Sini Pirnes-Karhu

Spermidine/spermine N 1-acetyltransferase in Mouse Hematopoiesis and Bone Remodeling and in Human Leukemias

Publications of the University of Eastern Finland Dissertations in Health Sciences

SINI PIRNES-KARHU

Spermidine/spermine N1-acetyltransferase in Mouse Hematopoiesis and Bone Remodeling and in Human Leukemias

To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in Tietoteknia Auditorium (TTA), Kuopio, on Saturday, December 14th 2013, at 1 pm

Publications of the University of Eastern Finland Dissertations in Health Sciences Number 207

Department of Biotechnology and Molecular Medicine A.I.Virtanen Institute for Molecular Sciences Faculty of Health Sciences University of Eastern Finland Tampere 2013

Juvenes Print – Suomen Yliopistopaino Oy Tampere, 2013 Series Editors: Professor Veli-Matti Kosma, M.D., Ph.D. Institute of Clinical Medicine, Pathology Faculty of Health Sciences Professor Hannele Turunen, Ph.D. Department of Nursing Science Faculty of Health Sciences Professor Olli Gröhn, Ph.D. A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences Professor Kai Kaarniranta, M.D., Ph.D. Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy Faculty of Health Sciences Distributor: University of Eastern Finland Kuopio Campus Library P.O.Box 1627 FI-70211 Kuopio, Finland http://www.uef.fi/kirjasto ISBN 978-952-61-1320-3 (print) ISBN 978-952-61-1321-0 (pdf) ISSN 1798-5706 (print) ISSN 1798-5714 (pdf) ISSN-L 1798-5706

III Author’s address:

Biotechnology and Molecular Medicine A.I.Virtanen Institute University of Eastern Finland KUOPIO FINLAND e-mail: [email protected]

Supervisors:

Professor Leena Alhonen, Ph.D. A.I.Virtanen Istitute University of Eastern Finland KUOPIO FINLAND Anne Uimari, Ph.D. A.I.Virtanen Institute University of Eastern Finland KUOPIO FINLAND

Reviewers:

Docent Jarmo Wahlfors, Ph.D. Research Council for Health Academy of Finland HELSINKI FINLAND Docent Juhani Sand, Ph.D. Division 2 Tampere University Hospital TAMPERE FINLAND

Opponent:

Professor Leif C. Andersson, MD, Ph.D. Haartman institute University of Helsinki HELSINKI FINLAND

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V Pirnes-Karhu, Sini. Spermidine/spermine N1-acetyltransferase in Mouse Hematopoiesis and Bone Remodeling and in Human Leukemias. University of Eastern Finland, Faculty of Health Sciences Publications of the University of Eastern Finland. Dissertations in Health Sciences 207. 2013. 56 p.

ISBN 978-952-61-1320-3 (print) ISBN 978-952-61-1321-0 (pdf) ISSN 1798-5706 (print) ISSN 1798-5714 (pdf) ISSN-L 1798-5706

ABSTRACT Polyamines are ubiquitous molecules essential for cell proliferation. Induction of the catabolic enzyme of polyamines, spermidine/spermine N1-acetyltransferase (SSAT), is successfully used to limit cancer cell growth in cell culture. In animal models, however, SSAT overexpression has been shown to increase susceptibility to cancer. In the present study, the role of enhanced polyamine catabolism in homeostasis of hematopoietic and bone systems were investigated in mice overexpressing SSAT (SSAT mice) and its association to hematopoietic malignancies was studied with patient samples. The basal levels of serum and hepatic inflammatory markers of SSAT mice were comparable to those encountered in wild-type littermates. When exposed to endotoxin SSAT mice displayed a slightly enhanced anti-inflammatory response but the survival and symptoms of the SSAT mice were similar to those of wild-type mice. Further studies of cells and tissues of the immune system revealed that the hematopoietic phenotype of SSAT mice fulfilled the criteria of mouse myeloproliferative disease. The myeloproliferation of SSAT mice resulted from both the intrinsic SSAT overexpression of hematopoietic cells and microenvironmental factors. Characterization of the bone phenotype of SSAT mice showed altered structure of bones of SSAT mice to provide larger niche for hematopoietic cells. SSAT mice also showed cell-autonomously impaired osteoblastogenesis and increased number of premature osteoblasts. The association between SSAT activity and myeloproliferation was supported by findings from human leukemia patients. The SSAT activity in peripheral blood mononuclear cells of these patients was associated with the leukocyte number in myeloid leukemias but not in lymphoid leukemia. Further investigation of SSAT mice provided evidence that the association between SSAT and myeloproliferation might be mediated through epigenetic factors. Our results show that enhanced SSAT activity leads to aberrant bone formation and subsequent changes in bone marrow microenvironment. In conjunction with enhanced intrinsic SSAT activity of hematopoietic cells, this results in a myeloproliferative disease in SSAT mice. The role of SSAT activity in myelopoiesis is further supported by the association between SSAT activity and the white blood cell count in human myeloid leukemias. National Library of Medicine Classification: QU 141, QU 475, WE 200, WH 140, WH 250, WH 380 Medical Subject Headings: Acetyltransferases/metabolism; Bone Marrow; Epigenesis, Genetic; Hematopoiesis; Human; Leukemia, Myeloid; Mice; Myeloproliferative Disorders; Osteogenesis; Oxidative stress; Polyamines

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VII Pirnes-Karhu Sini. Spermidiini/spermiini-N1-asetyylitransferaasi hiirten veren- ja luunmuodostuksessa sekä ihmisten leukemioissa. Itä-Suomen yliopisto, Terveystieteiden tiedekunta Publications of the University of Eastern Finland. Dissertations in Health Sciences 207. 2013. 56 s.

ISBN 978-952-61-1320-3 (print) ISBN 978-952-61-1321-0 (pdf) ISSN 1798-5706 (print) ISSN 1798-5714 (pdf) ISSN-L 1798-5706

TIIVISTELMÄ Polyamiinit ovat solujen kasvulle välttämättömiä molekyylejä. Niitä hajottavan entsyymin, spermidiini/spermiini-N1-asetyylitransferaasin (SSAT), indusointia on käytetty rajoittamaan viljeltyjen syöpäsolujen kasvua. Eläinmalleissa puolestaan SSAT-geenin yli-ilmentäminen on myös lisännyt alttiutta syövän synnylle. Tässä väitöskirjatutkimuksessa selvitettiin kohonneen polyamiinikatabolian roolia veren- ja luunmuodostuksessa SSAT-geeniä yliilmentävillä hiirillä (SSAT-hiiret) ja sen yhteyttä verisyöpiin potilasnäytteillä. SSAT-hiirten seerumista ja maksasta mitattujen tulehdusmarkkereiden perustasot olivat verrattavia villityypin hiirten markkereiden tasoon. SSAT-hiirten vaste endotoksiinille oli kuitenkin lievästi tulehdusta hillitsevä. Tästä huolimatta eloonjääminen ja oireet olivat verrattavia villityypin hiiren vastaaviin. Immunologisten solujen ja kudosten lisätutkimukset osoittivat että SSAT-hiirten ilmiasu täytti hiirten myeloproliferatiivisen sairauden kriteerit. SSAT-hiirten myeloproliferatio syntyi yhdessä valkosolujen sisäisen SSAT:n yliilmentämisen ja luuytimen mikroympäristön tekijöiden seurauksena. SSAT-hiirten luiden lähempi tarkastelu osoitti niiden muuttuneen rakenteen muodostavan suuremman tilan veren soluille. SSAT-hiirillä havaittiin lisäksi luuta muodostavien solujen (osteoblastien) erilaistumisen olevan ympäristöstä riippumattomasti häiriintynyt ja epäkypsien osteoblastien määrän kasvaneen. SSAT-aktiivisuuden ja myeloproliferaation välillä havaittua yhteyttä vahvisti ihmisen leukemianäytteiden tutkimus. Leukemiapotilaiden perifeerisen veren mononukleaaristen valkosolujen SSAT-aktiivisuus oli yhteydessä valkosolumäärään myeloista mutta ei lymfoista leukemiaa sairastavilla potilailla. Lisätutkimukset SSAT-hiirillä viittasivat epigeneettisten tekijöiden olevan yhteydessä SSAT-geenin ja myeloproliferaation välillä havaittuun suhteeseen. Saamamme tulokset osoittavat kohonneen SSAT-aktiivisuuden johtavan epänormaaliin luunmuodostukseen ja siitä johtuen poikkeavaan luuytimen mikroympäristöön. Yhdessä veren solujen kohonneen sisäisen SSAT-aktiivisuuden kanssa tämä johtaa myeloproliferatiivisen taudin kehittymiseen SSAT-hiirillä. SSAT-aktiivisuuden osuutta myelopoieesissa tukee lisäksi havainto SSAT-aktiivisuuden yhteydestä valkosolumäärään myeloista leukemiaa sairastavilla potilailla.

Yleinen Suomalainen asiasanasto: epigenetiikka; hiiret; ihmiset; immuunijärjestelmä; leukemia; luuydin; luusto; polyamiinit; veri

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Acknowledgements This work was carried out in the A.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, during the years 2009‒2013. I wish to express my sincere gratitude to my principal supervisor Professor Leena Alhonen, PhD, who offered me an interesting project to work with and has shown belief in my work ever since. My warm thanks belong also to my supervisor Anne Uimari, PhD, for her inexhaustible guidance and all the stimulating discussions both in science and in personal life. It has been great deal of fun to work with you! I wish to thank Docent Jarmo Wahlfors, PhD, and Docent Juhani Sand, PhD, the official reviewers of my thesis for their valuable comments to improve the manuscript. My thanks belong also to Ewen Macdonald, PhD, and Sara Wojciechowski, B.Sc., who revised the language of this manuscript. I wish to express my gratitude to all our collaborators whose expertise in their own fields has provided a strong foundation for my studies. I acknowledge Reijo Sironen, MD, PhD, for his rigorous work with my first manuscript. I am very thankful for Docent Esa Jantunen, MD, and Pentti Mäntymaa, MD, for their invaluable expertise in hematopoiesis and hematopoietic malignancies as well as for Jorma Määttä, PhD, and Mikko Finnilä, MSc, for their enthusiasm, work and advice with bone-related studies. I am grateful for Petri Mäkinen, PhD, and Sara Wojciechowski for their kind and knowledgeable assistance with flow cytometry. I wish to thank also Sohvi Hörkkö, MD, PhD, and Satu Mustjoki, MD, PhD, for their scientific effort on my second and third manuscripts. My warm thanks belong to all the present and former members of Leena’s laboratory with whom I have had the pleasure to work. Thank you Tuomo Keinänen, PhD, Mervi Hyvönen, PhD, Marko Pietilä, PhD, Taina Koponen, MSc, Marc Cerrada-Gimenez, PhD, Eija Pirinen, PhD, Anita Lampinen, MSc, Tekele Fashe, MSc, Susanna Vuohelainen, MSc, Maija Tusa, Lic.Phil., for helping me with my projects and making the working environment so pleasant. I wish to direct my special thanks to the technical personnel who participated in this study. Thank you, Anne Karppinen, Arja Korhonen, Marita Heikkinen, Sisko Juutinen and Tuula Reponen, for your indefatigable and skillful help in the laboratory and keeping the lab functional with all the practical essentials. In addition, I would like to acknowledge Eeva Hakala, Jouko Mäkäräinen, Riitta Sinervirta, Helena Pernu and all the personnel of National Laboratory Animal Center, especially Arja Konttinen, Teija Oinonen and Virve Immonen, for efficiently managing the practical details. My loving thanks belong to my family, especially to my husband Tero for making everyday life so much fun. I also admire your calm response to stressful situations and I am grateful for how you are able to make me calmer too. I am thankful for my parents, Maija and Veijo, for encouraging me to pursue a quality education and follow my own path. I am grateful for my beloved sisters Suvi, Säde and Sara and “brother” Teemu and my friends both inside and outside of the scientific world, Ansku, Hanna-Maija and Eini, for sharing the ups and downs of my work and personal life. I feel so privileged to have you all in my life! Lahti, November 2013

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List of the original publications

This dissertation is based on the following original publications:

I

Pirnes-Karhu S, Sironen R, Alhonen L and Uimari A. Lipopolysaccharideinduced anti-inflammatory acute phase response is enhanced in spermidine/spermine N1-acetyltransferase (SSAT) overexpressing mice. Amino Acids 42: 473-484, 2012.

II

Pirnes-Karhu S, Mäntymaa P, Sironen R, Mäkinen PI, Wojciechowski S, Juutinen S, Koistinaho J, Hörkkö S, Jantunen E, Alhonen L and Uimari A. Enhanced polyamine catabolism disturbs hematopoietic lineage commitment and leads to a myeloproliferative disease in mice overexpressing spermidine/spermine N1acetyltransferase. Amino Acids Epub Jul 9, 2013.

III

Pirnes-Karhu S, Jantunen E, Mäntymaa P, Mustjoki S, Alhonen L and Uimari A. Spermidine/spermine N1-acetyltransferase activity associates with white blood cell count in myeloid leukemias. Manuscript.

IV

Pirnes-Karhu S, Määttä J, Finnilä M, Alhonen L and Uimari A. Overexpression of Spermidine/spermine N1-acetyltransferase Impairs Osteoblastogenesis and Alters Mouse Bone Phenotype. Manuscript.

The following papers are referred to in the text by their Roman numeral. The publications were adapted with the permission of the copyright owners.

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Contents 1

INTRODUCTION ...................................................................................................................... 1

2

REVIEW OF THE LITERATURE ............................................................................................. 3

2.1

POLYAMINE METABOLISM ............................................................................................................................ 3

2.1.1

Biosynthesis of polyamines............................................................................................................................. 4

2.1.1.1

Ornithine decarboxylase ......................................................................................................................... 4

2.1.1.2

S-adenosylmethionine decarboxylase................................................................................................... 5

2.1.1.3

Spermidine and spermine synthases .................................................................................................... 5

2.1.2

Polyamine catabolism ...................................................................................................................................... 5

2.1.2.1

Spermidine/spermine N1-acetyltransferase.......................................................................................... 6

2.1.2.2

Acetylpolyamine oxidase and spermine oxidase ................................................................................ 6

2.1.3

Polyamine transport ........................................................................................................................................ 7

2.1.4

Cellular functions of polyamines and SSAT ................................................................................................. 9

2.1.5

Polyamine catabolism in disease .................................................................................................................. 10

2.1.6

Polyamine metabolism in hematopoiesis ................................................................................................... 11

2.1.7

Polyamine metabolism in bone remodeling ............................................................................................... 11

2.2

HEMATOPOIESIS .............................................................................................................................................. 11

2.2.1

Hematopoietic stem cells .............................................................................................................................. 13

2.2.2

Maintenance of self-renewal capacity of hematopoietic stem cells ......................................................... 13

2.2.3

Lineage commitment of hematopoietic cells .............................................................................................. 14

2.2.4

Regulation of myeloid and megakaryocyte-erythroid lineage commitment ......................................... 16

2.2.5

Aging of the hematopoietic system ............................................................................................................. 16

2.2.6 2.3

Bone marrow microenvironment ................................................................................................................. 17 BONE STRUCTURE, FUNCTION AND DEVELOPMENT ......................................................................... 18

2.3.1

Bone remodeling ............................................................................................................................................ 18

2.3.2

Osteoblasts and bone formation................................................................................................................... 19

2.3.3

Osteoclasts and bone resorption .................................................................................................................. 20

3

AIMS OF THE STUDY ............................................................................................................ 21

4

MATERIALS AND METHODS ............................................................................................ 23

4.1

PATIENT SAMPLES .......................................................................................................................................... 23

4.2

MICE .................................................................................................................................................................... 23

4.3

ISOLATION OF BONE MARROW CELLS FROM MICE............................................................................. 23

4.4

EXPANSION OF FACS-SORTED LSK CELLS ............................................................................................... 24

4.5

DIFFERENTIATION OF MESENCHYMAL STROMAL CELLS TO OSTEOBLASTS .............................. 24

4.6

DIFFERENTIATION OF HEMATOPOIETIC CELLS TO OSTEOCLASTS ................................................ 24

4.7

STATISTICAL ANALYSES ............................................................................................................................... 24

4.8

ANALYTICAL METHODS ............................................................................................................................... 24

5

RESULTS .................................................................................................................................... 27

5.1

EFFECTS OF ACTIVATED POLYAMINE CATABOLISM IN IMMUNE RESPONSE (I) ........................ 27

5.2

EFFECTS OF ACTIVATED POLYAMINE CATABOLISM ON HEMATOPOISIS IN MICE (II) ............ 28

5.2.1

Polyamine cycle is enhanced in hematopoietic cells of SSAT mice ......................................................... 28

5.2.2

SSAT mice exhibit myeloproliferative phenotype ..................................................................................... 28

5.2.3

The myeloproliferative phenotype of SSAT mice results from both the intrinsic factors of bone

marrow cells and the bone marrow microenvironment ........................................................................................ 29

XIV 5.3

SSAT ACTIVITY IN MYELOID AND LYMPHOID LEUKEMIAS AND EPIGENETICS IN

HEMATOPOIETIC CELLS OF SSAT OVEREXPRESSING MICE (III) .................................................................... 29 5.3.1

Polyamine metabolism of human leukemic blood cells is disturbed...................................................... 29

5.3.2

SSAT mice show epigenetic changes in hematopoietic cells and differential response to drugs

affecting epigenetic factors......................................................................................................................................... 29 5.4

EFFECTS OF ACTIVATED POLYAMINE CATABOLISM ON BONE REMODELING (IV) .................. 30

5.4.1

Polyamine metabolism of osteoblasts and osteoclasts is disturbed ........................................................ 30

5.4.2

SSAT overexpression affects osteoblastogenesis but not osteoclastogenesis cell-autonomously ....... 30

5.4.3

Induction of SSAT with α-methylspermidine disturbs wild-type osteoblastogenesis ......................... 31

5.4.4

Bone phenotype of SSAT mice reveals striking changes .......................................................................... 31

6

DISCUSSION ............................................................................................................................ 33

7

SUMMARY AND CONCLUSIONS ..................................................................................... 39

8

REFERENCES ............................................................................................................................ 41

ORIGINAL PUBLICATIONS I─IV .............................................................................................. 57

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Abbreviations AdoMet

S-adenosylmethionine

AGP

alpha-1-acid glycoprotein

ALAT

alanine aminotransferase

ALL

acute lymphoid leukemia

ALP

alkaline phosphatase

HAT

histone acetyltransferase

AML

acute myeloid leukemia

HDAC

histone deacetylase

APAO

acetylpolyamine oxidase

HMT

histone methyltransferase

AZ

antizyme

HSC

hematopoietic stem cell

AZI

antizyme inhibitor

IL-1β

interleukin-1beta

BMD

bone mass density

IL-6

interleukin-6

B.Pm

bone perimeter

IL-10

interleukin-10

BSP

bone sialoprotein

INF-γ

interferon gamma

BV

bone volume

LMPP

lymphoid-primed

C/EBP

CCAAT-enhancer-binding

CLP

GM-CSF

granulocyte-macrophage colony-stimulating factor

GMP

granulocyte-macrophage progenitors

multipotent progenitor

protein

LPS

lipopolysaccharide

common lymphoid

LSK

lineage-negative Sca-1+ c-Kit+

progenitor

cells

CML

chronic myeloid leukemia

LT-HSC

CMP

common myeloid progenitors

CRP

C-reactive protein

Cs.Th

cross-sectional thickness

CTSK

cathepsin K

MPP

multipotent progenitor

CTX

carboxy-terminal cross-

MSC

mesenchymal stromal cell

linking telopeptide

MTA

5’-deoxy-5’-

cell MEP

megakaryocyte-erythrocyte progenitors

dcAdoMet decarboxylated Sadenosylmethionine

long-term hematopoietic stem

methylthioadenosine MT-SSAT

spermidine/spermine N1-

DFMO

difluoromethylornithine

acetyltransferase under

DNMT

DNA methyltransferases

metallothionein promoter

XVI

NFATc1

nuclear factor of activated T-

SMI

structural model index

cells cytoplasmic 1

SMO

spermine oxidase

NF-κB

nuclear factor kappa-B

SRS

Snyder-Robinson syndrome

OC

osteocalcin

SSAT

spermidine/spermine N1-

ODC

ornithine decarboxylase

OPN

osteopontin

OSX

osterix

PBMC

peripheral blood

acetyltransferase SSATKO

spermidine/spermine N1acetyltransferase knockout

ST-HSC

mononuclear cell

short-term hematopoietic stem cell

PINP

procollagen type I propeptide

Tb.Th

trabecular thickness

Po.V

total volume of porosity

Tb.N

trabecular number

RUNX2

runt-related transcription

TNF-α

tumor necrosis factor alpha

factor 2

TPO

thrombopoietin

ROS

reactive oxygen species

TRAcP

tartrate-resistant acid

SAA

serum amyloid A

SAMDC

S-adenosylmethionine

TV

tissue volume

decarboxylase

WBC

white blood cell

SCF

stem cell factor

phosphatase

1 Introduction Cancers in general result from disturbances of several normal cellular functions at the same time. Tissue homeostasis is disrupted also during the normal aging process due to alterations occurring at the molecular level and these changes can predispose to cancer development. In addition to cell-autonomous factors, the surrounding stroma may contribute to the malignancy of neoplasias. Thus, elucidation of the function of factors predisposing to cancer represents one way to prevent cancer development. All blood cells, including platelets, red blood cells and white blood cells, are derived from multi-potent hematopoietic stem cells. Platelets, which originate from megakaryocytes, are involved in blood coagulation process whereas the function of red blood cells is to bind oxygen and deliver it to tissues. White blood cells, further subdivided into myeloid and lymphoid lineages, are responsible for the immune defense of body. In the hematopoietic system the disruption of tissue homeostasis with aging leads not only to an increased propensity to anemia but also to the attenuation of lymphoid lineage and further to dominance by myeloid lineages (Woolthuis et al. 2011). The domination of myeloid lineages is connected to the increased incidence of myeloid leukemias observed in the elderly. In addition to cell-intrinsic factors, recently much attention has been focused on the role of the micro-environment in the development of hematological malignancies (Askmyr et al. 2011; Carlesso and Cardoso 2010). Bone is a connective tissue consisting of cellular components as well as of an organic and inorganic matrix. Osteoblasts, osteoclasts and osteocytes are the cellular components of bone. Bone serves a variety of functions e.g. providing structural support and protection for internal organs as well as a suitable environment where hematopoiesis can take place. Bone is constantly being renewed by a process termed remodeling (Eriksen 2010; Kular et al. 2012). This is carried out by bone-resorbing osteoclasts and bone-forming osteoblasts. The osteoclasts break down the old or damaged bone and osteoblasts re-fill the gap in the bone matrix. In healthy young bone the process of bone resorption and bone formation are tightly coupled to maintain homeostasis and overall bone mass. Thus, a defect in either of the events can evoke an imbalance in bone structure, leading to a disease state. Polyamines are small cationic molecules which have been conserved during the course of evolution. Due to their cationic nature, polyamines interact with negatively charged macromolecules (Igarashi and Kashiwagi 2010; Watanabe et al. 1991). Through these interactions, polyamines are involved in many cellular functions, with the best known being their function in cell proliferation. Polyamines have been linked also to neoplastic growth as biosynthesis of polyamines is enhanced in cancer cells as compared to healthy tissue (Wallace et al. 2003). In cell cultures, the induction of the activity of the catabolic enzyme of polyamines, spermidine/spermine N1-acetyltransferase (SSAT), has been successfully used to limit cancer cell growth (Kee et al. 2004b; Vujcic et al. 2000). In animal models, however, also opposite results are found; SSAT overexpression has been reported to enhance the animal’s susceptibility to skin and intestinal cancer (Coleman et al. 2002;

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Tucker et al. 2005). Additionally, human breast cancer patients have been shown to exhibit increased SSAT activity in their tumor cells compared to healthy tissue (Wallace et al. 2000). This study aimed to reveal the role of disturbed polyamine metabolism in tissue homeostasis of the hematopoietic system. Thereby, polyamine levels and activity of their metabolic enzymes were measured from leukemia patient samples. Furthermore, the immunological and hematopoietic phenotypes of SSAT overexpressing mice were examined. These mice were shown to develop a myeloproliferative disease with the bone marrow microenvironment partly contributing to its development. Thus, in order to clarify how the microenvironmental factors could affect hematopoiesis the bone remodeling status of SSAT mice were also characterized.

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2 Review of the literature 2.1 POLYAMINE METABOLISM Polyamines are amino acid-derived aliphatic molecules found in all eukaryotes and nearly all prokaryotes (Cohen 1998). The three polyamines present in mammalian cells, putrescine, spermidine and spermine, contain two to four amino groups and at physiological pH they are fully protonated and possess cationic characteristics (Figure 1). Unlike the divalent inorganic cations, like Ca2+, the positive charge in polyamines is distributed along the carbon backbone. Thus, polyamines bind readily to negatively charged macromolecules such as DNA, RNA and proteins and therefore the proportion of free cellular polyamines is small (Igarashi and Kashiwagi 2010; Watanabe et al. 1991). Spermidine and spermine interact more strongly with macromolecules than putrescine. The ratio of spermidine and spermine is claimed to be important for cellular functions (Pegg and Michael 2010). The cellular content of spermine equals or exceeds that of spermidine only in animal tissues whereas not all prokaryotic species contain spermine at all. Putrescine is often considered as the precursor molecule and spermine as a reservoir molecule for spermidine. However, both of these molecules have been shown to possess their own individual functions. In view of their ability to form ionic bonds with other molecules and, additionally, to serve as precursors for other molecules, polyamines are involved in many cellular functions, such as transcription, translation and cell signaling. As a result of their important role in these functions, it is recognized that polyamines are essential for normal cell growth and development of organisms (Nishimura et al. 2002; Pendeville et al. 2001). The cellular polyamine content and the activity of their biosynthetic enzymes increase rapidly when proliferation is induced (Wallace 2009). The depletion of the cellular polyamine content triggers growth arrest by reducing synthesis of nucleic acids and proteins whereas high concentrations of polyamines may be toxic to cells (Davis et al. 1992; Persson 2009). For this reason, cellular concentrations of polyamines are very tightly regulated. In addition to their requirement in cell proliferation, the conservation of polyamines across evolution is also evidence emphasizing biological significance of these molecules (Wallace et al. 2003).

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Figure 1 Mammalian polyamines, putrescine, spermidine and spermine. Polyamines are small aliphatic molecules with two to four cationic amino groups. Positively charged amino groups enable interaction with negatively charged macromolecules such as DNA and RNA.

2.1.1 Biosynthesis of polyamines The major source of polyamines in mammals is via de novo biosynthesis, whereas dietary polyamines and polyamine production by gut microflora make a smaller contribution (Wallace 2009). The biosynthesis of polyamines uses two amino acids, methionine and arginine, as precursors (Cohen 1998). Methionine is converted to S-adenosylmethionine (AdoMet) and L-arginine to L-ornithine and both are then further decarboxylated by Sadenosylmethionine decarboxylase (SAMDC) and ornithine decarboxylase (ODC), respectively. The decarboxylation of L-ornithine results in the formation of the diamine putrescine. Spermidine is synthesized from putrescine, and spermine further from spermidine, by transfer of aminopropyl group which is provided by decarboxylated Sadenosylmethionine. The enzymes which catalyze the transfer are spermidine synthase and spermine synthase. The metabolic pathway of polyamines is presented in Figure 2. 2.1.1.1 Ornithine decarboxylase Ornithine decarboxylase (ODC) is a pyridoxal phosphate -dependent enzyme which catalyzes the decarboxylation of L-ornithine to form the diamine putrescine. ODC is one of the two rate-limiting enzymes of polyamine biosynthesis. The knockout of the gene and subsequent loss of intracellular polyamine pools is lethal in mice (Pendeville et al. 2001). ODC functions as a homodimer with two active sites (Coleman et al. 1994). The association between the two ODC subunits is relatively weak and the dimers exist in rapid equilibrium with the inactive monomers. ODC has a very rapid turnover rate for a mammalian enzyme. The depletion of cellular polyamine pools stabilizes the enzyme whereas in excess of polyamines ODC is directed to degradation. The degradation is brought about by 26S proteasome but instead of ubiquitination ODC associates with protein termed antizyme (AZ) that directs it to proteosomal degradation. AZ is synthesized and its degradation is

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inhibited in response to an increase in cellular polyamine content. It binds the ODC monomer preventing formation of dimer and, thus, it inhibits enzymatic activity. On the other hand, AZ activity is regulated by antizyme inhibitor (AZI) which binds AZ more tightly than ODC and thereby releases ODC from ODC-AZ complex (Murakami et al. 1996). ODC protein levels are controlled also by high cellular polyamine content through translational regulation. 2.1.1.2 S-adenosylmethionine decarboxylase The second rate-limiting enzyme of biosynthesis of polyamines is S-adenosylmethionine decarboxylase (SAMDC) (Nishimura et al. 2002). Inactivation of mouse SAMDC gene and the subsequent loss of intracellular polyamine pools is lethal at early stage of development. SAMDC is synthesized as proenzyme which after intramolecular cleavage reactions forms α and β subunits (Pegg 2009). In mammals, SAMDC functions as a tetramer of two pairs of α and β subunits and, additionally, it contains a covalently bound prosthetic group, pyruvate. SAMDC catalyzes the decarboxylation of AdoMet producing an aminopropyl donor for synthesis of higher polyamines (Pegg et al. 1998). Decarboxylated AdoMet (dcAdoMet) is required in polyamine biosynthesis whereas the non-decarboxylated form of AdoMet is needed as a methyl group donor in methyl transfer reactions. The decarboxylated form is not suitable for this purpose. For this reason, the steady-state level of dcAdoMet is kept very low and SAMDC activity is highly regulated by polyamines at the transcriptional, translational and protein levels. Putrescine enhances the proenzyme processing and catalytic activity of SAMDC whereas high concentrations of spermidine and spermine suppress transcription and translation and accelerate turn-over rate of SAMDC (Kameji and Pegg 1987; Pegg 2009). SAMDC is degraded by 26S proteasome after polyubiquitination. 2.1.1.3 Spermidine and spermine synthases Spermidine and spermine synthases are aminopropyl transferases that function as homodimers and use dcAdoMet as a donor of aminopropyl group (Ikeguchi et al. 2006; Pegg and Michael 2010; Wu et al. 2007). Mammalian aminopropyltransferases are highly specific for their acceptor molecules, putrescine and spermidine, from which spermidine and spermine are formed, respectively. The by-product of the process, 5’-deoxy-5’methylthioadenosine (MTA), strongly inhibits both spermidine and spermine synthase (Ikeguchi et al. 2006; Wu et al. 2008). However, MTA is normally rapidly degraded to adenosine and methionine and, thus, does not have a great inhibitory effect in vivo. Spermidine and spermine synthases are expressed constitutively and, unlike ODC and SAMDC, not readily induced (Ikeguchi et al. 2006; Pegg and Michael 2010). Rather their enzymatic activity is limited by the availability of their common substrate, dcAdoMet. 2.1.2 Polyamine catabolism Spermine is converted to spermidine and spermidine to putrescine by the concerted action of two enzymes, spermidine/spermine N1-acetyltransferase (SSAT) and acetylpolyamine oxidase (APAO) (Casero and Pegg 1993). SSAT adds acetyl groups to aminopropyl end(s) of spermidine and spermine. The acetylated forms are then either exported or oxidized by APAO which cleaves the molecules to generate N-acetylaminopropanal and the smaller polyamine. Additionally, spermine can be converted directly to spermidine by spermine

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oxidase (SMO) (Vujcic et al. 2002). Due to its ability to directly convert spermine to spermidine, SMO can be considered as a way to recycle the higher polyamines (Wallace 2009). The spermidine/spermine N1-acetyltransferase (SSAT) pathway, on the other hand, can be considered to be mainly utilized for depletion of the polyamines as acetylpolyamines produced by SSAT are readily excreted. For this reason, acetylpolyamines are only rarely found in normal cells, however, these molecules can be found in high concentrations in cancer cells (Kingsnorth and Wallace 1985). Furthermore, acetylation of polyamines reduces their charge and thus decreases their functional ability and potency to bind anionic molecules. High levels of SSAT have been shown to increase polyamine flux (Janne et al. 2006; Kee et al. 2004b). The activity of SSAT decreases polyamine levels and to compensate the polyamine depletion ODC and SAMDC are induced. The increased flux increases consumption of ATP and acetyl-CoA in addition to increased production of toxic compounds such as H2O2. 2.1.2.1 Spermidine/spermine N1-acetyltransferase Spermidine/spermine N1-acetyltransferase is considered as a key catabolic enzyme of polyamines (Pegg 2008). SSAT is a part of the GCN5-related N-acetyltransferase (GNAT) family. It is primarily a cytosolic enzyme, although there is evidence of its localization in mitochondria and nucleus as well (Holst et al. 2008; Uimari et al. 2009). SSAT is active as a homodimer. Using acetyl-CoA as a donor for acetyl group SSAT is able to acetylate N1position of spermidine and either end of spermine as spermine is a symmetrical molecule (Matsui and Pegg 1981). In addition, other molecules with the aminopropyl structure are excellent substrates for SSAT, whereas for example, putrescine with its aminobutyl group does not (Della Ragione and Pegg 1983). SSAT is stringently regulated by polyamines and other factors at many levels, e.g. transcription, mRNA processing, translation and protein stability (Casero and Pegg 2009; Pegg 2008). Polyamine analogues, several molecules (growth factors, toxic compounds, drugs) and various pathophysiological stimuli affect SSAT either directly or indirectly through the regulation of polyamine levels. In the presence of high levels of polyamines, transcription and translation of SSAT are increased whereas degradation of the protein and incorrect splicing of the mRNA are reduced (Pegg 2008). Cellular SSAT activity is very low under resting conditions but it can be readily induced by several factors and the stability of the otherwise short-lived protein can be enhanced (Matsui and Pegg 1981; Persson and Pegg 1984). When polyamines and polyamine analogues bind the SSAT protein, its half-life is greatly prolonged since this prevents the ubiquitination and subsequent degradation by the 26S proteasome (Coleman and Pegg 1997). Many polyamine analogs have strong effects on the regulation of SSAT and as they are not substrates for SSAT, their content is not reduced in response to its induction (Pegg 2008). 2.1.2.2 Acetylpolyamine oxidase and spermine oxidase APAO is a peroxisomal FAD-dependent enzyme in the polyamine catabolic pathway catalyzing the turn-over of N1-acetylated polyamines back to spermidine or putrescine (Casero and Pegg 2009). Spermine can be converted back to spermidine also directly, without the acetylation step, through the action of SMO (Vujcic et al. 2002). Both reactions, oxidation by APAO or SMO, produce highly toxic by-products, hydrogen peroxide (H2O2)

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and 3-acetoamidopropanal or 3-aminopropanal, respectively. APAO is located in peroxisomes and this means that any oxidizing compounds produced by APAO activity may be immediately detoxified by peroxisomal catalase. SMO, however, is located in the cytosol and nucleus. Thus, the reactive oxygen species (ROS) produced by its activity can be potentially more harmful to the cell (Murray-Stewart et al. 2008). APAO is constitutively expressed and its activity is limited by the availability of its substrates, the acetylated forms of polyamines, which are produced by action of SSAT (Holtta 1977; Vujcic et al. 2003). APAO has very poor ability to oxidize non-acetylated polyamines. SMO, on the other hand, is highly inducible and all of the analogues inducing SSAT also induce SMO expression (Casero and Pegg 2009). APAO activity has been shown to be low in breast cancer patients in comparison to healthy controls, thus partly contributing to the accumulation of cellular acetylated polyamines (Kingsnorth and Wallace 1985; Wallace et al. 2000). 2.1.3 Polyamine transport In addition to biosynthesis and catabolism, polyamine uptake and export are important pathways in the regulation of intracellular polyamine content. If polyamine synthesis is prevented, then the transport system becomes critical for normal cellular growth. The polyamine transporters have been well documented in prokaryotes but the transport mechanisms in mammalian cells are not well characterized and no mammalian transporter genes have been cloned yet. Food contains large quantities of polyamines which are absorbed from the intestine and can be transported for use by cells throughout the body (Bardocz 1993). Additionally intestinal bacteria produce and excrete polyamines which can be taken up and utilized (Wallace et al. 2003). Due to the positive charge of polyamines, they require a transport system to permit exogenous uptake. Thus, cells take up polyamines from the environment through an active, energy-requiring, transport system (Seiler et al. 1996). Two transport systems for uptake of polyamines have been recognized: a sodiumdependent form that has a preference for putrescine (although it can transport also spermidine and spermine) and a sodium-independent form that is capable of transporting spermidine and spermine. Additionally, there has been postulated to be an endocytosismediated mechanism for polyamine transport (Soulet et al. 2002). The uptake and export of polyamines seem to be mediated by different transporters since polyamine uptake-deficient mutant cells are able to excrete polyamines (Hyvonen et al. 1994). However, both systems are regulated by the same factor, AZ, which also functions as an ODC inhibitor (Sakata et al. 2000). The main polyamines to be exported are putrescine and acetylated polyamines (Seiler et al. 1996). Acetylated polyamines do not function as substrates for the polyamine uptake system and thus once exported, they are not reaccumulated into cells (Byers and Pegg 1989). Exported acetylpolyamines are transported to the kidneys and excreted in urine. In many cancers, it is known that there are elevated urinary acetylpolyamine levels (Seiler et al. 1981).

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Figure 2 Mammalian polyamine metabolism. Polyamine synthesis uses amino acids as precursors. The polyamine metabolism can be considered as a cycle (red arrows). The higher polyamines are produced from the smaller forms (spermidine from putrescine and spermine from spermidine) by the action of biosynthetic enzymes, ODC, SAMDC, SPDSy, SPMSy, and can again be converted back to the smaller forms by catabolic enzymes SMO, SSAT, APAO. The polyamine cycle consumes metabolites such as Ac-Co and ATP and produces toxic compounds (ROS and reactive aldehydes). Ac-coA, acetyl-coenzyme A; APAO, acetylpolyamineoxidase; ARG, arginine; ATP, adenosine triphosphate; AZ, antizyme; dcAdoMet, decarboxylated Sadenosylmethionine; MAT, methionine adenosyltransferase; MTA, methylthioadenosine; ODC, ornithine decarboxylase; ROS, reactive oxygen species; S-AdoMet, S-adenosylmethionine; SAMDC, S-adenosylmethionine decarboxylase; SMO, spermine oxidase; SPDSy, spermidine synthase; SPMSy, spermine synthase; SSAT, spermidine/spermine N1-acetyltransferase

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2.1.4 Cellular functions of polyamines and SSAT Polyamines can stabilize DNA and protect it from damage that is caused by for example oxidation and radiation (Ha et al. 1998b; Newton et al. 1996; TABOR 1962). Additionally, among the polyamines, spermine has been reputed to exert protective roles also during inflammation as it inhibits post-transcriptionally pro-inflammatory cytokine production in human mononuclear cells (Ha et al. 1998a; Zhang et al. 1997b). Moreover, spermine has been shown to reduce carrageenan-induced edema and partially protect from cecal ligation and puncture -induced sepsis in mice (Zhang et al. 1997b; Zhu et al. 2009). Polyamines modify gene activation and gene silencing by modulating the transition of DNA from the Bto the Z-conformation, inducing chromosome condensation by neutralizing the charges on histone and chromatin phosphate and by regulating activity of the histone acetyltransferases (Fredericq et al. 1991; Hougaard et al. 1987; Thomas and Messner 1986). Polyamines are known to be able to influence protein synthesis at various stages by changing the structure of mRNA, tRNA and rRNA [reviewed in (Igarashi and Kashiwagi 2010)]. Furthermore, polyamines can change protein conformation by undergoing interactions with acidic protein structures thereby disturbing their functions. Polyamines regulate also membrane potential of cells by interacting with glutamate receptors and ion channels (Donevan and Rogawski 1995; Williams 1997). The best recognized function of polyamines is their role in cell proliferation. Similar to the content of known cell cycle regulators, cyclins and cyclin-dependent kinases, also polyamine concentration and activities of polyamine metabolic enzymes fluctuate during the cell cycle (Oredsson 2003). The activity of ODC, and subsequently the polyamine levels, peak at G1 and G2 phases whereas AZ and SSAT expressions are up-regulated during the G2/M phase. The dramatic changes in putrescine concentration during cell cycle suggest that this divalent polyamine directly drives cells through G1 restriction point to S phase (Bettuzzi et al. 1999). Additionally, a specific role of spermidine in eukaryotic cell proliferation is its posttranscriptional protein modification function in the formation of eukaryotic elongation factor 5A, eIF5A. Spermidine serves as the sole precursor of hypusine, a rare amino acid, which is required in the maturation of eIF5A (Park et al. 1981). The maturation of eIF5A is prevented not only by depleted spermidine pools but also by accumulated putrescine (Tome et al. 1997). Polyamines have a bivalent role in the regulation of cellular functions as, in addition to cell proliferation, they can regulate also apoptosis. The studies covering their role in inducing apoptosis are, however, contradictory. The depletion of polyamines has been shown to both induce and prevent apoptosis (Schipper et al. 2000). One can conclude from these studies that it is essential for cell survival to maintain cellular polyamine content within a narrow range. In addition to its central role in polyamine catabolism, the SSAT enzyme also possess other functions. SSAT is known to autoacetylate its own lysine residue (Bewley et al. 2006). Furthermore, SSAT can acetylate the deoxyhypusine/hypusine residue of eIF5A and, thereby, regulate its activity (Lee et al. 2011). It is possible that also other molecules function as substrates for SSAT. A recent study revealed SSAT to have an additional role in repression of expression of other proteins, however, the repression was restricted to exogenous proteins and only transiently transfected SSAT was able to exert any effects (Lee et al. 2010). The mechanism, however, is not yet known but the effect of SSAT action does not seem to depend on acetylpolyamines or on the depletion of polyamines. In addition, SSAT activity and the concomitant depletion of local polyamines is believed to induce

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inward rectifier potassium channel Kir4.2 (Chen et al. 2004; deHart et al. 2008). Furthermore, SSAT binds α9β1 integrin and increased SSAT activity enhances cell migration through colocalization of α9β1 integrin and Kir4.2 (Chen et al. 2004). Furthermore, SSAT is linked to inflammatory responses through tumor-necrosis factor-α (TNF-α) and non-steroidal anti-inflammatory drugs (NSAIDs) which are able to induce SSAT activity (Babbar et al. 2007). Activation of SSAT will lead to polyamine depletion and hence to a reduction in cell growth and triggering of apoptosis. This may be beneficial in conditions of inflammatory stress. 2.1.5 Polyamine catabolism in disease Studies of transgenic animals overexpressing or deficient for SSAT have pointed to a role for SSAT in many disease states where the effects of disturbed polyamine metabolism are mediated differently depending on the diseased tissue. A rapid depletion of spermidine and spermine increases the susceptibility to pancreatitis (Alhonen et al. 2000; Hyvonen et al. 2007). The accumulation of putrescine in hair follicles has evoked hair loss and skin problems in SSAT overexpressing mice (Pietila et al. 1997; Pietila et al. 2001). A similar polyamine profile and skin changes has been reported also in a patient with keratosis follicularis spinulosa decalvans (KSFD) (Gimelli et al. 2002). A lower consumption of acetylCoA as a result of reduced SSAT enzyme activity has caused insulin resistance in SSAT knockout mice (Jell et al. 2007; Niiranen et al. 2006) whereas SSAT overexpressing mice exhibited an increased consumption of acetyl-CoA and ATP and, thus, they represented the opposite phenotype (Pirinen et al. 2007). Increased production of ROS as a consequence of increased polyamine catabolism has been implicated in pathogenesis of ischemia/reperfusion injury in stroke and acute renal tubular necrosis (Tomitori et al. 2005; Zahedi et al. 2010). Since polyamines display such a strong positive correlation with cell growth, it is not surprising that the growth rate of cancer cells is also closely linked to the polyamine content of the cell. An increase in ODC activity and the subsequent increase in cellular polyamine pools have been considered to be an early event in several cancer cell types (Kingsnorth et al. 1984a; Kingsnorth et al. 1984b). It might be anticipated that inhibition of biosynthesis or enhancing the catabolism of polyamines would impair neoplastic growth. Decreased tumor incidence was indeed found in a tumorigenesis initiation study with mice overexpressing SSAT (Pietila et al. 2001). Furthermore, crossbreeding SSAT overexpressing mice with TRAMP mice, which develop prostate tumors reduced the incidence of tumors (Kee et al. 2004a). However, SSAT overexpression has been linked also to increased susceptibility to tumor growth. The tumor cells of breast cancer patients have increased SSAT activity and decreased APAO activity in comparison to normal tissue (Wallace et al. 2000). The mouse model of K6/SSAT mice overexpressing SSAT only in skin exhibited increased tumor incidence in response to a two-stage tumorigenesis initiation protocol (Coleman et al. 2002). In a study where APCmin mice were crossed with SSAT overexpressing mice, the SSAT overexpression was associated with a susceptibility towards intestinal tumor growth (Tucker et al. 2005). However, in such a situation the effect of SSAT seemed to be indirect since prolonged exposure of intestine to increased levels of bile acid is known to predispose to carcinogenesis (Reddy et al. 1977) and the SSAT mice have been shown to suffer from an increased excretion of bile acids (Pirinen et al. 2010).

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2.1.6 Polyamine metabolism in hematopoiesis In addition to other neoplasias , polyamine metabolism has also been linked to leukemias. The erythrocyte spermine content has prognostic value in childhood acute lymphoid leukemia (ALL) (Bergeron et al. 1997). In addition, the ODC activity in peripheral blood mononuclear cells from common myeloid leukemia (CML) patients has been shown to reflect the neoplastic proliferation activity of leukemic cells (Tripathi et al. 2002). Furthermore, the catabolic side of polyamine metabolism has been implicated in leukemias since SSAT is differentially expressed during different stages of CML pointing to an involvement of SSAT in disease transformation (Janssen et al. 2005). Additionally, SSAT expression is increased in the transition phase of acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia, common in individuals with Down syndrome (Lightfoot et al. 2004). Furthermore, SSAT is super-induced in response to treatment with 12-0tetradecanoylphorbol-13-acetate (TPA), a compound which triggers the differentiation of human myeloblastic leukemia cell line ML-1. Thus, SSAT was speculated to be an important regulator of myeloid differentiation (Wang et al. 1998). 2.1.7 Polyamine metabolism in bone remodeling There are a few experiments describing the role of polyamines in bone formation (Tjabringa et al. 2008) and bone resorption (Yamamoto et al. 2012). Tjabringa et al (2008) showed that addition of spermine could enhance osteoblastogenesis in cultured mesenchymal stromal cells (MSC). Yamamoto et al. (2012) found spermidine or spermine treatment to prevent bone loss in ovariectomized mice. The effect of administered polyamines was mediated through disruption of osteoclast maturation. Polyamines did not have any substantial effect on osteoblasts in that study. Polyamine metabolism has been lined to bone homeostasis also in a human disorder called Snyder-Robinson syndrome (SRS) which is associated with mutations in the spermine synthase gene (Becerra-Solano et al. 2009; Cason et al. 2003; de Alencastro et al. 2008). SRS patients were reported to suffer from osteoporosis and the authors claimed that it resembled osteogenesis imperfecta, although, the diagnosis was not confirmed (Arena et al. 1996; Becerra-Solano et al. 2009; de Alencastro et al. 2008). SRS patients exhibit only a modest reduction in spermine but their spermidine content is greatly elevated (Cason et al. 2003). Thus, the spermine:spermidine ratio is reduced and the total polyamine content increased with the putrescine level being decreased. Additionally, mice lacking spermine synthase activity, termed gyro (Gy) mice, have been described to have a similar cellular spermine:spermidine ratio as SRS patients and also to suffer a defect in bone development in addition to other symptoms (Lyon et al. 1986; Mackintosh and Pegg 2000; Meyer et al. 1998). However, Gy mice have a deletion of a part of the X chromosome that inactivates not only the spermine synthase gene but also Phex, a gene that regulates phosphate metabolism. Therefore, caution is necessary in the use of Gy mice to study the role of spermine in bone formation (Wang et al. 2004). 2.2 HEMATOPOIESIS Hematopoietic stem cells (HSC), like all other multi-potent stem cells in the body, are able to both self-renew and differentiate into more mature cells. Self-renewal is essential for maintaining the HSC compartment and hence is a prerequisite for lifelong hematopoiesis. HSC divisions can be either symmetrical or asymmetrical (Blank et al. 2008). During

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symmetrical cell division, both daughter cells retain stem cell properties whereas in asymmetrical cell division one of the resulting daughter cells maintains stem cell properties but the other commits to a more differentiated outcome. As a result of symmetrical cell division, the stem cell pool is expanded. On the other hand, asymmetrical cell division maintains the HSC compartment. The more differentiated progeny progressively lose their self-renewal potential and become more restricted in their differentiation capacity. All circulating blood cells, including platelets, red blood cells and white blood cells, are descendants of multi-potent hematopoietic stem cells. Platelets (also called thrombocytes) are important participants in the blood coagulation process. They are derived by budding from large precursors called megakaryocytes. Red blood cells, or erythrocytes, bind oxygen and deliver it to tissues. During differentiation erythrocytes undergo denucleation in order to gain space for hemoglobin, the molecule able used for binding and transporting oxygen. White blood cells, also called leukocytes, are responsible for the immune defense in the body. They are further subdivided into myeloid and lymphoid lineages which both have several subpopulations with different specialized tasks in immune defense. The commitment of hematopoietic lineages is presented in Figure 3.

Figure 3 Origin of hematopoietic and bone cells. Hematopoietic and bone cells both originate from the bone marrow. All the mature hematopoietic cells (right side of figure) develop from myeloid and lymphoid progenitors that differentiated from the hematopoietic stem cells. The bone destructing osteoclasts also have a hematopoietic origin whereas the bone forming osteoblasts derive from the mesenchymal stromal cells. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; HSC, hematopoietic stem cell; LMPP, lymphoid-primed multipotent progenitor; MEP, megakaryocyteerythrocyte progenitor; MPP, multipotent progenitor; MSC, mesenchymal stromal cell; OB, osteoblast; OC, osteoclast

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2.2.1 Hematopoietic stem cells The prevailing definition for murine hematopoietic stem cells is Lineage- Sca-1+ c-Kit+, designated as LSK cells (Ikuta and Weissman 1992; Li and Johnson 1995) (Figure 4.). These cells lack the lineage-associated surface markers but the expression of stem cell antigen 1 (Sca-1) and c-Kit (also termed receptor for stem cell factor or CD117) is high. HSCs can be further classified into most primitive self-renewing stem cells, termed long-term hematopoietic stem cells (LT-HSC), and into more differentiated short-term hematopoietic stem cells (ST-HSC) and multipotent progenitors (MPP). LT-HSCs are able to reconstitute the bone marrow of a transplanted animal. The ST-HSCs and MPPs, however, are capable to only short-term reconstitution. The LT-HSCs reside in Thy1.1lo, CD34-, CD38+, Flt3- or Hoechstlo fraction of the LSK population (Adolfsson et al. 2001; Goodell et al. 1996; Morrison and Weissman 1994; Osawa et al. 1996; Randall et al. 1996). There seems to be no clear-cut phenotypic definition, however, to distinguish ST-HSCs and MPPs from each other (Iwasaki and Akashi 2007a). HSC compartment is heterogenous in respect of the lineage and self-renewal potential of the cells and HSCs comprise distinct lineage biased clonal subtypes (Beerman et al. 2010b). These HSCs with differential lineage potential can be purified based on the expression of CD150 (Slamf1) (Beerman et al. 2010a). The human HSCs are defined by the expression of surface markers CD34 and CD38, the most primitive HSCs (LT-HSC) being lineage- CD34+ CD38- rhodaminelow (McKenzie et al. 2007). 2.2.2 Maintenance of self-renewal capacity of hematopoietic stem cells Self-renewal capacity, i.e. a daughter cell maintaining its stem cell properties, is sustained by both the intrinsic factors present in the stem cells and the extrinsic cues of the microenvironment surrounding the cells. Many transcription factors, such as Gfi-1, Pbx1, interferon regulatory factor-2, thioredoxin-interacting protein and Nurr-1 are known to be important regulators of HSC quiescence (Li 2011). For example, Gfi-1 knockout in mice leads to an increase in the numbers of cycling cells within the HSC pool and the function of HSCs in bone marrow transplantation experiments is decreased (Hock et al. 2004). The homeobox (Hox) transcription factors, such as Hoxb4 and Hoxa9, are widely studied transcription factors indispensable for hematopoiesis. They are expressed preferentially in HSC and immature progenitor cell populations and are down-regulated during the differentiation of hematopoietic cells (Argiropoulos and Humphries 2007). The Hox family transcription factors have overlapping functions and the loss of one gene may be compensated for by the other members of this family. Quiescent HSCs have also a higher expression of GATA-2, although its exact role in quiescence regulation in vivo is not clear (Li 2011; Venezia et al. 2004). Additionally, p53 is important in ensuring that HSCs remain quiescent and thus the increased expression of p53 is related to aging of hematopoietic cells (Dumble et al. 2007). Several cytokines, such as IL-3, IL-6, IL-11, Flt-3 ligand, thrombopoietin (TPO) and stem-cell factor (SCF, c-kit ligand), have been proposed as regulators of HSC self-renewal and used in the in vitro expansion of HSCs (Blank et al. 2008). However, only studies with TPO and SCF have provided sufficient evidence to support their important role as positive regulators of stem cell pool. The receptors for TPO and SCF, c-mpl and c-kit respectively, are expressed on HSCs. Many signaling pathways have been reported to regulate HSC self-renewal or promote expansion of these cells in vitro, but nonetheless, their function in vivo has proved to be less pronounced. This indicates that there are overlapping pathways to secure the control of HSC self-renewal.

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The maintenance of the self-renewal capacity of HSCs and the promotion of differentiation of committed cells are regulated also by epigenetic factors, such as DNA methylation, histone modifications and non-coding RNA (Cedar and Bergman 2011; Scholz and Marschalek 2012). DNA methyltransferases (DNMTs) catalyze the addition of a methyl group to the 5’ position of the cytosine ring of DNA, especially to CpG dinucleotide sequences. The N-terminal histone tails are subject to a vast array of covalent modifications, including acetylation, methylation, ubiquitination, sumolation and phosphorylation. The acetylation of histones H3 and H4 by histone acetyltransferases (HAT) promotes the activation of transcription which is opposed by histone deacetylases (HDACs) that remove acetyl groups, resulting in the formation of heterochromatin. Histone methylation by histone methyltransferases (HMTs) can be a signal for either eu- or heterochromatin (active or inactive chromatin). Methylation of H3K4 is known to signal for euchromatin whereas methylation of H3K9 and/or H3K27 promotes the association of repressive proteins, such as polycomb-group (PcG) proteins. The PcG proteins repress the genes that are involved in cell-cycle regulation and differentiation (Radulovic et al. 2013). Bmi1 is one of the best studied members of PcG family. It is specifically expressed in non-differentiated hematopoietic cells and is required for HSC self-renewal. Although, there is no evidence that Bmi1 overexpression could itself induce leukemia, it is believed to be an important collaborating factor in leukemic transformation. Furthermore, other participants in epigenetics have been shown to take part in leukemogenesis (Plass et al. 2008). Thus, therapeutic agents targeting the enzymes of DNA methylation (DNMT inhibitors such as decitabine (5-aza-2'-deoxycytidine)) and histone modifications (HDAC inhibitors such as trichostatin A) are being actively developed, with some already in clinical use. Since HSCs reside in an environment where the oxygen tension is very low, the maintenance of low intrinsic oxidant levels has been proposed to be an important way of keeping HSCs quiescent. The ROS level of HSCs is lower than the ROS level of committed progenitor cells (Tothova et al. 2007). Forkhead transcription factors, FoxOs, have been reported to protect HSCs from oxidative stress and triple knockout of FoxO1, FoxO3 and FoxO4 resulted in an accelerated cycling of HSCs in mice (Tothova et al. 2007). Additionally, mice deficient for ATM, a cell-cycle checkpoint regulator directly downstream of FoxO3A, were shown to display increased ROS levels in HSCs which led to a defect in maintenance of HSC quiescence (Ito et al. 2004). 2.2.3 Lineage commitment of hematopoietic cells The expression of Sca-1 decreases in the more differentiated cell populations being, low in common lymphoid progenitors (CLP) and negative in common myeloid progenitors (CMP, Figure 4). The receptor for interleukin 7 (IL-7Rα) can be used to distinguish these two populations from each other. IL-7Rα, an essential cytokine for T and B cell development, is up-regulated in CLPs and absent in CMPs (Akashi et al. 2000; Bhatia et al. 1995; Kondo et al. 1997). The mature blood cells of lymphoid lineage consist of small, round cells: T, B and natural killer (NK) cells. Early lymphoid progenitors reside in bone marrow but the true maturation of T cell lineage takes place in the thymus. Myeloid progenitors can be further divided into three subsets on the basis of the expression of CD34 and FcγRII/III: CD34+FcγRII/IIIlo CMPs, CD34-FcγRII/IIIlo megakaryocyte-erythrocyte progenitors (MEPs) and CD34+FcγRII/IIIhi granulocyte-macrophage progenitors (GMPs). CMPs can produce all types of myeloid colonies whereas MEPs and GMPs, which differentiate from the CMPs,

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can generate only granulocytes (i.e. neutrophils, basophils and eosinophils), mast and macrophage-dendritic cells or megakaryocytes and erythrocytes, respectively (Arinobu et al. 2005; Iwasaki et al. 2005a). Dendritic cells can be derived also from CLPs (Manz et al. 2001). The monopotent megakaryocyte lineage-committed progenitors, giving rise to platelets, have been identified in the CD9+ MEP fraction whereas the surface markers of monopotent erythrocyte progenitors, giving rise to red blood cells, have not been characterized (Iwasaki and Akashi 2007b; Nakorn et al. 2003). Although the above definition for the distinct common lymphoid and myeloid progenitors derived from MPPs is widely used, there is some evidence that the differentiation routes of hematopoietic cells may be more complex [reviewed in (Iwasaki and Akashi 2007a)]. The postulated model implies that myeloid differentiation can occur not only through MPP-CMP pathway but also through lymphoid-primed multipotent progenitors (LMPP) (Adolfsson, Mansson et al. 2005, Lai, Kondo 2006). LMPPs, defined by the expression of Fms-like tyrosine kinase 3 (Flt3) and vascular cell adhesion protein 1 (VCAM-1), are lymphoid-biased but possess a granulocyte-macrophage potential. The myeloid-potential is gradually down-regulated and eventually silenced during the course of lymphocyte differentiation. The commitment to the lymphoid lineage is finally established when the cell loses all of its abilities to produce cells with a myeloid lineage, i.e. at the CLP stage. Megakaryocyte-erythrocyte differentiation is traditionally thought to occur through the MPP-CMP pathway but there is also evidence that MEPs can be generated directly from HSC-MPP compartment (Adolfsson et al. 2005; Friedman 2007; Iwasaki et al. 2005b). LMPPs lack megakaryocyte-erythrocyte potential.

Figure 4 Differentiation path of murine hematopoietic stem and progenitor cells. The hematopoietic stem cells and the myeloid and lymphoid progenitors can be identified and divided into subtypes based on the expression levels of the surface markers c-Kit, Sca-1, CD34, IL-7R and FcγRII/III. CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; LT-HSC, long-term hematopoietic stem cell; MEP, megakaryocyte-erythrocyte progenitor; MPP, multipotent progenitor; ST-HSC, short-term hematopoietic stem cell

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2.2.4 Regulation of myeloid and megakaryocyte-erythroid lineage commitment The lineage commitment is controlled by transcription factors that selectively activate or silence a set of genes. The order in which certain transcription factors become expressed and the expression level of transcription factor can decide the lineage outcome (Graf and Enver 2009). The expression level and timing of transcription factors depend on both intrinsic and extrinsic signals. Additionally, transcription factors may undergo crossantagonistic interaction with each other, inactivating transcription factors that direct the differentiation of other cell types (Dahl et al. 2003; Nerlov et al. 2000; Rekhtman et al. 2003; Zhang et al. 1999). PU.1 is one of the most important regulators of hematopoietic lineage commitment. It is highly expressed in myelomonocytic cells, B cells and at a lower level in their precursors, such as HSCs, CMPs and CLPs (Akashi et al. 2000). PU.1 regulates myelolymphoid differentiation and thereby its deficiency impairs the development of granulocytes, macrophages and lymphocytes but does not have any impact on the development of megakaryocytes or erythrocytes (McKercher et al. 1996; Scott et al. 1994). Conditional knock-out models have demonstrated PU.1 to be necessary for the maintenance of HSCs and indispensable in allowing MPPs to proceed to CMPs, GMPs and CLPs (Dakic et al. 2005; Iwasaki et al. 2005b). Moreover, PU.1 expression favors myeloid over lymphoid development and monopoiesis over granulopoiesis (Dahl et al. 2003; DeKoter and Singh 2000). C/EBPs are expressed predominantly in granulocytes, monocytes and eosinophils and C/EBPα also in HSCs, CMPs and GMPs but not in CLPs or MEPs (Friedman 2007; Traver et al. 2001). Mice deficient in C/EBPα lack neutrophils and eosinophils whereas this deficiency does not impact on lymphoid or megakaryocyte-erythrocyte development (Zhang et al. 1997a; Zhang et al. 2004). Moreover, C/EBPα is required to allow CMPs to proceed to GMPs as mice with a conditional deficiency of C/EBPα lack GMPs but the development of CMPs, MEPs and CLPs is normal. However, C/EBPα is not absolutely required for GMPs to differentiate into mature granulocyte-macrophages as demonstrated by disruption of C/EBPα at GMP stage (Zhang et al. 2004). Disruption of PU.1 at GMP stage, on the other hand, results in differentiation arrest (Iwasaki et al. 2005b). GATA factors are essential transcription factors for the development of megakaryocytes and erythrocytes (Iwasaki and Akashi 2007a). Enforced expression of GATA-1 in HSCs has been shown to lead to an increase in megakaryocyte-erythrocyte differentiation (Zhang et al. 2004). For further lineage specification, bipotent megakaryocyte-erythrocyte progenitors require the presence of additional signals. Erythroid Krüppel-like factor (Kfl-1) and friend leukemia integration 1 (Fli-1) have been identified as transcription factors directing erythrocyte and megakaryocyte commitment, respectively (Mancini et al. 2012). GATA-2 is crucial for the maintenance and proliferation of HSCs and multipotent progenitors (Vicente et al. 2012). It is normally down-regulated as hematopoiesis proceeds and it is likely repressed by GATA-1 (Crispino 2005). 2.2.5 Aging of the hematopoietic system Similar to the homeostasis of other cellular systems, the cellular homeostasis of hematopoietic system becomes disrupted with age. The most striking feature of an aging hematopoietic system is the skewing towards a myeloid-biased output. The lymphoid output, on the contrary, is decreased and adaptive immunity concomitantly diminished. The incidence of myeloproliferative diseases increases with age whereas lymphoid

17

leukemias are more common in the young (Beerman et al. 2010b; Woolthuis et al. 2011). Elderly humans and mice have also a greater propensity to anemia. The age-related changes of hematopoietic system begins from HSCs whose population expands in advancing age and the repopulation capacity diminishes (Beerman et al. 2010a; Rossi et al. 2005). During aging, the myeloid-biased (CD150high) HSC population expands whereas the proportion of HSCs with balanced lineage output (CD150low) diminishes. A comparison of old mice with their young counterparts has additionally revealed that the GMP population expands with age whereas the frequencies of CMPs and MEPs do not differ from those of young mice (Rossi et al. 2005). The gene expression profile of hematopoietic stem cells changes with age and epigenetic factors have been postulated as being important in this phenomenon (Woolthuis et al. 2011). 2.2.6 Bone marrow microenvironment In recent years, it has become evident that the stromal cells surrounding solid tumors are in close contact with the tumor cells and they can promote the transformation of these cells into a malignant form. Hence, it is not surprising that also the microenvironment around hematopoietic cells has been claimed to play a role in development of hematological malignancies (Askmyr et al. 2011; Carlesso and Cardoso 2010). There is also evidence that leukemic cells are able to influence bone cells and thereby may further contribute to the severity of the hematopoietic disease (Edwards et al. 2008; Frisch et al. 2012). Early in life, hematopoiesis occurs in the yolk sac, the aorta-gonadal-mesonephros region and the liver. After bone forming cells (osteoblasts) have initiated the mineralization of the bone extracellular matrix, the hematopoietic stem cells move to the bone environment and in adults, hematopoiesis is localized in bone marrow. It is directed there by the calciumsensing receptor expressed on hematopoietic stem cells that enables cells to respond to extra-cellular ionic calcium concentrations (Adams et al. 2006). The bone marrow microenvironment is formed not only of the bone mineral matrix but also of cellular components, such as mesenchymal stromal cells, bone forming and destroying cells, perivascular cells and sympathetic neurons. Cells of the nervous system have been shown to support HSC quiescence by influencing the molecular and cellular components of the microenvironment (Katayama et al. 2006; Yamazaki et al. 2011). The vasculature of bone marrow is also recognized as being an important factor for hematopoietic homeostasis (Kopp et al. 2005). The most primitive HSCs are thought to line the endosteal walls of bones whereas the more differentiated progenitors reside in close proximity to the vasculature (Kopp et al. 2005; Zhang et al. 2003). HSCs from elderly individuals have been found to localize more distantly from endosteum due to their reduced adhesion to stromal cells (Kohler et al. 2009). Osteoblasts, lining the endosteal walls of bones, have been reported to be one of the key cellular components maintaining hematopoiesis (Calvi et al. 2003). The ablation of these cells leads to the concomitant loss of bone marrow cells (Visnjic et al. 2004). Osteoblasts maintain HSCs by secreting thrombopoietin (TPO) and by expressing angiopoietin-1 (Ang-1), stem cell factor (SCF, c-kit ligand) and Jagged-1 on their surface (Blank et al. 2008; Li 2011). Additionally, the adhesion molecules present in osteoblasts such as N-cadherin, integrins and osteopontin anchor HSCs to the osteoblastic niche and regulate the cell-cycle quiescence of HSCs. Adipocytes, on contrary, have been considered to be negative regulators of hematopoiesis (Naveiras et al. 2009). In aging organisms, adipocytes seem to displace the hematopoietic cells in the bone marrow.

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2.3 BONE STRUCTURE, FUNCTION AND DEVELOPMENT Bone serves a variety of functions providing, e.g. structural support and protection of internal organs. In addition, it maintains the body’s mineral homeostasis and serves as environment for hematopoiesis. Based on its structure, bone can be divided into cortical and trabecular compartments (Bilezikian et al. 2008). Cortical bone is called also compact bone due to its dense structure. It forms the hard outer layer of bones. Trabecular bone, on the other hand, is a porous network inside the area that cortical bone has bordered. As a result of its appearance, trabecular bone is often called spongy bone. In the long bones of the limbs, trabeculae can be found at the ends of the bones (epiphyses). Bone is a connective tissue consisting of cellular components, organic matrix and inorganic elements (Bilezikian et al. 2008). Osteoblasts, osteoclasts and osteocytes are the cellular components of bone, the latter accounting for 90 percent of bone cellularity. The most abundant protein in the organic bone matrix, representing over 90% of all the bone proteins, is type I collagen which is involved in the aligning of the mineral matrix. In order to assemble collagen molecules into fibrils, procollagen needs to be cleaved by specific proteinases. This process releases two propeptides, the amino-terminal PINP and carboxyterminal PICP whose serum concentrations reflect the rate of synthesis of type I collagen and therefore have been used as markers of bone formation (Pollmann et al. 2007; Rissanen et al. 2008). In addition, bone has also several non-collagenous proteins, such as osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC). In fact, circulating levels of OC can be used as a specific marker for osteoblastic activity and bone formation (Bilezikian et al. 2008). Bone proteins are bound to hydroxyapatite crystals, the inorganic calcium phosphate constituent of the bone (accounting for approximately 60% of bone), and they serve important functions in the regulation of mineralization. During growth, bone formation occurs via two distinct mechanisms: intramembranous and endochondral ossification (Clarke 2008). The flat bones are formed by intramembranous ossification, a mechanism of direct bone formation. In endochondral ossification, on the other hand, the pre-existing cartilage template is replaced by the mineralized bone matrix. The cartilage growth plate has an integral role in the postnatal linear growth of the long bones, nevertheless, the long bones are formed by combination of these two mechanisms. The peak bone mass is achieved in adulthood, but thereafter bone is lost as a result of the normal aging process. This means that the bone formation exceeds bone loss in early life but the situation is reversed in the elderly. 2.3.1 Bone remodeling After growth, bone appears to be a static organ. However, it is being constantly moulded and renewed by a process termed remodeling (Eriksen 2010; Kular et al. 2012). Bone remodeling allows repair of microdamage and replacement of old bone with new. It is generally thought that the purpose of bone remodeling is to maintain the mechanical properties of the skeleton as well as regulating mineral homeostasis. Remodeling is carried out by the bone-resorbing osteoclasts, breaking down the old or damaged bone, and the bone-forming osteoblasts, re-filling the gaps in the bone matrix. Signals from osteoclasts and the products of the resorbed matrix partly regulate osteoblastogenesis and bone formation (Martin and Sims 2005). However, human patients and mice with osteopetrosis due to impaired osteoclast function have normal or increased numbers of functional

19

osteoblasts, thus indicating that it is the number of osteoclasts, rather than their activity which is more crucial for osteoblastic bone formation (Karsenty 2000). Mesenchyme derived cells, such as osteoblasts and osteocytes, are important regulators of osteoclastogenesis and there are many regulatory factors influencing osteoclast formation. Some of these influence the cells indirectly by stimulating expression of osteoblastic/osteocytic receptor activator of nuclear factor kappa B ligand (RANKL). Osteoblasts regulate osteoclastogenesis additionally by secreting macrophage colony stimulating factor (M-CSF) (Udagawa et al. 1990). Previously, osteoblasts were thought to be the major regulatory cell type involved in osteoclastogenesis but in recent years, the important role of osteocytes has also been recognized (Xiong and O'Brien 2012). Additionally, osteocytes secrete sclerostin, a protein which inhibits bone formation (van Bezooijen et al. 2004). In healthy young bone, the process of resorption and formation are tightly coupled to maintain homeostasis and overall bone mass. Thus, a defect in either will cause an imbalance in bone structure leading to a disease state. An imbalance in cellular processes of bone remodeling is involved in serious bone diseases such as osteoporosis, osteosclerosis, osteopetrosis and Paget’s disease of bone (Kular et al. 2012). Osteosclerosis and osteopetrosis are diseases characterized by a marked increase in bone mass due to stimulated osteoblast activity and osteoclast dysfunction, respectively. Osteoporosis, the most common bone disease, on the other hand, is characterized by decreased bone mineral density and by deranged levels and forms of bone proteins. Osteoporosis is classified into primary and secondary diseases, with the primary form being further classified into postmenopausal (in women) and age-related (both women and men) osteoporosis. The pathophysiology of senile bone loss is characterized by bone formation exceeding bone resorption on the periosteal surface and bone resorption exceeding bone formation on the endosteal surface (Bilezikian et al. 2008; Clarke 2008). Consequently, bones increase in diameter and the marrow space expands in the elderly. Osteoporosis is preceded by a state called osteopenia in which there is a reduced bone mass, but not so extensive to fulfill the criteria of osteoporosis. 2.3.2 Osteoblasts and bone formation Osteoblasts are cells of mesenchymal origin (Dennis et al. 1999; Pittenger et al. 1999) (Figure 3). They are responsible for initial bone formation during development and later in bone remodeling. The maturation of osteoblasts from mesenchymal progenitors consists of three distinct phases: proliferation, extra-cellular matrix maturation and matrix mineralization. After mineralization, the majority of osteoblasts undergo apoptosis whereas the remainder of the population differentiates into osteocytes or quiescent bone lining cells. Osteocytes are cells that become trapped inside the bone-matrix. They are able to sense a mechanical load and subsequently send signals which cause bone to be either resorbed or formed. The runt-related transcription factor 2 (Runx2) is viewed as the master regulator in embryonic bone development as well as in postnatal differentiation and bone formation of osteoblasts (see (Ducy et al. 1999; Komori 2003; Stein et al. 2004) for a review). Mice lacking Runx2 do not produce any osteoblasts. Despite the essential role of Runx2 in osteoblast commitment, it is not needed for the maintenance of the major bone matrix genes in mature osteoblasts. Instead, it seems to function as a negative regulator of the terminal differentiation of osteoblasts (Liu et al. 2001; Marie 2008; Maruyama et al. 2007). Among

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many other signaling pathways, Wnt/β-catenin pathway has been shown to be an important promoter of osteoblastogenesis through the stimulation of Runx2 (Gaur et al. 2005). The bone cell phenotype is further reinforced by transcription factor Osterix (OSX) (Nakashima et al. 2002). OSX is believed to act downstream of Runx2 during osteoblast differentiation since it is not expressed in Runx2-deficient embryos but Runx2 is expressed in Osx-deficient mice. After proliferation, the expression of bone-matrix genes, such as type I collagen and fibronectin, increase in pre-osteoblasts and these cells turn into bone-matrixsecreting osteoblasts. The expression of bone-matrix proteins osteocalcin, octeopontin and bone sialoprotein is increased in the mineralization phase in response to Runx2 promotion (Ganss et al. 1999; Neve et al. 2013; Sodek et al. 2000). These bone-matrix proteins have high affinity for the mineral constituent of bone, the hydroxyapatite crystal. The mineralization of the secreted matrix requires the presence of tissue non-specific alkaline phosphatase (TNAP/ALP), whose expression is increased along with the genes encoding bone-matrix proteins (Orimo 2010). ALP hydrolyzes pyrophosphate and provides the inorganic phosphate needed for hydroxyapatite crystallization. The hydroxyapatite crystals are formed within the matrix vesicles of osteoblasts (and chondrocytes) and deposited between collagen fibrils. Deactivating mutations in the TNAP gene are responsible for poorly mineralized cartilage and bones and also the accumulation of pyrophosphate. 2.3.3 Osteoclasts and bone resorption Osteoclasts are multinucleated gigantic cells derived from the monocyte-macrophage lineage of hematopoietic cells (Walker 1975) (Figure 3). Despite sharing some cell-surface markers with macrophages, osteoclasts differ clearly from these cells, most distinctively by their ability to excavate bone (Chambers 2000). Upon reaching the bone matrix, mononuclear precursors fuse to form multinucleated osteoclasts which then become polarized. These polarized osteoclasts generate an isolated environment (sealing zone) between themselves and the bone and begin the degradation with the resorbing membrane (ruffled border) (Vaananen and Horton 1995). Osteoclasts secrete protons to lower the pH and, thus, are able to dissolve minerals from the bone-matrix (Bilezikian et al. 2008). Additionally, the matrix osteoclasts secrete enzymes, such as matrix metalloproteinases (MPPs) and cysteine proteinases, especially cathepsin K (CTSK), which degrade the organic components. When CTSK degrades the bone-matrix, collagen fragments, such as carboxyterminal cross-linking telopeptide (CTX), are released and thus the serum concentration of CTX reflects the resorbing activity of osteoclasts. The bone degradation products are transcytosed into vesicles via the resorbing osteoclasts from the ruffled border to the basal membrane. Tartrate-resistant acid phosphatase (TRAcP) is believed to function in the vesicles to undertake the final steps in the destruction of matrix components. The serum concentration of TRAcP5b has been shown to reflect the number of osteoclasts (Alatalo et al. 2004). After resorption, osteoclasts are subjected to apoptosis. The master transcriptional regulator of osteoclastogenesis is nuclear factor of activated T cells cytoplasmic 1 (NFATc1) (Zhao et al. 2010). NFATc1 regulates the expression of genes encoding adhesion molecules, proton pumps and bone-matrix degradation proteins, thus, modulating osteoclast migration, adhesion, differentiation and activation. NFATc1 is activated by RANKL signaling which leads to the activation of several downstream molecules, for example NFκB, c-jun and c-fos.

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3 Aims of the study The main purpose of this study was to investigate the effects of activated polyamine catabolism on hematopoiesis and bone remodeling, and to evaluate the interplay between these two processes.

The specific aims were:

   

To investigate the response of SSAT overexpressing mice to an immunological challenge To characterize the hematopoietic phenotype of SSAT overexpressing mice and to assess the factors affecting the phenotype To assess the role of SSAT activity in human lymphoid and myeloid leukemias To characterize the bone phenotype of SSAT overexpressing mice and to elucidate the molecular mechanisms underlying the changes

22

23

4 Materials and methods 4.1 PATIENT SAMPLES Peripheral blood mononuclear cells (PBMCs) were obtained from 21 CML, 28 AML and 33 ALL patients at time of diagnosis. Blood samples from AML and ALL patients were collected in the Department of Medicine at Kuopio University hospital in the years 2001─2004. PBMCs were isolated (CPTTM tubes, BD Biosciences, Franklin Lakes, NJ) and stored at the Laboratory of Eastern Finland. Blood samples from CML patients were collected at the Helsinki University Central Hospital (Helsinki, Finland) during years 2004─2011. PBMCs from CML patients were separated with Ficoll centrifugation (GE Healthcare Bio-Sciences, San Jose, CA, USA). Control PBMCs were obtained from healthy donors (n=5─12). The use of mononuclear cells taken at diagnosis in AML and ALL patients was accepted by the Ethical Committee of the North Savo Hospital District and by the National Supervisory Authority for Welfare and Health. The collection of samples from CML patients was approved by the Helsinki University Central Hospital Ethics. Written informed consents were obtained from all patients prior to sample collection. 4.2 MICE Mice overexpressing the SSAT gene under its endogenous promoter (SSAT mice), their wild-type littermates (wt) and mice deficient in SSAT (SSATKO mice) were used in the experiments (Niiranen et al. 2006; Pietila et al. 1997). SSAT and wild-type mice were in C57BL/6JOlaHsd and SSATKO in C57BL/6J background. All animal experiments were approved by the National Animal Experiment Board of Finland. In the LPS experiments, mice were given a single intraperitoneal injection of lipopolysaccharide (LPS, E. coli strain 0111:B4, Sigma-Aldrich, 2 mg/kg as the non-lethal dose, 15 mg/kg or 30 mg/kg as the lethal dose) or saline (controls), n=3─10/group. Mice were monitored once every 1 to 2 hours and sacrificed at different time-points (and if in a moribund state). In bone marrow transplantation, the recipient mice (n=8─10/group) were lethally irradiated twice with a dose of 5.5 Gy (fractions 3 hours apart) one day before the transplantation. Donor bone marrow cells (5x106 cells/ 200 µl of PBS-2% FBS) were injected into the tail vein of the recipient mice under anesthesia. In cell proliferation studies, 1 mg of BrdU (BrdU Flow Kit, 557891, BD Biosciences, San Jose, CA) in PBS was administered intraperitoneally ten to eleven hours before sacrifice (n=6/group). In studies treating mice with drugs affecting epigenetic factors, the animals were subjected to daily intraperitoneal injections of decitabine (0.2 mg/kg, 5-Aza-2’-deoxycytidine, Sigma-Aldrich, St. Louis, MO, n=5─11) or trichostatin A (1 mg/kg, Selleck Chemicals, Houston, TX, n=7─9) for seven and ten days, respectively. Control animals received intraperitoneal PBS at a dose of 10 ml/kg. 4.3 ISOLATION OF BONE MARROW CELLS FROM MICE Bone marrow cells were isolated from the bone marrow cavities of the hind legs by flushing with PBS-10% FBS. The cells were filtered through 40 µm strainers before being used in

24

flow cytometry (n=6─8 mice/group) or bone cell (osteoblasts and osteoclasts) differentiation experiments. 4.4 EXPANSION OF FACS-SORTED LSK CELLS Sorted LSK cells pooled from six to eight mice were grown for 16 hours at 37ºC in 5% CO2 in IMDM (Iscove’s modified Dulbeccos medium) supplemented with 1% BSA, 0.1 mM 2mercaptoethanol, 2 mM glutamine, human transferrin (200 µg/ml), bovine pancreatic insulin (10 µg/ml), low density lipoproteins (40 µg/ml), human Flt-3 ligand (100 ng/ml), human IL-11(100 ng/ml) and murine SCF (50 ng/ml). 4.5 DIFFERENTIATION OF MESENCHYMAL STROMAL CELLS TO OSTEOBLASTS Osteoblast differentiation from mesenchymal stromal cells was conducted as described previously (Heino and Hentunen 2008). Shortly, bone marrow cells isolated from 4‒5 weeks old mice (n=4─6) were cultured in αMEM supplemented with 15% FBS, 10 mM HEPES, 50 µg/ml gentamycin and 10 nM dexamethasone for one week at 37°C with 5 % CO 2. Freefloating cells were removed at day two of culture. Adherent cells, enriched for mesenchymal stromal cells, were detached by trypsin treatment and plated in six-well plates with 150 000 cells /well or in 24-well plates with 30 000 cells /well. Differentiation of osteoblasts was initiated by adding 10 mM Na-β-glycerophosphate (Sigma-Aldrich, St. Louis, MO) and 70 µg/ml ascorbate-2-phosphate (Sigma-Aldrich, St. Louis, MO). Dexamethasone was omitted on the third day of differentiation. The medium was changed on every third day and the cells were differentiated for up to 21 days. 4.6 DIFFERENTIATION OF HEMATOPOIETIC CELLS TO OSTEOCLASTS Non-adherent bone marrow cells isolated from 4‒5 weeks old mice (n=4─6) were cultured in αMEM supplemented with 10% FBS, 50 µg/ml gentamycin and 5 ng/ml M-CSF (Merck Millipore, Billerica, MA) at 37°C with 5 % CO2. After 24 hours, the medium was changed to contain 30 ng/ml M-CSF. After an additional 48 hours, the cells were plated in equal numbers and osteoclast formation was induced by addition of 35 ng/ml RANKL (Merck Millipore, Billerica, MA). 4.7 STATISTICAL ANALYSES Data are presented as mean ± SD when applicable. GraphPad Prism 5.03 software package (GraphPad Software Inc., LaJolla, CA) was used to perform the unpaired two-tailed Student’s t test and Pearson’s correlation analyses. P value < 0.05 was considered to be statistically significant. 4.8 ANALYTICAL METHODS Peripheral blood samples were collected from hind leg saphenous vein or directly from the hearts of sacrificed mice into EDTA (ethylenediaminetetraacetic acid) -coated or serum

25

separator tubes (BD Diagnostic, Franklin Lakes, NJ). Table 1 lists the methods used to measure blood and serum analytes in the original publications I─IV. Table 2 summarizes the methods used to analyze tissue and cell samples as described in detail in the original publications (indicated in the table). Table 1. Analyses of blood and serum samples in original publications I─IV Analyte/ analysis

Reference/kit/instrument

Method

Publ

Blood count

Sysmex k-4500 haematology analyser

Flow cytometry

II, III

PINP

Rat/mouse PINP EIA (IDS)

EIA

IV

TRAcP5b

Mouse TRAP Assay ELISA kit (IDS)

ELISA

IV

OC

Mouse osteocalcin RIA kit (Biomedical technologies Inc)

RIA

IV

CTX

RatLaps (CTX-I) EIA kit (IDS)

EIA

IV

ALAT

ALAT (GPT) FS* (IFFC mod.) kit (DiaSys)

Spectrophotometry

I

Creatinine

Merckotest Creatinine kit (Merck)

Spectrophotometry

I

IL-1β

Bioplex Pro mouse cytokine kit (Bio-Rad)

BioPlex 200 System

I

IL-6

Bioplex Pro mouse cytokine kit (Bio-Rad)

BioPlex 200 System

I

IL-10

Bioplex Pro mouse cytokine kit (Bio-Rad)

BioPlex 200 System

I

INF-γ

Bioplex Pro mouse cytokine kit (Bio-Rad)

BioPlex 200 System

I

TNF-α

Bioplex Pro mouse cytokine kit (Bio-Rad)

BioPlex 200 System

I

IgA

(Kankaanpaa et al. 2009; Nissinen et al. 2012)

Chemiluminescent immunoassay

II

IgM

(Kankaanpaa et al. 2009; Nissinen et al. 2012)

Chemiluminescent immunoassay

II

IgG

(Kankaanpaa et al. 2009; Nissinen et al. 2012)

Chemiluminescent immunoassay

II

26 Table 2. Methods used to analyze samples in the original publications Method/analysis

Tissue/cells

Reference/kit

Publ

Cell smear

PB, BM

REASTAIN Quick-Diff kit (Reagena)

II

Histology

L, K, S, Th, LN, BM

von Kossa staining

OB

Bone length and diameter

F, T

IV

Ash weight

T

IV

Micro-computed tomography

T

(Bouxsein et al. 2010; Maatta et al. 2013)

IV

Biomechanical analysis

F, T

(Jamsa et al. 2001)

IV

ALP activity

OB

(Leskela et al. 2003)

IV

Polyamine concentration (HPLC)

L, K, S, LN, BM, PBMC, OB, OC

(Hyvonen et al. 1992)

I─IV

SSAT activity

L, K, S, LN, BM, PBMC, OB, OC

(Bernacki et al. 1995)

I─IV

ODC activity

L, K, S, LN, BM, PBMC, OB, OC

(Janne and Williams-Ashman 1971)

I─IV

RNA analysis

L

TRIzol reagent (Invitrogen), High Capacity cDNA Reverse Transcription Kit (AB)

I

Quantitative real-time PCR

LSK

RNeasy Mini Kit (Qiagen), High Capacity cDNA Reverse Transcription Kit (AB)

II

Quantitative real-time PCR

OB, OC

TRIzol reagent (Invitrogen), High Capacity cDNA Reverse Transcription Kit (AB)

IV

Western blot

PBMC

Protein concentration

L, K, S, LN, BM, LSK, PBMC, OB, OC

Protein assay kit (Bio-Rad)

I─IV

Magnetic separation

BM

Mouse hematopoietic progenitor cell enrichment kit (Stemcell Technologies)

II

Flow cytometry

PB, S, BM, LSK

I─II (Leskela et al. 2003)

IV

III

II

PB, peripheral blood; BM, bone marrow; L, liver; K, kidney; S, spleen; Th, thymus; LN, lymph node; F, femur; T, tibia; OB, osteoblast; OC, osteoclast; PBMC, peripheral blood mononuclear cell; LSK, LineageSca-1+cKit+ hematopoietic stem cell

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5 Results 5.1 EFFECTS OF ACTIVATED POLYAMINE CATABOLISM IN IMMUNE RESPONSE (I) To monitor whether the immune response of SSAT mice was altered compared to wild-type mice, the immune system of these mice was challenged with lipopolysaccharide (LPS). LPS is a structural constituent of the outer membrane of gram-negative bacteria. It activates immune and liver cells to produce pro- and anti-inflammatory mediators and ultimately causes endotoxemia leading to septic shock (Pinsky 2004). SSAT mice and wild-type mice had similar survival rates after administration of a lethal dose of LPS (I, Fig 1). However, SSAT mice begun to show early signs of inflammation (e.g. diarrhea and lethargy) slightly before their wild-type littermates. The histopathological changes observed in liver and kidneys after a non-lethal LPS challenge were similar in SSAT and wild-type mice (I, Fig 3 and 4). The creatinine levels were also similar in both genotypes whereas serum ALAT activities were significantly higher in SSAT mice in comparison with the wild-type littermates (I, Fig 2). Thus, the liver of SSAT mice was affected to a greater extent than the liver of the wild-type mice to the LPS challenge. Serum concentrations of pro-inflammatory cytokines TNF-α and IL-6 were similar in SSAT and wild-type mice after a non-lethal LPS challenge whereas the increase in IL-1β and INFγ concentrations was greater in the wildtype than in the SSAT mice (I, Fig 5). On the other hand, the concentration of the antiinflammatory cytokine IL-10 was higher in SSAT mice than in wild-type mice. The expression levels of hepatic acute-phase proteins C-reactive protein (CRP), haptoglobin and alpha-1-acid glycoprotein (AGP1) were increased in SSAT mice after a non-lethal LPS challenge whereas the level of expression of serum amyloid A (SAA1) was comparable to that of wild-type mice (I, Fig 6). In the basal state, levels of serum cytokines and hepatic acute-phase proteins showed no differences between genotypes. The lethal LPS challenge increased ODC activity in kidneys and liver in both genotypes (I, Tables 1 and 3). SSAT activity, on the other hand, was enhanced only in SSAT mice. Polyamine levels changed in accordance with the enzyme activities, i.e. putrescine was increased in both tissues in both genotypes, spermidine was decreased in liver and spermine in kidneys of SSAT mice. A non-lethal LPS challenge increased hepatic ODC activity and renal SSAT activity in SSAT mice (I, Table 2 and 4). The wild-type mice displayed no major changes in enzyme levels in either tissue. (Renal ODC activity was not analysed.) However, putrescine accumulated in both tissues in both genotypes. In addition, wild-type mice showed an increased level of spermidine in both tissues after the LPS challenge. N1-acetylspermidine accumulated mainly only in SSAT mice.

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5.2 EFFECTS OF ACTIVATED POLYAMINE CATABOLISM ON HEMATOPOISIS IN MICE (II) 5.2.1 Polyamine cycle is enhanced in hematopoietic cells of SSAT mice Polyamine metabolism was disturbed in SSAT overexpressing mice in all of the hematopoietic tissues investigated, i.e. bone marrow, spleen, peripheral blood mononuclear cells, lymph nodes and macrophages (II, Fig 4, bone marrow). SSAT activity was significantly elevated as also was the activity of the biosynthetic enzyme ODC. Putrescine became highly accumulated and, uncharacteristically for mammalian cells, its concentration exceeded that of spermidine and spermine. In fact, the spermidine and spermine concentrations were decreased. Furthermore, the activity of SSAT increased N1acetylspermidine levels. The level of bone marrow SSAT activity was observed to be associated with the number of cells in bone marrow and with the weight of spleen. 5.2.2 SSAT mice exhibit myeloproliferative phenotype The blood count of SSAT mice started to exhibit changes at sexual maturation and the changes became more drastic at an older age (II, Table 1). The number of mature neutrophils increased with age at the expense of lymphocytes which resulted in an elevated proportion of neutrophils. The number of platelets was significantly elevated after two months of age and in old SSAT mice there was evidence of anemia, due to a decreased number of erythrocytes. The splenic proportion of T cells (CD3+) was decreased and the proportion of B cells (B220+) was increased in SSAT mice (II, Table 2). Additionally, serum IgG levels were increased in SSAT mice compared to the wild-type. The ratio of CD4positive helper T cells to CD8-positive cytotoxic T cells (CD4+/CD8+) was significantly reduced and the proportion of regulatory T cells (CD4+CD25+FoxP3+) from all T cells was significantly higher in the peripheral blood and spleen of SSAT mice. Additionally, spleens were enlarged in SSAT mice (II, Fig 3). However, the histological sections of spleen, thymus and liver revealed no morphological differences between SSAT and wild-type mice and no infiltration of leukocytes or extramedullary hematopoiesis was observed in the liver of these mice. Bone marrow cellularity was significantly higher in SSAT mice and their bone marrow exhibited enhanced myelopoiesis and thrombocytopoiesis (II, Fig 2 and 3). The bone marrow architecture, however, was normal and no fibrosis was detected. The proportion of blast cells in the bone marrow remained unchanged. The numbers and proportion of common myeloid progenitors (CMP) and megakaryocyte-erythrocyte progenitors (MEP) from all bone marrow cells were significantly decreased whereas the numbers and proportion of granulocyte-macrophage progenitors (GMP) were elevated in SSAT mice (II, Fig 6). The number and proportion of LSK cells (including hematopoietic stem cells and multipotent progenitors) and more specifically, long-term hematopoietic stem cells (LT-HSC) also increased in SSAT mice. Proliferation of LSK cells was found to be increased in SSAT mice. However, there were no changes in the phases of cell cycle or in apoptosis of sorted LSK cells expanded in cell culture for 4 days. The expression of HSC self-renewal regulators Gfi1, Bmi1, GATA-2 and HoxB4 was comparable between wild-type and SSAT LSK cells as was the expression of a myeloid lineage commitment regulator C/EBPα. The expression of the megakaryocyte-erythrocyte commitment factor GATA-1 was increased whereas that of the myelo-lymphoid commitment regulator PU.1 declined in LSK cells.

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5.2.3

The myeloproliferative phenotype of SSAT mice results from both the intrinsic factors of bone marrow cells and the bone marrow microenvironment Blood count analysis revealed that after short-term transplantation (up to ten weeks) wildtype and SSAT recipients injected with SSAT bone marrow as well as the SSAT recipients receiving wild-type cells displayed an increased proportion of neutrophils compared with the wild-type animals transplanted with wild-type bone marrow (II, Fig 5). Neutrophilia remained present in all SSAT recipients after long-term reconstitution whereas the wildtype environment attenuated blood count in long-term reconstitution in wild-type mice that received SSAT bone marrow. The most prominent phenotype was observed in those SSAT mice transplanted with SSAT bone marrow. The number of bone marrow cells was increased only in the SSAT recipients at the time of sacrifice (26 weeks). However, the size of the spleen was significantly increased in all SSAT recipients as well as in wild-type recipients transplanted with SSAT bone marrow in comparison to wild-type mice transplanted with wild-type bone marrow. The bone marrow cells of SSAT recipients and the wild-type mice transplanted with SSAT bone marrow had increased SSAT activity, an increased amount of putrescine as well as reduced levels of spermidine compared to wildtype mice transplanted with wild-type bone marrow. 5.3 SSAT ACTIVITY IN MYELOID AND LYMPHOID LEUKEMIAS AND EPIGENETICS IN HEMATOPOIETIC CELLS OF SSAT OVEREXPRESSING MICE (III) 5.3.1 Polyamine metabolism of human leukemic blood cells is disturbed Levels of spermidine and spermine were significantly increased in peripheral blood mononuclear cells (PBMCs) of acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and acute lymphoid leukemia (ALL) (III, Fig 1) patients compared with the healthy controls and the levels correlated positively with peripheral blood white blood cell (WBC) count and blast percentage. Additionally, the activity of SSAT was increased in all leukemia types. Interestingly, in myeloid leukemia patients (AML and CML), but not ALL patients, with the peripheral WBC count above the reference range SSAT activity was increased compared with the patients with normal or low peripheral blood WBC count. The SSAT activity correlated positively with the peripheral blood WBC count. After treatment, both the PBMC SSAT activity and WBC count of the follow-up samples of CML patients declined to the level of healthy controls. The activity of ODC and the level of putrescine were elevated only in ALL patients and the ODC activity correlated negatively with WBC count and blasts percentage. 5.3.2

SSAT mice show epigenetic changes in hematopoietic cells and differential response to drugs affecting epigenetic factors The amount of methylation was significantly increased in tri-methylated lysine 36, 9 and 4 residues and also in di-methylated lysine 79 residue of histone 3 in the bone marrow cells of SSAT mice compared to wild-type mice (III, Fig 4). The acetylation of histone 3 in lysine 9 and lysine 14 residues and the level of histone deacetylase 1 (HDAC1) were also slightly increased in the bone marrow cells of SSAT mice compared to wild-type mice. Seven day treatment with decitabine, a DNA methyltransferase inhibitor used as an antileukemic drug in the therapy of human myeloid malignancies, evened up the changes in the peripheral

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blood granulocytic and lymphoid cells between SSAT and wild-type littermates (III, Fig 2). In addition, the bone marrow cell number of SSAT mice was equal to that of the wild-type mice after treatment with decitabine. The SSAT activity of the bone marrow cells was not affected by the treatment. Ten day treatment with trichostatin A, a histone deacetylase inhibitor, did not affect WBCs of SSAT mice but significantly decreased the proportion and number of lymphocytes and increased the proportion of neutrophils in wild-type mice (III, Fig 3). The treatment also reduced the total WBC count in wild-type animals whereas in SSAT mice, the WBC count was unaltered. 5.4 EFFECTS OF ACTIVATED POLYAMINE CATABOLISM ON BONE REMODELING (IV) 5.4.1 Polyamine metabolism of osteoblasts and osteoclasts is disturbed Polyamine metabolism of in vitro differentiated osteoblasts of SSAT overexpressing mice showed significantly increased SSAT activity, accumulation of putrescine and a decreased level of spermidine (IV, Fig 5). The spermine level was unaltered and there was no accumulation of the acetylated forms of polyamines. Interestingly, ODC activity was reduced in SSAT osteoblasts differentiated for 14 days but it increased as differentiation proceeded further. In SSATKO osteoblasts, ODC activity was decreased, the putrescine level was significantly reduced whereas spermidine and spermine levels remained unaltered when compared to wild-type osteoblasts. Polyamine metabolism of in vitro differentiated SSAT osteoclasts showed increased SSAT and ODC activities and accumulation of putrescine (unpublished data). Spermidine and spermine levels were unchanged and no acetylated forms of polyamines were detected. 5.4.2

SSAT overexpression affects osteoblastogenesis but not osteoclastogenesis cellautonomously Mesenchymal stromal cells of SSAT mice differentiated into osteoblasts in cell culture showed decreased ALP enzyme activity and a decreased number of mineralized calcium nodules as compared to osteoblasts of wild-type littermates indicating impairment in osteoblastogesis (IV, Fig 4). In contrast to SSAT osteoblasts, SSATKO osteoblasts showed increased osteoblastogenesis. The expression of the osteoblastogenic regulator OSX as well as of the osteoblastogenic marker genes ALP, OC, OPN and BSP were decreased in SSAT osteoblasts. On the other hand, expression of RUNX2, the master regulator of osteoblastogenesis, was not changed. However, as differentiation proceeded, the expression of RUNX2 and ALP increased, whereas that of the other genes was still significantly lower in SSAT osteoblasts as compared to the level of expression in wild-type cells. In vivo, the serum concentration of PINP, a marker of collagen deposition and thus bone formation, was slightly elevated in SSAT mice compared to wild-type mice (IV, Fig 3). The serum concentration of OC, a protein binding bone mineral matrix, on the other hand, was reduced in SSAT mice. The numbers of osteoclasts, differentiated in vitro from bone marrow cells, were comparable in wild-type and SSAT mice. The expression of the osteoclastogenic regulator NFATC1 was significantly increased in the osteoclasts of SSAT mice (IV, Fig 4). However, that did not reflect to the expression levels of the osteoclastogenic markers, TRACP and CTSK, as their levels were unaltered. In vivo, the concentration of the bone resorption marker reflecting the number of osteoclasts, TRACP5b,

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was decreased in SSAT mice whereas the concentration of a marker of osteoclast activity, CTX, was comparable with that found in wild-type mice (IV, Fig 3). 5.4.3

Induction of SSAT with α-methylspermidine disturbs wild-type osteoblastogenesis Treatment of SSAT osteoblasts with α-methylspermidine did not have any effect on their defective differentiation potential (IV, Fig 6). The ALP activity of SSATKO osteoblasts was slightly, but statistically significantly, decreased after treatment with the analogue. Nevertheless, it remained markedly increased as compared to ALP activity of wild-type and SSAT osteoblasts. ALP activity and the number of mineralized nodules of wild-type osteoblasts, however, decreased to the level of those in SSAT osteoblasts after treatment with the analogue. α-Methylspermidine induced SSAT activity in wild-type and SSAT cells but not in SSATKO cells lacking a functional SSAT gene. The activity of ODC was decreased similarly in all three genotypes compared to their untreated counterparts. The levels of natural polyamines decreased drastically in all three genotypes and they were replaced by the analogue. 5.4.4 Bone phenotype of SSAT mice reveals striking changes The skeleton of SSAT mice started to show changes at sexual maturation revealing kyphosis (hunched back). The diameter of femurs and tibiae were larger in SSAT mice (IV, Table 1). The length of the bones was increased only in males at the age of 5 months. The diameter and length of long bones of SSATKO mice, however, were unaltered. The bone mineral content of tibiae was increased in young SSAT mice but comparable between the three genotypes at older age. The bones of SSAT mice broke more easily than the bones of wild-type mice when subjected to compression whereas biomechanical studies showed that in response to a bending force, the bones of female SSAT mice had greater stiffness and strength (IV, Fig 2). The bones of male SSAT mice showed increased tibial but not femoral stiffness and strength. In contrast to SSAT mice, the bone strength of SSATKO mice in response to bending was slightly decreased in the tibiae in females and in the femur necks of the males. An investigation of tibiae of SSAT and SSATKO mice by computational microtomography revealed drastic differences in their structure compared to wild-type mice (IV, Fig 1). The endosteal tissue volume (TV) of cortical bone was significantly larger in SSAT mice and the BV/TV ratio was significantly decreased. Additionally, the bone perimeter (B.Pm) showed a significant increase in SSAT mice whereas the cross-sectional thickness (Cs.Th) was reduced. The trabecular thickness (Tb.Th) was reduced and total volume of porosity (Po.V) was larger in SSAT mice. Additionally, in the 7 month old SSAT mice, the trabecular number (Tb. N) was increased. In SSATKO mice, both the cortical BV and TV were reduced resulting in a BV/TV ratio which was comparable to that in the wild-type mice. B.Pm and Cs.Th were unaltered in SSATKO mice. The Tb. N was reduced in SSATKO mice compared to wild-type. The bone mass density (BMD) was similar in SSAT and wildtype mice whereas it was decreased in SSATKO mice when compared to wild-type mice.

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6 Discussion SSAT is the key catabolic enzyme of polyamines. Activation of SSAT in mice concurrently enhances the whole polyamine cycle in many tissues which in addition to affecting polyamine levels, influences the availability of metabolites shared with other metabolic pathways and increases the production of toxic metabolites. The present work has characterized the role of enhanced polyamine catabolism in immune defense, hematopoiesis and bone remodeling. I Inflammatory challenge of SSAT mice Neutrophils are the first cells recruited to acute inflammatory sites. They bind surface structures of pathogens, such as lipopolysaccharide (LPS) of gram-negative bacteria. In consequence, neutrophils begin to produce cytokines to recruit other immune cells as well as enzymes and reactive oxygen species (ROS) to kill ingested bacteria. During the early stages of endotoxic shock neutrophils are known to be essential for suppression of bacteremia and the preservation of tissue function (Hoesel et al. 2005). However, in the later stages of endotoxic shock, neutrophils are themselves significant contributors to tissue damage and organ failure. These present results show that after exposing animals to a lipopolysaccharide (LPS) -induced endotoxic shock, the anti-inflammatory actions were enhanced and pro-inflammatory actions inhibited earlier in SSAT mice than in wild-type mice. Although, SSAT mice revealed an enhanced anti-inflammatory response at the molecular level during the early stage of LPS challenge, this had no effect on the mortality of these mice as it was comparable to their wild-type counterparts. The increased neutrophil numbers of SSAT mice (II) and the harmful effects of neutrophils at later stages of endotoxic shock could explain the end result of the inflammatory challenge. In further support of this proposal, SSAT mice had increased alanine aminotransferase (ALAT) values at 24 hours after the LPS-challenge, evidence of greater liver damage in the late stage of endotoxic shock. II-IV SSAT in myeloproliferation and association of hematopoiesis and bone phenotype in SSAT mice These present studies demonstrated that overexpression of SSAT in mice evoked a myeloproliferative phenotype that emerged at sexual maturity and progressed with age. The hematological phenotype of SSAT mice fulfilled the criteria of a myeloproliferative disease, according to the Bethesda proposal for classification of murine nonlymphoid hematopoietic neoplasms (Kogan et al. 2002). This was supported by the immunological studies which revealed that the basal levels of serum cytokines and acute phase protein expressions were comparable between SSAT and wild-type mice (I), thus suggesting that SSAT mice do not suffer from a systemic chronic inflammation. Furthermore, the hematological phenotype of SSAT mice was shown to originate partly from the intrinsic SSAT overexpression of hematopoietic cells as demonstrated by transplantation experiments. However, the bone marrow microenvironment was also shown to contribute to the development of myeloproliferation in SSAT mice with the most prominent hematopoietic phenotype being encountered when both the SSAT bone marrow cells and

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the SSAT microenvironment were present. Nevertheless, the changes seen in bone structure and in bone forming cells of SSAT mice were in line with the increased hematopoiesis (IV). The myeloproliferative phenotype of SSAT mice implied that there had been changes in lineage commitment. This was indeed apparent already at the hematopoietic stem cell (HSC) level as the expression level of the lineage marker PU.1 was decreased and that of GATA-1 increased when compared to the expression levels in wild-type HSCs. PU.1 is an important regulator of the myelolymphoid lineage commitment and GATA-1 an essential transcription factor for the lineage commitment of megakaryocytes and erythrocytes at the HSC level (Iwasaki et al. 2005b; Iwasaki and Akashi 2007b). Mature granulocytic cells were extensively accumulated in the hematopoietic tissues of SSAT mice at the expense of lymphoid cells. This myeloid domination was apparent already at the progenitor level as the numbers of granulocyte-macrophage progenitors (GMPs) were elevated in SSAT mice. In addition to granulocytic accumulation, SSAT overexpression led to an increase in the number of platelets in SSAT mice as compared to wild-type mice whereas the numbers of MEPs, the common progenitor for megakaryocytes and red blood cells, were reduced as were those of mature erythrocytes. The findings from the mouse model were complemented by the studies of polyamine metabolism in human leukemia samples which revealed that the SSAT activity was associated with the white blood cell count in patients with myeloid leukemias (III). In contrast in lymphoid leukemia patients, the increased peripheral blood mononuclear cell SSAT activity did not correlate with the white blood cell count. Furthermore, the follow-up samples from treated chronic myeloid leukemia (CML) patients exhibited a normalized peripheral blood white blood cell count and additionally normalized peripheral blood mononuclear cell SSAT activity. An earlier study by Janssen et al (Janssen et al. 2005) has also pointed to a role for SSAT in myeloid leukemias as its expression was shown to differ during the different stages of CML (being highest in the chronic phase in comparison to the diagnosis or that in a blast crisis). Moreover, the expression of SSAT has been shown to be super-induced in response of 12-0tetradecanoylphorpol-13-acetate (TPA) induced differentiation of human myeloblastic leukemia cell line ML-1 (Wang et al. 1998). Interestingly, increased SSAT expression has also been related to megakaryocyte malignancy in a study which characterized genes in the transition phase of acute megakaryoblastic leukemia (AMKL), a form of acute myeloid leukemia in children with Down syndrome (Lightfoot et al. 2004). Taken together, the earlier studies and these present findings from mouse and human studies indicate that increased SSAT activity is linked to myeloid and megakaryocyte differentiation. Polyamines have been shown to play essential roles in the differentiation of many cell types (Facchini et al. 2012; Pietila et al. 2005; Vuohelainen et al. 2010). In SSAT mice, the accumulation of putrescine has been reported to disturb the differentiation of keratinocytes (Pietila et al. 2005). Also the hematopoietic cells of SSAT mice accumulated high levels of putrescine whereas the levels of spermidine and spermine were reduced. Despite the marked changes in the polyamine levels of hematopoietic cells, the polyamines do not seem to substantially affect hematopoietic cell differentiation or the hematopoietic phenotype of SSAT mice. Firstly, in a preliminary study, where wild-type and SSAT mice were treated for three months with 2% difluoromethylornithine (DFMO, an ODC inhibitor) to decrease the biosynthesis of polyamines, there were no changes detected in blood counts or bone marrow cell counts, i.e. SSAT mice still exhibited neutrophilia as compared to wild-type mice (unpublished data). However, DFMO treatment did reduce the levels of putrescine

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and spermidine as well as the activity of SSAT in bone marrow cells of both genotypes. Nonetheless, the SSAT activity of the cells of SSAT mice remained higher than that of wildtype mice. Secondly, the blood counts and spleen weights of SSATKO mice were evaluated but these mice did not exhibit any notable hematopoietic phenotype despite the alterations in their hematopoietic cell polyamine levels (decreased putrescine and increased spermidine concentrations, unpublished data). Thus, these results suggest that increased SSAT activity influences the myeloproliferation in SSAT mice which is in line with the present findings from studies of human leukemia samples described in earlier chapter. SSAT has been reported to possess also other functions in addition to regulation of cellular polyamine levels. For example, SSAT is able to bind integrin α9β1 and binding of SSAT with integrin α9β1 could potentially influence the differentiation of hematopoietic cells of SSAT mice as increased SSAT activity has been shown to enhance cell migration through binding of this integrin (Chen et al. 2004). Integrin α9β1 is widely expressed in many cell types, including neutrophils and hematopoietic stem cells (Grassinger et al. 2009; Hoye et al. 2012). Mice lacking integrin α9β1 have been shown to exhibit a dramatic impairment in neutrophil differentiation due to a defect in granulocyte colony-stimulating factor (G-CSF) mediated signaling (Chen et al. 2006). The numbers of granulocyte precursor cells and mature neutrophils are reduced in these mice. The results of Chen et al (2006), however, indicate that SSAT binding does not affect the interaction of integrin and G-CSF. Preliminary data from granulocytic cells of SSAT mice indicated that apoptosis of these cells seemed to be decreased as compared to wild-type granulocytes (unpublished data). Interestingly, in addition to playing a role in migration and differentiation, α9β1 is known to regulate cell survival of neutrophils by inhibiting apoptosis through vascular cell adhesion protein 1 (VCAM-1) binding (Ross et al. 2006). Thus, it is plausible that hematopoietic phenotype of SSAT mice is partly influenced by the interplay between integrin α9β1 and enhanced SSAT activity either through increased migration potential or decreased apoptosis. The enhanced catabolism in hematopoietic cells of SSAT mice enhanced the whole polyamine metabolic cycle as, in addition to increased SSAT activity, also the ODC activity was increased in the cells. The enhanced polyamine cycle could influence the availability of metabolites, such as acetyl-CoA, which are shared with other metabolic pathways and in this way it could affect hematopoietic cell differentiation (Jell et al. 2007; Kee et al. 2004a; Pegg 2008). The consumption of acetyl-coA by SSAT could potentially interfere with the acetylation processes conducted by histone acetyltransferases. However, it was found that the increased activity of SSAT did not decrease the acetylation status of H3 lysine 9/14 in bone marrow cells of SSAT overexpressing mice but rather increased it slightly (III). The protein level of histone deacetylase 1 (HDAC1), was also increased in bone marrow cells of SSAT mice compared to wild-type mice and the cells did show slight changes in their histone methylation profile. Additionally, the myeloproliferative phenotype of SSAT mice was attenuated when they were exposed to decitabine, a DNA methyltransferase drug that is in clinical use. These data indicate that the myeloproliferation found in SSAT overexpressing mice is related to epigenetic changes in the hematopoietic cells. The enhanced polyamine cycle and concomitantly the increased acetylpolyamine oxidase activity lead to the production of ROS which could potentially increase the level of oxidative stress in SSAT mice. As a matter of fact, recently our group reported mice overexpressing SSAT under the control of a metallothionein promoter (MT-SSAT mice) had an

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increased cellular protein carbonyl content which was interpreted as an indication of enhanced oxidative stress (Cerrada-Gimenez et al. 2011). Additionally, overall MT-SSAT mice have a very similar phenotype to the SSAT mice used in the present experiments, including similar changes in their blood cell count (unpublished data). Interestingly, ROS have been implicated in hematopoietic malignancies as there is evidence for the presence of chronic oxidative stress in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) (Ahmad et al. 2008; Sallmyr et al. 2008). Furthermore, in normal hematopoiesis, ROS are indicated to have signaling functions being linked especially to differentiation of myeloid lineage cells, e.g. ROS are involved in promoting differentiation of Drosophila hematopoietic cells akin to the mammalian myeloid progenitors (Owusu-Ansah and Banerjee 2009). In addition, in murine myeloid progenitors, the ROS level has been shown to be higher than that of hematopoietic stem cells and increased levels of intracellular ROS in hematopoietic cells led to a myeloproliferative phenotype in mice which had a conditional deletion of all FoxO isoforms (Tothova et al. 2007). In addition to differentiation of myeloid cells, ROS have been shown to contribute to the lineage commitment of megakaryocytes (Eliades et al. 2012; Sardina et al. 2010). Thus, increased oxidative stress, if present in SSAT mice, could represent a possible mechanism to account the increased myeloproliferation seen in these mice. The bone marrow microenvironment is implicated to be associated with myeloid lineage commitment of hematopoietic cells. In mouse models it has been shown that the bone marrow microenvironment contributes to the development of myeloproliferation (Askmyr et al. 2011; Walkley et al. 2007). Moreover, humans with myeloid diseases suffer from related bone defects. However, the exact cell type within the microenvironment influencing the myeloproliferation is not known in either case. The key component supporting HSC, however, is known to be the osteoblastic cell (Calvi et al. 2003; Zhang et al. 2003). The first experiments highlighting the importance of osteoblasts in HSC maintenance was conducted with mice having a conditional inactivation of bone morphogenetic protein receptor type IA (BMPRIA) (Calvi et al. 2003). These mice show, similarly to the situation in SSAT mice, an increase in their LT-HSC numbers (II) which was evoked by an increase in the numbers of early osteoblastic cells. Furthermore, a subsequent study by Raaijmakers et al (Raaijmakers et al. 2010; Zhang et al. 2003) emphasized the importance of osteoprogenitors in sustaining hematopoiesis. Interestingly, although osteoblasts of SSAT mice had an impaired osteogenic potential, the serum concentration of a bone formation marker PINP was increased, thus, indicating that the number of osteoblastic cells was increased in vivo in SSAT mice. In support of this proposal, SSAT mice had also an increased number of trabeculae which are the main sites for osteoblastic cells. SSAT HSCs showed increase in proliferation rate compared to wild-type HSCs and decrease in expression of PU.1 which is not only a differentiation marker but also a regulator of HSC maintenance (Iwasaki et al. 2005b). However, no changes were observed between wild-type and SSAT mice in other self-renewal markers, GFI, BMI, GATA-2 or HOXB4, or in the proportion of apoptotic HSCs. Thus, the expansion of the HSC pool in SSAT mice was not due to promotion of selfrenewal or to a block of apoptosis. All of these findings indicate that it is the osteoblastic cells, rather than some intrinsic mechanisms which are likely to be responsible for the increase in HSC number in SSAT mice. Furthermore, osteoblasts may take part also in the regulation of lineage commitment in these mice.

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IV Bone remodeling and structure in SSAT mice SSAT overexpression elicited striking changes in the structure of bones in SSAT mice. Outwardly the most apparent skeletal change was the hunched back. The long bones of SSAT mice were larger in perimeter and the cortex became strikingly thin with age. The bones of old SSAT mice snapped more easily than those of wild-type or SSATKO mice when subjected to compression. The striking diminution of cortex explains the fragility to compression. However, SSAT bones tolerated bending slightly better than wild-type or SSATKO mice, which is understandable as the cortical bone of SSAT mice was larger in the perimeter. SSAT osteoblasts were indicated to have a defect in the mineralization steps of bone formation. However, there were no differences in the total bone mineral content of old wild-type, SSAT and SSATKO mice but since the bones of SSAT mice were of a different size than those of wild-type or SSATKO mice and the bone volume (BV) was increased in these mice, there may be differences in the relative mineral density between the genotypes. Thereby, since the bone mineral density of SSAT mice could not be confirmed, the bone phenotype of these mice could not be designated as osteopenic/osteoporotic. The osteoblastogenesis of SSAT mice was cell-autonomously impaired as the activity of alkaline phosphatase (ALP), an enzyme required for generation of inorganic phosphate for hydroxyapatite crystallization, was decreased in in vitro differentiated SSAT osteoblasts. Additionally, calcium nodules were nearly absent and there was reduced gene expression of late osteogenic markers. The osteoblasts of mice deficient for SSAT showed opposite features, thus, supporting the view that the level of SSAT activity affected osteoblastogenesis. Serum markers supported the in vitro results of impaired mineralization steps of bone formation in SSAT mice as there was a reduction in the concentration of osteocalcin, a protein which binds the hydroxyapatite mineral of bone matrix. However, even though the maturation of osteoblasts was impaired, the circulating PINP was slightly increased. PINP is a byproduct of collagen cleavage and thus a marker of bone formation rate. The increased activity of PINP, thereby, indicated the numbers of osteoblastic cells were increased in SSAT mice. SSAT osteoclasts displayed no notable changes in their number in cell culture, but the reduced serum tartrate-resistant acid phosphatase (TRAcP5b) concentration indicated that their number in vivo was decreased (Alatalo et al. 2004). Despite the decreased number of osteoclasts, their activity, as measured by a marker of collagen degradation (CTX), remained comparable between wild-type and SSAT mice. This indicated that the relative activity of osteoclasts (activity per osteoclast) was increased in SSAT mice. The expression of the master transcriptional regulator of osteoclastogenesis, NFATc1, was increased in SSAT osteoclasts. However, this was not reflected in the expressions of either TRAcP or cathepsin K (CTSK). The lack of effect on osteoclastogenic markers may have been due to the early time-point used in the experiment. Taken together, the bone phenotype of SSAT mice resulted, for the most part, from an impaired function of osteoblasts. A recent study has shown that polyamines enhance osteoblastogenesis (Lee et al. 2013). However in SSAT mice, although the osteoblastic concentration of spermidine was reduced, the mechanism leading to defective osteoblastogenesis appeared to be related to the induction of SSAT enzyme activity. First of all, the treatment of SSAT osteoblasts with a spermidine analogue did not improve osteoblastogenesis in the SSAT mice. Secondly, SSATKO osteoblasts showed a significantly higher osteoblastogenic potential than that of SSAT mice, even though their polyamine levels did not dramatically differ from each other.

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Finally, the osteoblastogenesis in the wild-type osteoblasts was decreased to the level of SSAT osteoblasts in response to addition of α-methylspermidine to the culture media and the common feature with the analogue-treated wild-type and SSAT osteoblasts was the high enzymatic activity of SSAT. In contrast to the situation in hematopoietic cells, the increased SSAT activity did not affect the acetylation status of histone protein 3 in the osteoblasts of SSAT overexpressing mice (data not shown). Thus, the effect of polyamine catabolism on osteoblastogenesis could be mediated through increased oxidative stress. The MT-SSAT mice, which have been reported to have increased oxidative stress, express also increased levels of p53 and age prematurely (Cerrada-Gimenez et al. 2011). Similarly to SSAT mice, MT-SSAT mice display skeletal changes such as kyphosis (unpublished data). Age-related changes in the bone phenotype, such as expansion of marrow space and thinning of cortex, are known to result from increased oxidative stress in mice (Almeida et al. 2007a; Levasseur et al. 2003). Aging mice suffer defects in osteoblastogenesis and, additionally, the number of osteoclasts declines with age (Almeida et al. 2007b). Additionally in humans, osteoblastogenic potential of mesenchymal stromal cells (MSCs) is reduced with age (D'Ippolito et al. 1999). Moreover, oxidative stress has been demonstrated to impair osteoblastogenesis also in rabbit MSCs and gamma-glutamyl transpeptidase (GGT) deficient mice (Bai et al. 2004; Levasseur et al. 2003). Therefore, premature aging and oxidative stress would seem to be plausible explanations for the changes seen in osteoblastogenesis and in bone phenotype of SSAT mice.

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7 Summary and conclusions Not much is known about the role of polyamines, let alone about SSAT, in the immune response, hematopoiesis and bone formation. These present studies have shed some light on the role of enhanced polyamine catabolism in these processes. The results demonstrated that it was the presence of enhanced polyamine catabolism, rather than the polyamines themselves, which disturbed bone formation in SSAT overexpressing mice. Aberrant bone formation evoked changes in the bone marrow microenvironment which, together with intrinsic SSAT overexpression of hematopoietic cells, resulted in a myeloproliferative disease in these mice. The observation that SSAT activity was associated with the white blood cell count in human myeloid leukemias is further support for the view that SSAT activity is connected to myeloid cell development. I SSAT mice displayed no difference in their response to endotoxic shock with respect to survival or symptoms. However, at the molecular level, SSAT mice showed a slightly enhanced anti-inflammatory response. The basal levels of serum cytokines and acute-phase protein expressions of SSAT mice were comparable to those in their wild-type littermates which indicated SSAT mice did not suffer from a systemic chronic inflammation in the basal state. II The investigation of hematopoietic phenotype of SSAT mice revealed an enhanced blood leukocyte count dominated by mature neutrophils at the expense of lymphocytes. The blood platelet number was also increased whereas the red blood cell count decreased with age, causing anemia. In addition, the spleens of SSAT mice were larger than those of their wild-type counterparts and the bone marrow cellularity was increased revealing the presence of enhanced myelopoiesis and thrombocytopoiesis. The numbers of long-term hematopoietic stem cells and granulocyte-macrophage progenitors were increased in the bone marrows of SSAT mice whereas the number of megakaryocyte-erythrocyte progenitors was reduced. Altogether, the hematopoietic phenotype of SSAT mice fulfilled the criteria of mouse myeloproliferative disease. The myeloproliferative phenotype of SSAT mice resulted from both the intrinsic SSAT overexpression and from microenvironmental factors. The myeloproliferative phenotype of SSAT mice indicated that the enhanced SSAT activity was participating in myeloid differentiation. III Patients with chronic myeloid leukemia, acute myeloid leukemia and acute lymphoid leukemia showed increased levels of spermidine and spermine and enhanced SSAT activity in their peripheral blood mononuclear cells. The increased SSAT activity was associated with the increased number of white blood cells in myeloid leukemias but not in lymphoid leukemia. This supported the findings from SSAT mice that SSAT activity was associated with myeloproliferation. The myeloproliferation of these mice was considered to be connected to epigenetic factors as the SSAT mice showed a differential response to human leukemia drugs affecting epigenetics and the acetylation and methylation patterns of bone marrow cells differed from those found in their wild-type controls. IV The bones of SSAT mice broke more easily than those of wild-type mice when compressed. The long bones of SSAT mice were enlarged at the perimeter yet had a strikingly thinned cortical wall. The thickness of the trabeculae was also decreased in SSAT

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mice but their number was increased. Osteoblastogenesis was cell-autonomously impaired in SSAT mice. Nevertheless, there appeared to be a greater number of premature osteoblasts. Altogether, the cellular and structural changes in the bones meant that there was a larger niche for hematopoietic cells. Thus, the bone phenotype studies supported the view that the myeloproliferation of SSAT mice was partly due to bone marrow microenvironmental factors. The study also indicated that SSAT activity impaired osteoblastogenesis through some mechanism other than depletion of polyamine levels. This mechanism could potentially be through excess production of ROS. These findings increase our understanding of the role of polyamine metabolism in hematopoiesis and bone remodeling. Together with earlier studies, these present findings emphasize the important role of SSAT in myeloid cell differentiation. In addition, these studies contribute to the body of research investigating the interplay between hematopoietic cells and bone microenvironment. Importantly, they also highlight the contribution of the catabolic part of the polyamine cycle in pathophysiological processes. In particular, highlighting the possibility that functions other than polyamine depletion may be responsible for the disturbed cellular homeostasis in bone forming cells and perhaps also in hematopoietic cells.

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Sini Pirnes-Karhu Spermidine/spermine N 1-acetyltransferase in Mouse Hematopoiesis and Bone Remodeling and in Human Leukemias

Polyamines are ubiquitious molecules essential for cell proliferation. However, the role of polyamine metabolism in the immune response, hematopoiesis, and bone remodeling is largely unknown and was thus addressed in this study. The study showed that enhanced activity of spermidine/ spermine N1-acetyltransferase (SSAT), a catabolic regulator of polyamines, was associated with disturbed hematopoiesis and bone remodeling in mice and myeloid leukemias in humans.

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