in the medium. Nature 309(5971):746-747.

Dijkers PF, Medema RH, Lammers JW, Koenderman L, Coffer PJ. 2000. Expression of the pro-apoptotic Bcl-2 family member Bim is regulated by the forkhead transcription factor FKHR-L1. Curr Biol 10(19):1201-1204.

Doench JG, Petersen CP, Sharp PA. 2003. siRNAs can function as miRNAs. Genes & development 17(4):438-442.

REFERENCES

78

Eliasson P, Karlsson, R. and Jönsson, J.I. 2006. Hypoxia expands primitive hematopoietic progenitor cells from mouse bone marrow during in vitro culture and preserves the colony-forming ability. J Stem Cells 1(4):247-257.

Engström M, Karlsson R, Jönsson JI. 2003. Inactivation of the forkhead transcription factor FoxO3 is essential for PKB-mediated survival of hematopoietic progenitor cells by kit ligand. Experimental hematology 31(4):316-323.

Eskes R, Desagher S, Antonsson B, Martinou JC. 2000. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Molecular and cellular biology 20(3):929-935.

Essers MA, de Vries-Smits LM, Barker N, Polderman PE, Burgering BM, Korswagen HC. 2005. Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science (New York, NY 308(5725):1181-1184.

Essers MA, Weijzen S, de Vries-Smits AM, Saarloos I, de Ruiter ND, Bos JL, Burgering BM. 2004. FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. The EMBO journal 23(24):4802-4812.

Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L, La Starza R, Diverio D, Colombo E, Santucci A, Bigerna B, Pacini R, Pucciarini A, Liso A, Vignetti M, Fazi P, Meani N, Pettirossi V, Saglio G, Mandelli F, Lo-Coco F, Pelicci PG, Martelli MF. 2005. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. The New England journal of medicine 352(3):254-266.

Feldser D, Agani F, Iyer NV, Pak B, Ferreira G, Semenza GL. 1999. Reciprocal positive regulation of hypoxia-inducible factor 1alpha and insulin-like growth factor 2. Cancer research 59(16):3915-3918.

Fire A, Albertson D, Harrison SW, Moerman DG. 1991. Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development (Cambridge, England) 113(2):503-514.

Forsberg EC, Serwold T, Kogan S, Weissman IL, Passegue E. 2006. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors. Cell 126(2):415-426.

Fraser CC, Szilvassy SJ, Eaves CJ, Humphries RK. 1992. Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability. Proceedings of the National Academy of Sciences of the United States of America 89(5):1968-1972.

Gallardo D, de la Camara R, Nieto JB, Espigado I, Iriondo A, Jimenez-Velasco A, Vallejo C, Martin C, Caballero D, Brunet S, Serrano D, Solano C, Ribera JM, de la Rubia J, Carreras E. 2009. Is mobilized peripheral blood comparable with bone marrow as a source of hematopoietic stem cells for allogeneic transplantation from HLA-identical sibling donors? A case-control study. Haematologica 94(9):1282-1288.

Gardner LB, Li F, Yang X, Dang CV. 2003. Anoxic fibroblasts activate a replication checkpoint that is bypassed by E1a. Molecular and cellular biology 23(24):9032-9045.

Gardner LB, Li Q, Park MS, Flanagan WM, Semenza GL, Dang CV. 2001. Hypoxia inhibits G1/S transition through regulation of p27 expression. The Journal of biological chemistry 276(11):7919-7926.

Gerber HP, Malik AK, Solar GP, Sherman D, Liang XH, Meng G, Hong K, Marsters JC, Ferrara N. 2002. VEGF regulates haematopoietic stem cell survival by an internal autocrine loop mechanism. Nature 417(6892):954-958.

REFERENCES

79

Ghaffari S. 2008. Oxidative stress in the regulation of normal and neoplastic hematopoiesis. Antioxidants & redox signaling 10(11):1923-1940.

Gilliland DG, Griffin JD. 2002. The roles of FLT3 in hematopoiesis and leukemia. Blood 100(5):1532-1542.

Goda N, Ryan HE, Khadivi B, McNulty W, Rickert RC, Johnson RS. 2003. Hypoxia-inducible factor 1alpha is essential for cell cycle arrest during hypoxia. Molecular and cellular biology 23(1):359-369.

Gogvadze V, Orrenius S, Zhivotovsky B. 2008. Mitochondria in cancer cells: what is so special about them? Trends in cell biology 18(4):165-173.

Goldberg MA, Dunning SP, Bunn HF. 1988. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science (New York, NY 242(4884):1412-1415.

Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. 1996. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. The Journal of experimental medicine 183(4):1797-1806.

Gorlach A, Diebold I, Schini-Kerth VB, Berchner-Pfannschmidt U, Roth U, Brandes RP, Kietzmann T, Busse R. 2001. Thrombin activates the hypoxia-inducible factor-1 signaling pathway in vascular smooth muscle cells: Role of the p22(phox)-containing NADPH oxidase. Circulation research 89(1):47-54.

Green DR, Reed JC. 1998. Mitochondria and apoptosis. Science (New York, NY 281(5381):1309-1312.

Greenbaum D, Jansen R, Gerstein M. 2002. Analysis of mRNA expression and protein abundance data: an approach for the comparison of the enrichment of features in the cellular population of proteins and transcripts. Bioinformatics (Oxford, England) 18(4):585-596.

Greer EL, Brunet A. 2005. FOXO transcription factors at the interface between longevity and tumor suppression. Oncogene 24(50):7410-7425.

Griffith J, Black J, Faerman C, Swenson L, Wynn M, Lu F, Lippke J, Saxena K. 2004. The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell 13(2):169-178.

Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, Lundkvist J, Ruas JL, Poellinger L, Lendahl U, Bondesson M. 2005. Hypoxia requires notch signaling to maintain the undifferentiated cell state. Developmental cell 9(5):617-628.

Guyton AC, Hall JE. 1996. Textbook of Medical Physiology. Mansfield: W.B. Saunders company.

Harrison DE, Astle CM, Lerner C. 1988. Number and continuous proliferative pattern of transplanted primitive immunohematopoietic stem cells. Proceedings of the National Academy of Sciences of the United States of America 85(3):822-826.

Harrison JS, Rameshwar P, Chang V, Bandari P. 2002. Oxygen saturation in the bone marrow of healthy volunteers. Blood 99(1):394.

Hattori K, Heissig B, Tashiro K, Honjo T, Tateno M, Shieh JH, Hackett NR, Quitoriano MS, Crystal RG, Rafii S, Moore MA. 2001. Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells. Blood 97(11):3354-3360.

Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Rafii S. 2002. Recruitment of stem and progenitor cells from the

REFERENCES

80

bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109(5):625-637.

Hengst L, Reed SI. 1996. Translational control of p27Kip1 accumulation during the cell cycle. Science (New York, NY 271(5257):1861-1864.

Hermitte F, Brunet de la Grange P, Belloc F, Praloran V, Ivanovic Z. 2006. Very low O2 concentration (0.1%) favors G0 return of dividing CD34+ cells. Stem cells (Dayton, Ohio) 24(1):65-73.

Ho AD. 2005. Kinetics and symmetry of divisions of hematopoietic stem cells. Experimental hematology 33(1):1-8.

Hock H, Hamblen MJ, Rooke HM, Schindler JW, Saleque S, Fujiwara Y, Orkin SH. 2004. Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells. Nature 431(7011):1002-1007.

Hoogeboom D, Burgering BM. 2009. Should I stay or should I go: beta-catenin decides under stress. Biochimica et biophysica acta 1796(2):63-74.

Ikuta K, Weissman IL. 1992. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proceedings of the National Academy of Sciences of the United States of America 89(4):1502-1506.

Iscove NN, Yan XQ. 1990. Precursors (pre-CFCmulti) of multilineage hemopoietic colony-forming cells quantitated in vitro. Uniqueness of IL-1 requirement, partial separation from pluripotential colony-forming cells, and correlation with long term reconstituting cells in vivo. J Immunol 145(1):190-195.

Ito K, Hirao A, Arai F, Matsuoka S, Takubo K, Hamaguchi I, Nomiyama K, Hosokawa K, Sakurada K, Nakagata N, Ikeda Y, Mak TW, Suda T. 2004. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature 431(7011):997-1002.

Ito K, Hirao A, Arai F, Takubo K, Matsuoka S, Miyamoto K, Ohmura M, Naka K, Hosokawa K, Ikeda Y, Suda T. 2006. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nature medicine 12(4):446-451.

Ivanovic Z, Bartolozzi B, Bernabei PA, Cipolleschi MG, Rovida E, Milenkovic P, Praloran V, Dello Sbarba P. 2000. Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors. British journal of haematology 108(2):424-429.

Ivanovic Z, Belloc F, Faucher JL, Cipolleschi MG, Praloran V, Dello Sbarba P. 2002. Hypoxia maintains and interleukin-3 reduces the pre-colony-forming cell potential of dividing CD34(+) murine bone marrow cells. Experimental hematology 30(1):67-73.

Ivanovic Z, Hermitte F, Brunet de la Grange P, Dazey B, Belloc F, Lacombe F, Vezon G, Praloran V. 2004. Simultaneous maintenance of human cord blood SCID-repopulating cells and expansion of committed progenitors at low O2 concentration (3%). Stem cells (Dayton, Ohio) 22(5):716-724.

Jang YY, Sharkis SJ. 2007. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood 110(8):3056-3063.

Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. 2006. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 443(7110):421-426.

Jogi A, Ora I, Nilsson H, Lindeheim A, Makino Y, Poellinger L, Axelson H, Pahlman S. 2002. Hypoxia alters gene expression in human neuroblastoma cells toward an immature

REFERENCES

81

and neural crest-like phenotype. Proceedings of the National Academy of Sciences of the United States of America 99(10):7021-7026.

Jones RJ, Collector MI, Barber JP, Vala MS, Fackler MJ, May WS, Griffin CA, Hawkins AL, Zehnbauer BA, Hilton J, Colvin OM, Sharkis SJ. 1996. Characterization of mouse lymphohematopoietic stem cells lacking spleen colony-forming activity. Blood 88(2):487-491.

Josko J, Gwozdz B, Jedrzejowska-Szypulka H, Hendryk S. 2000. Vascular endothelial growth factor (VEGF) and its effect on angiogenesis. Med Sci Monit 6(5):1047-1052.

Jönsson M, Engstrom M, Jönsson JI. 2004. FLT3 ligand regulates apoptosis through AKT-dependent inactivation of transcription factor FoxO3. Biochemical and biophysical research communications 318(4):899-903.

Kay HE. 1965. How Many Cell-Generations? Lancet 2(7409):418-419. Ke Q, Costa M. 2006. Hypoxia-inducible factor-1 (HIF-1). Molecular pharmacology

70(5):1469-1480. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. 1993. A C. elegans mutant that lives

twice as long as wild type. Nature 366(6454):461-464. Kertesz Z, Vas V, Kiss J, Urban VS, Pozsonyi E, Kozma A, Paloczi K, Uher F. 2006. In vitro

expansion of long-term repopulating hematopoietic stem cells in the presence of immobilized Jagged-1 and early acting cytokines. Cell biology international 30(5):401-405.

Kiel MJ, Acar M, Radice GL, Morrison SJ. 2009. Hematopoietic stem cells do not depend on N-cadherin to regulate their maintenance. Cell stem cell 4(2):170-179.

Kiel MJ, Morrison SJ. 2008. Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol 8(4):290-301.

Kiel MJ, Radice GL, Morrison SJ. 2007. Lack of evidence that hematopoietic stem cells depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. Cell stem cell 1(2):204-217.

Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ. 2005. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121(7):1109-1121.

Kikushige Y, Yoshimoto G, Miyamoto T, Iino T, Mori Y, Iwasaki H, Niiro H, Takenaka K, Nagafuji K, Harada M, Ishikawa F, Akashi K. 2008. Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival. J Immunol 180(11):7358-7367.

Kim JW, Tchernyshyov I, Semenza GL, Dang CV. 2006. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell metabolism 3(3):177-185.

Kim KT, Baird K, Ahn JY, Meltzer P, Lilly M, Levis M, Small D. 2005. Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood 105(4):1759-1767.

Kirito K, Fox N, Kaushansky K. 2003. Thrombopoietin stimulates Hoxb4 expression: an explanation for the favorable effects of TPO on hematopoietic stem cells. Blood 102(9):3172-3178.

Knapper S. 2007. FLT3 inhibition in acute myeloid leukaemia. British journal of haematology 138(6):687-699.

Knapper S, Mills KI, Gilkes AF, Austin SJ, Walsh V, Burnett AK. 2006. The effects of lestaurtinib (CEP701) and PKC412 on primary AML blasts: the induction of

REFERENCES

82

cytotoxicity varies with dependence on FLT3 signaling in both FLT3-mutated and wild-type cases. Blood 108(10):3494-3503.

Knoblich JA. 2001. Asymmetric cell division during animal development. Nature reviews 2(1):11-20.

Kohl TM, Hellinger C, Ahmed F, Buske C, Hiddemann W, Bohlander SK, Spiekermann K. 2007. BH3 mimetic ABT-737 neutralizes resistance to FLT3 inhibitor treatment mediated by FLT3-independent expression of BCL2 in primary AML blasts. Leukemia 21(8):1763-1772.

Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, Deng X, Zhai D, Shi YX, Sneed T, Verhaegen M, Soengas M, Ruvolo VR, McQueen T, Schober WD, Watt JC, Jiffar T, Ling X, Marini FC, Harris D, Dietrich M, Estrov Z, McCubrey J, May WS, Reed JC, Andreeff M. 2006. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer cell 10(5):375-388.

Konopleva M, Konoplev S, Hu W, Zaritskey AY, Afanasiev BV, Andreeff M. 2002. Stromal cells prevent apoptosis of AML cells by up-regulation of anti-apoptotic proteins. Leukemia 16(9):1713-1724.

Kops GJ, Dansen TB, Polderman PE, Saarloos I, Wirtz KW, Coffer PJ, Huang TT, Bos JL, Medema RH, Burgering BM. 2002. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature 419(6904):316-321.

Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, Huang LE. 2004. HIF-1alpha induces cell cycle arrest by functionally counteracting Myc. The EMBO journal 23(9):1949-1956.

Kottaridis PD, Gale RE, Frew ME, Harrison G, Langabeer SE, Belton AA, Walker H, Wheatley K, Bowen DT, Burnett AK, Goldstone AH, Linch DC. 2001. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials. Blood 98(6):1752-1759.

Krishnamurthy J, Torrice C, Ramsey MR, Kovalev GI, Al-Regaiey K, Su L, Sharpless NE. 2004a. Ink4a/Arf expression is a biomarker of aging. J Clin Invest 114(9):1299-1307.

Krishnamurthy P, Ross DD, Nakanishi T, Bailey-Dell K, Zhou S, Mercer KE, Sarkadi B, Sorrentino BP, Schuetz JD. 2004b. The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme. The Journal of biological chemistry 279(23):24218-24225.

Kubota Y, Takubo K, Suda T. 2008. Bone marrow long label-retaining cells reside in the sinusoidal hypoxic niche. Biochemical and biophysical research communications 366(2):335-339.

Kuroda J, Puthalakath H, Cragg MS, Kelly PN, Bouillet P, Huang DC, Kimura S, Ottmann OG, Druker BJ, Villunger A, Roberts AW, Strasser A. 2006. Bim and Bad mediate imatinib-induced killing of Bcr/Abl+ leukemic cells, and resistance due to their loss is overcome by a BH3 mimetic. Proceedings of the National Academy of Sciences of the United States of America 103(40):14907-14912.

Lacorazza HD, Yamada T, Liu Y, Miyata Y, Sivina M, Nunes J, Nimer SD. 2006. The transcription factor MEF/ELF4 regulates the quiescence of primitive hematopoietic cells. Cancer cell 9(3):175-187.

Laiosa CV, Stadtfeld M, Graf T. 2006. Determinants of lymphoid-myeloid lineage diversification. Annu Rev Immunol 24:705-738.

REFERENCES

83

Larochelle A, Vormoor J, Hanenberg H, Wang JC, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE. 1996. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nature medicine 2(12):1329-1337.

Levesque JP, Winkler IG, Hendy J, Williams B, Helwani F, Barbier V, Nowlan B, Nilsson SK. 2007. Hematopoietic progenitor cell mobilization results in hypoxia with increased hypoxia-inducible transcription factor-1 alpha and vascular endothelial growth factor A in bone marrow. Stem cells (Dayton, Ohio) 25(8):1954-1965.

Levis M, Small D. 2003. FLT3: ITDoes matter in leukemia. Leukemia 17(9):1738-1752. Levis M, Small D. 2005. FLT3 tyrosine kinase inhibitors. International journal of hematology

82(2):100-107. Levis M, Tse KF, Smith BD, Garrett E, Small D. 2001. A FLT3 tyrosine kinase inhibitor is

selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations. Blood 98(3):885-887.

Li CL, Johnson GR. 1995. Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood 85(6):1472-1479.

Li H, Zhu H, Xu CJ, Yuan J. 1998. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94(4):491-501.

Li J, Zhang X, Sejas DP, Pang Q. 2005. Negative regulation of p53 by nucleophosmin antagonizes stress-induced apoptosis in human normal and malignant hematopoietic cells. Leukemia research 29(12):1415-1423.

Li W, Johnson SA, Shelley WC, Yoder MC. 2004. Hematopoietic stem cell repopulating ability can be maintained in vitro by some primary endothelial cells. Experimental hematology 32(12):1226-1237.

Liang Y, Van Zant G, Szilvassy SJ. 2005. Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells. Blood 106(4):1479-1487.

Lisovsky M, Braun SE, Ge Y, Takahira H, Lu L, Savchenko VG, Lyman SD, Broxmeyer HE. 1996. Flt3-ligand production by human bone marrow stromal cells. Leukemia 10(6):1012-1018.

Liu Y, Elf SE, Miyata Y, Sashida G, Liu Y, Huang G, Di Giandomenico S, Lee JM, Deblasio A, Menendez S, Antipin J, Reva B, Koff A, Nimer SD. 2009. p53 regulates hematopoietic stem cell quiescence. Cell stem cell 4(1):37-48.

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods (San Diego, Calif 25(4):402-408.

Lo Celso C, Fleming HE, Wu JW, Zhao CX, Miake-Lye S, Fujisaki J, Cote D, Rowe DW, Lin CP, Scadden DT. 2009. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457(7225):92-96.

Locksley RM, Killeen N, Lenardo MJ. 2001. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104(4):487-501.

Lyman SD, James L, Vanden Bos T, de Vries P, Brasel K, Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR, et al. 1993. Molecular cloning of a ligand for the flt3/flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75(6):1157-1167.

Mancini SJ, Mantei N, Dumortier A, Suter U, MacDonald HR, Radtke F. 2005. Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation. Blood 105(6):2340-2342.

REFERENCES

84

Mantel C, Luo Z, Canfield J, Braun S, Deng C, Broxmeyer HE. 1996. Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo. Blood 88(10):3710-3719.

Matsunaga T, Kato T, Miyazaki H, Ogawa M. 1998. Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. Blood 92(2):452-461.

Matsuzaki Y, Nakayama K, Nakayama K, Tomita T, Isoda M, Loh DY, Nakauchi H. 1997. Role of bcl-2 in the development of lymphoid cells from the hematopoietic stem cell. Blood 89(3):853-862.

Matthews W, Jordan CT, Gavin M, Jenkins NA, Copeland NG, Lemischka IR. 1991. A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit. Proceedings of the National Academy of Sciences of the United States of America 88(20):9026-9030.

Maxwell PH, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, Wykoff CC, Pugh CW, Maher ER, Ratcliffe PJ. 1999. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis. Nature 399(6733):271-275.

Mayani H, Dragowska W, Lansdorp PM. 1993. Lineage commitment in human hemopoiesis involves asymmetric cell division of multipotent progenitors and does not appear to be influenced by cytokines. Journal of cellular physiology 157(3):579-586.

McCulloch EA, Siminovitch L, Till JE, Russell ES, Bernstein SE. 1965. The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl-Sld. Blood 26(4):399-410.

McNiece IK, Bertoncello I, Kriegler AB, Quesenberry PJ. 1990. Colony-forming cells with high proliferative potential (HPP-CFC). International journal of cell cloning 8(3):146-160.

Mead AJ, Linch DC, Hills RK, Wheatley K, Burnett AK, Gale RE. 2007. FLT3 tyrosine kinase domain mutations are biologically distinct from and have a significantly more favorable prognosis than FLT3 internal tandem duplications in patients with acute myeloid leukemia. Blood 110(4):1262-1270.

Metcalf D. 1998. Lineage commitment and maturation in hematopoietic cells: the case for extrinsic regulation. Blood 92(2):345-347; discussion 352.

Michallet M, Archimbaud E, Bandini G, Rowlings PA, Deeg HJ, Gahrton G, Montserrat E, Rozman C, Gratwohl A, Gale RP. 1996. HLA-identical sibling bone marrow transplantation in younger patients with chronic lymphocytic leukemia. European Group for Blood and Marrow Transplantation and the International Bone Marrow Transplant Registry. Annals of internal medicine 124(3):311-315.

Miller CL, Eaves CJ. 1997. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proceedings of the National Academy of Sciences of the United States of America 94(25):13648-13653.

Miyamoto K, Araki KY, Naka K, Arai F, Takubo K, Yamazaki S, Matsuoka S, Miyamoto T, Ito K, Ohmura M, Chen C, Hosokawa K, Nakauchi H, Nakayama K, Nakayama KI, Harada M, Motoyama N, Suda T, Hirao A. 2007. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell stem cell 1(1):101-112.

Mizuki M, Fenski R, Halfter H, Matsumura I, Schmidt R, Muller C, Gruning W, Kratz-Albers K, Serve S, Steur C, Buchner T, Kienast J, Kanakura Y, Berdel WE, Serve H. 2000. Flt3

REFERENCES

85

mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways. Blood 96(12):3907-3914.

Mizuki M, Schwable J, Steur C, Choudhary C, Agrawal S, Sargin B, Steffen B, Matsumura I, Kanakura Y, Bohmer FD, Muller-Tidow C, Berdel WE, Serve H. 2003. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations. Blood 101(8):3164-3173.

Morrison SJ, Csete M, Groves AK, Melega W, Wold B, Anderson DJ. 2000. Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J Neurosci 20(19):7370-7376.

Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. 1996. The aging of hematopoietic stem cells. Nature medicine 2(9):1011-1016.

Morrison SJ, Weissman IL. 1994. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1(8):661-673.

Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. 1994. Development of hematopoietic stem cell activity in the mouse embryo. Immunity 1(4):291-301.

Månsson R, Hultquist A, Luc S, Yang L, Anderson K, Kharazi S, Al-Hashmi S, Liuba K, Thoren L, Adolfsson J, Buza-Vidas N, Qian H, Soneji S, Enver T, Sigvardsson M, Jacobsen SE. 2007. Molecular evidence for hierarchical transcriptional lineage priming in fetal and adult stem cells and multipotent progenitors. Immunity 26(4):407-419.

Nakano K, Vousden KH. 2001. PUMA, a novel proapoptotic gene, is induced by p53. Mol Cell 7(3):683-694.

Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K, Sonoda Y, Fujimoto T, Misawa S. 1996. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 10(12):1911-1918.

Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. 1999. Vascular endothelial growth factor (VEGF) and its receptors. Faseb J 13(1):9-22.

Nishi H, Nakada T, Kyo S, Inoue M, Shay JW, Isaka K. 2004. Hypoxia-inducible factor 1 mediates upregulation of telomerase (hTERT). Molecular and cellular biology 24(13):6076-6083.

Nygren JM, Bryder D. 2008. A novel assay to trace proliferation history in vivo reveals that enhanced divisional kinetics accompany loss of hematopoietic stem cell self-renewal. PloS one 3(11):e3710.

Ogawa M. 1993. Differentiation and proliferation of hematopoietic stem cells. Blood 81(11):2844-2853.

Ohneda O, Fennie C, Zheng Z, Donahue C, La H, Villacorta R, Cairns B, Lasky LA. 1998. Hematopoietic stem cell maintenance and differentiation are supported by embryonic aorta-gonad-mesonephros region-derived endothelium. Blood 92(3):908-919.

Opferman JT, Iwasaki H, Ong CC, Suh H, Mizuno S, Akashi K, Korsmeyer SJ. 2005. Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science (New York, NY 307(5712):1101-1104.

Orford KW, Scadden DT. 2008. Deconstructing stem cell self-renewal: genetic insights into cell-cycle regulation. Nat Rev Genet 9(2):115-128.

Osawa M, Hanada K, Hamada H, Nakauchi H. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science (New York, NY 273(5272):242-245.

REFERENCES

86

Pagano M, Tam SW, Theodoras AM, Beer-Romero P, Del Sal G, Chau V, Yew PR, Draetta GF, Rolfe M. 1995. Role of the ubiquitin-proteasome pathway in regulating abundance of the cyclin-dependent kinase inhibitor p27. Science (New York, NY 269(5224):682-685.

Panayiotidis P, Jones D, Ganeshaguru K, Foroni L, Hoffbrand AV. 1996. Human bone marrow stromal cells prevent apoptosis and support the survival of chronic lymphocytic leukaemia cells in vitro. British journal of haematology 92(1):97-103.

Papandreou I, Cairns RA, Fontana L, Lim AL, Denko NC. 2006. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell metabolism 3(3):187-197.

Parcells BW, Ikeda AK, Simms-Waldrip T, Moore TB, Sakamoto KM. 2006. FMS-like tyrosine kinase 3 in normal hematopoiesis and acute myeloid leukemia. Stem cells (Dayton, Ohio) 24(5):1174-1184.

Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF. 2003. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 423(6937):302-305.

Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD. 2007. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proceedings of the National Academy of Sciences of the United States of America 104(13):5431-5436.

Pearce DJ, Bonnet D. 2007. The combined use of Hoechst efflux ability and aldehyde dehydrogenase activity to identify murine and human hematopoietic stem cells. Experimental hematology 35(9):1437-1446.

Pedersen M, Lofstedt T, Sun J, Holmquist-Mengelbier L, Pahlman S, Ronnstrand L. 2008. Stem cell factor induces HIF-1alpha at normoxia in hematopoietic cells. Biochemical and biophysical research communications 377(1):98-103.

Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T. 2002. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nature immunology 3(7):687-694.

Pierre-Louis O, Clay D, de la Grange PB, Blazsek I, Desterke C, Guerton B, Blondeau C, Malfuson JV, Prat M, Bennaceur-Griscelli A, Lataillade JJ, Le Bousse-Kerdiles MC. 2009. Dual SP/ALDH Functionalities Refine The Human Hematopoietic Lin(-) CD34(+) CD38(-) Stem/Progenitor Cell Compartment. Stem cells (Dayton, Ohio).

Piloto O, Wright M, Brown P, Kim KT, Levis M, Small D. 2007. Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways. Blood 109(4):1643-1652.

Pinto FO, Roberts I. 2008. Cord blood stem cell transplantation for haemoglobinopathies. British journal of haematology 141(3):309-324.

Ploemacher RE, van der Sluijs JP, van Beurden CA, Baert MR, Chan PL. 1991. Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse. Blood 78(10):2527-2533.

Polyak K, Lee MH, Erdjument-Bromage H, Koff A, Roberts JM, Tempst P, Massague J. 1994. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78(1):59-66.

Qian H, Buza-Vidas N, Hyland CD, Jensen CT, Antonchuk J, Mansson R, Thoren LA, Ekblom M, Alexander WS, Jacobsen SE. 2007. Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells. Cell stem cell 1(6):671-684.

REFERENCES

87

Ramsfjell V, Borge OJ, Veiby OP, Cardier J, Murphy MJ, Jr., Lyman SD, Lok S, Jacobsen SE. 1996. Thrombopoietin, but not erythropoietin, directly stimulates multilineage growth of primitive murine bone marrow progenitor cells in synergy with early acting cytokines: distinct interactions with the ligands for c-kit and FLT3. Blood 88(12):4481-4492.

Rana TM. 2007. Illuminating the silence: understanding the structure and function of small RNAs. Nature reviews 8(1):23-36.

Rasko JE, Metcalf D, Rossner MT, Begley CG, Nicola NA. 1995. The flt3/flk-2 ligand: receptor distribution and action on murine haemopoietic cell survival and proliferation. Leukemia 9(12):2058-2066.

Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, Hintz L, Nusse R, Weissman IL. 2003. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423(6938):409-414.

Reya T, Morrison SJ, Clarke MF, Weissman IL. 2001. Stem cells, cancer, and cancer stem cells. Nature 414(6859):105-111.

Richard DE, Berra E, Gothie E, Roux D, Pouyssegur J. 1999. p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1. The Journal of biological chemistry 274(46):32631-32637.

Rizo A, Vellenga E, de Haan G, Schuringa JJ. 2006. Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Human molecular genetics 15 Spec No 2:R210-219.

Rombouts EJ, Pavic B, Lowenberg B, Ploemacher RE. 2004. Relation between CXCR-4 expression, Flt3 mutations, and unfavorable prognosis of adult acute myeloid leukemia. Blood 104(2):550-557.

Rosnet O, Buhring HJ, Marchetto S, Rappold I, Lavagna C, Sainty D, Arnoulet C, Chabannon C, Kanz L, Hannum C, Birnbaum D. 1996. Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells. Leukemia 10(2):238-248.

Rosnet O, Mattei MG, Marchetto S, Birnbaum D. 1991. Isolation and chromosomal localization of a novel FMS-like tyrosine kinase gene. Genomics 9(2):380-385.

Rosnet O, Schiff C, Pebusque MJ, Marchetto S, Tonnelle C, Toiron Y, Birg F, Birnbaum D. 1993. Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells. Blood 82(4):1110-1119.

Rossi DJ, Bryder D, Weissman IL. 2007. Hematopoietic stem cell aging: mechanism and consequence. Experimental gerontology 42(5):385-390.

Sacchetti B, Funari A, Michienzi S, Di Cesare S, Piersanti S, Saggio I, Tagliafico E, Ferrari S, Robey PG, Riminucci M, Bianco P. 2007. Self-Renewing Osteoprogenitors in Bone Marrow Sinusoids Can Organize a Hematopoietic Microenvironment. Cell 131(2):324-336.

Sauvageau G, Iscove NN, Humphries RK. 2004. In vitro and in vivo expansion of hematopoietic stem cells. Oncogene 23(43):7223-7232.

Scandura JM, Boccuni P, Massague J, Nimer SD. 2004. Transforming growth factor beta-induced cell cycle arrest of human hematopoietic cells requires p57KIP2 up-regulation. Proceedings of the National Academy of Sciences of the United States of America 101(42):15231-15236.

REFERENCES

88

Scheijen B, Ngo HT, Kang H, Griffin JD. 2004. FLT3 receptors with internal tandem duplications promote cell viability and proliferation by signaling through Foxo proteins. Oncogene.

Schofield R. 1978. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 4(1-2):7-25.

Screaton G, Xu XN. 2000. T cell life and death signalling via TNF-receptor family members. Current opinion in immunology 12(3):316-322.

Seagroves TN, Ryan HE, Lu H, Wouters BG, Knapp M, Thibault P, Laderoute K, Johnson RS. 2001. Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells. Molecular and cellular biology 21(10):3436-3444.

Semenza GL. 2007. Oxygen-dependent regulation of mitochondrial respiration by hypoxia-inducible factor 1. The Biochemical journal 405(1):1-9.

Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P, Giallongo A. 1996. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. The Journal of biological chemistry 271(51):32529-32537.

Semenza GL, Nejfelt MK, Chi SM, Antonarakis SE. 1991. Hypoxia-inducible nuclear factors bind to an enhancer element located 3' to the human erythropoietin gene. Proceedings of the National Academy of Sciences of the United States of America 88(13):5680-5684.

Sherr CJ, Roberts JM. 1999. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & development 13(12):1501-1512.

Shima H, Takubo K, Iwasaki H, Yoshihara H, Gomei Y, Hosokawa K, Arai F, Takahashi T, Suda T. 2008. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice. Biochemical and biophysical research communications.

Small D. 2006. FLT3 mutations: biology and treatment. Hematology / the Education Program of the American Society of Hematology American Society of Hematology:178-184.

Small D, Levenstein M, Kim E, Carow C, Amin S, Rockwell P, Witte L, Burrow C, Ratajczak MZ, Gewirtz AM, et al. 1994. STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells. Proceedings of the National Academy of Sciences of the United States of America 91(2):459-463.

Somervaille TC, Cleary ML. 2006. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer cell 10(4):257-268.

Spangrude GJ, Brooks DM. 1992. Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1-negative subset. Blood 80(8):1957-1964.

Spangrude GJ, Heimfeld S, Weissman IL. 1988. Purification and characterization of mouse hematopoietic stem cells. Science (New York, NY 241(4861):58-62.

Stacchini A, Fubini L, Severino A, Sanavio F, Aglietta M, Piacibello W. 1996. Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts. Leukemia 10(10):1584-1591.

Stier S, Cheng T, Dombkowski D, Carlesso N, Scadden DT. 2002. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood 99(7):2369-2378.

Stirewalt DL, Radich JP. 2003. The role of FLT3 in haematopoietic malignancies. Nat Rev Cancer 3(9):650-665.

REFERENCES

89

Stone RM, DeAngelo DJ, Klimek V, Galinsky I, Estey E, Nimer SD, Grandin W, Lebwohl D, Wang Y, Cohen P, Fox EA, Neuberg D, Clark J, Gilliland DG, Griffin JD. 2005. Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412. Blood 105(1):54-60.

Suda T, Suda J, Ogawa M. 1984. Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors. Proceedings of the National Academy of Sciences of the United States of America 81(8):2520-2524.

Sugiyama T, Kohara H, Noda M, Nagasawa T. 2006. Maintenance of the Hematopoietic Stem Cell Pool by CXCL12-CXCR4 Chemokine Signaling in Bone Marrow Stromal Cell Niches. Immunity 25(6):977-988.

Sutherland HJ, Eaves CJ, Eaves AC, Dragowska W, Lansdorp PM. 1989. Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74(5):1563-1570.

Suzuki T, Yokoyama Y, Kumano K, Takanashi M, Kozuma S, Takato T, Nakahata T, Nishikawa M, Sakano S, Kurokawa M, Ogawa S, Chiba S. 2006. Highly efficient ex vivo expansion of human hematopoietic stem cells using Delta1-Fc chimeric protein. Stem cells (Dayton, Ohio) 24(11):2456-2465.

Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. 1990. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proceedings of the National Academy of Sciences of the United States of America 87(22):8736-8740.

Tabe Y, Konopleva M, Munsell MF, Marini FC, Zompetta C, McQueen T, Tsao T, Zhao S, Pierce S, Igari J, Estey EH, Andreeff M. 2004. PML-RARalpha is associated with leptin-receptor induction: the role of mesenchymal stem cell-derived adipocytes in APL cell survival. Blood 103(5):1815-1822.

Tallman MS, Gilliland DG, Rowe JM. 2005. Drug therapy for acute myeloid leukemia. Blood 106(4):1154-1163.

Tanaka M, Kirito K, Kashii Y, Uchida M, Watanabe T, Endo H, Endoh T, Sawada K, Ozawa K, Komatsu N. 2001. Forkhead family transcription factor FKHRL1 is expressed in human megakaryocytes. Regulation of cell cycling as a downstream molecule of thrombopoietin signaling. The Journal of biological chemistry 276(18):15082-15089.

Tavor S, Petit I, Porozov S, Avigdor A, Dar A, Leider-Trejo L, Shemtov N, Deutsch V, Naparstek E, Nagler A, Lapidot T. 2004. CXCR4 regulates migration and development of human acute myelogenous leukemia stem cells in transplanted NOD/SCID mice. Cancer research 64(8):2817-2824.

TeKippe M, Harrison DE, Chen J. 2003. Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice. Experimental hematology 31(6):521-527.

Thiede C, Koch S, Creutzig E, Steudel C, Illmer T, Schaich M, Ehninger G. 2006. Prevalence and prognostic impact of NPM1 mutations in 1485 adult patients with acute myeloid leukemia (AML). Blood 107(10):4011-4020.

Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U, Wermke M, Bornhauser M, Ritter M, Neubauer A, Ehninger G, Illmer T. 2002. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 99(12):4326-4335.

REFERENCES

90

Thomas ED, Lochte HL, Jr., Lu WC, Ferrebee JW. 1957. Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. The New England journal of medicine 257(11):491-496.

Till JE, Mc CE. 1961. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213-222.

Tothova Z, Kollipara R, Huntly BJ, Lee BH, Castrillon DH, Cullen DE, McDowell EP, Lazo-Kallanian S, Williams IR, Sears C, Armstrong SA, Passegue E, DePinho RA, Gilliland DG. 2007. FoxOs are critical mediators of hematopoietic stem cell resistance to physiologic oxidative stress. Cell 128(2):325-339.

Tsujimura T, Hashimoto K, Kitayama H, Ikeda H, Sugahara H, Matsumura I, Kaisho T, Terada N, Kitamura Y, Kanakura Y. 1999. Activating mutation in the catalytic domain of c-kit elicits hematopoietic transformation by receptor self-association not at the ligand-induced dimerization site. Blood 93(4):1319-1329.

Uchida N, Weissman IL. 1992. Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. The Journal of experimental medicine 175(1):175-184.

Umemoto T, Yamato M, Nishida K, Yang J, Tano Y, Okano T. 2005. p57Kip2 is expressed in quiescent mouse bone marrow side population cells. Biochemical and biophysical research communications 337(1):14-21.

Wada T, Penninger JM. 2004. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23(16):2838-2849.

van der Horst A, Burgering BM. 2007. Stressing the role of FoxO proteins in lifespan and disease. Nature reviews 8(6):440-450.

van der Loo JC, Ploemacher RE. 1995. Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors. Blood 85(9):2598-2606.

van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ. 2005. High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 11(18):6520-6527.

Vanhaesebroeck B, Alessi DR. 2000. The PI3K-PDK1 connection: more than just a road to PKB. The Biochemical journal 346 Pt 3:561-576.

Varnum-Finney B, Xu L, Brashem-Stein C, Nourigat C, Flowers D, Bakkour S, Pear WS, Bernstein ID. 2000. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nature medicine 6(11):1278-1281.

Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ. 2001. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science (New York, NY 292(5517):727-730.

Veiby OP, Jacobsen FW, Cui L, Lyman SD, Jacobsen SE. 1996. The flt3 ligand promotes the survival of primitive hemopoietic progenitor cells with myeloid as well as B lymphoid potential. Suppression of apoptosis and counteraction by TNF-alpha and TGF-beta. J Immunol 157(7):2953-2960.

Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. 1993. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75(2):229-240.

REFERENCES

91

Weisberg E, Barrett R, Liu Q, Stone R, Gray N, Griffin JD. 2009. FLT3 inhibition and mechanisms of drug resistance in mutant FLT3-positive AML. Drug Resist Updat 12(3):81-89.

Weisberg E, Boulton C, Kelly LM, Manley P, Fabbro D, Meyer T, Gilliland DG, Griffin JD. 2002. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer cell 1(5):433-443.

Wenger RH. 2002. Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression. Faseb J 16(10):1151-1162.

Wenger RH, Stiehl DP, Camenisch G. 2005. Integration of oxygen signaling at the consensus HRE. Sci STKE 2005(306):re12.

Wilson A, Murphy MJ, Oskarsson T, Kaloulis K, Bettess MD, Oser GM, Pasche AC, Knabenhans C, Macdonald HR, Trumpp A. 2004. c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes & development 18(22):2747-2763.

Wilson A, Trumpp A. 2006. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol 6(2):93-106.

Vivanco I, Sawyers CL. 2002. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2(7):489-501.

Wognum AW, Eaves AC, Thomas TE. 2003. Identification and isolation of hematopoietic stem cells. Archives of medical research 34(6):461-475.

Wright DE, Wagers AJ, Gulati AP, Johnson FL, Weissman IL. 2001. Physiological migration of hematopoietic stem and progenitor cells. Science (New York, NY 294(5548):1933-1936.

Xie Y, Yin T, Wiegraebe W, He XC, Miller D, Stark D, Perko K, Alexander R, Schwartz J, Grindley JC, Park J, Haug JS, Wunderlich JP, Li H, Zhang S, Johnson T, Feldman RA, Li L. 2009. Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457(7225):97-101.

Yagi M, Ritchie KA, Sitnicka E, Storey C, Roth GJ, Bartelmez S. 1999. Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin. Proceedings of the National Academy of Sciences of the United States of America 96(14):8126-8131.

Yamamoto Y, Kiyoi H, Nakano Y, Suzuki R, Kodera Y, Miyawaki S, Asou N, Kuriyama K, Yagasaki F, Shimazaki C, Akiyama H, Saito K, Nishimura M, Motoji T, Shinagawa K, Takeshita A, Saito H, Ueda R, Ohno R, Naoe T. 2001. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 97(8):2434-2439.

Yamazaki S, Iwama A, Takayanagi S, Morita Y, Eto K, Ema H, Nakauchi H. 2006. Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells. The EMBO journal 25(15):3515-3523.

Yang L, Bryder D, Adolfsson J, Nygren J, Mansson R, Sigvardsson M, Jacobsen SE. 2005. Identification of Lin(-)Sca1(+)kit(+)CD34(+)Flt3- short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 105(7):2717-2723.

Yilmaz OH, Kiel MJ, Morrison SJ. 2006. SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. Blood 107(3):924-930.

REFERENCES

92

Yoshida K, Kirito K, Yongzhen H, Ozawa K, Kaushansky K, Komatsu N. 2008. Thrombopoietin (TPO) regulates HIF-1alpha levels through generation of mitochondrial reactive oxygen species. International journal of hematology 88(1):43-51.

Yoshihara H, Arai F, Hosokawa K, Hagiwara T, Takubo K, Nakamura Y, Gomei Y, Iwasaki H, Matsuoka S, Miyamoto K, Miyazaki H, Takahashi T, Suda T. 2007. Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell stem cell 1(6):685-697.

You H, Mak TW. 2005. Crosstalk between p53 and FOXO transcription factors. Cell cycle (Georgetown, Tex 4(1):37-38.

You H, Pellegrini M, Tsuchihara K, Yamamoto K, Hacker G, Erlacher M, Villunger A, Mak TW. 2006. FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal. The Journal of experimental medicine 203(7):1657-1663.

Yu H, Yuan Y, Shen H, Cheng T. 2006. Hematopoietic stem cell exhaustion impacted by p18 INK4C and p21 Cip1/Waf1 in opposite manners. Blood 107(3):1200-1206.

Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. 2004. In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nature cell biology 6(5):436-442.

Yun Z, Maecker HL, Johnson RS, Giaccia AJ. 2002. Inhibition of PPAR gamma 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Developmental cell 2(3):331-341.

Zeng Y, Yi R, Cullen BR. 2003. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proceedings of the National Academy of Sciences of the United States of America 100(17):9779-9784.

Zeng Z, Shi YX, Samudio IJ, Wang RY, Ling X, Frolova O, Levis M, Rubin JB, Negrin RR, Estey EH, Konoplev S, Andreeff M, Konopleva M. 2009. Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML. Blood 113(24):6215-6224.

Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, Lodish HF. 2006a. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nature medicine 12(2):240-245.

Zhang CC, Lodish HF. 2005. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood 105(11):4314-4320.

Zhang J, Grindley JC, Yin T, Jayasinghe S, He XC, Ross JT, Haug JS, Rupp D, Porter-Westpfahl KS, Wiedemann LM, Wu H, Li L. 2006b. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature 441(7092):518-522.

Zhang J, Niu C, Ye L, Huang H, He X, Tong WG, Ross J, Haug J, Johnson T, Feng JQ, Harris S, Wiedemann LM, Mishina Y, Li L. 2003. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 425(6960):836-841.

Zhang L, Ming L, Yu J. 2007. BH3 mimetics to improve cancer therapy; mechanisms and examples. Drug Resist Updat 10(6):207-217.

Zhang S, Broxmeyer HE. 1999. p85 subunit of PI3 kinase does not bind to human Flt3 receptor, but associates with SHP2, SHIP, and a tyrosine-phosphorylated 100-kDa protein in Flt3 ligand-stimulated hematopoietic cells. Biochemical and biophysical research communications 254(2):440-445.

Zhang S, Fukuda S, Lee Y, Hangoc G, Cooper S, Spolski R, Leonard WJ, Broxmeyer HE. 2000. Essential role of signal transducer and activator of transcription (Stat)5a but not

REFERENCES

93

Stat5b for Flt3-dependent signaling. The Journal of experimental medicine 192(5):719-728.

Zhang W, Konopleva M, Ruvolo VR, McQueen T, Evans RL, Bornmann WG, McCubrey J, Cortes J, Andreeff M. 2008. Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway. Leukemia 22(4):808-818.

Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP. 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nature medicine 7(9):1028-1034.

Zong WX, Lindsten T, Ross AJ, MacGregor GR, Thompson CB. 2001. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes & development 15(12):1481-1486.

Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90(3):405-413.

Zou H, Li Y, Liu X, Wang X. 1999. An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. The Journal of biological chemistry 274(17):11549-11556.

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