Doctor of Philosophy in Clinical/Bioanalytical Chemistry Cleveland State University

A CASEIN KINASE 2 INHIBITOR IS A POTENT ANTI-CANCER DRUG CANDIDATE

ALIETA CIOCEA

DIPLOMA ENGINEER IN CHEMICAL ENGINEERING Babes-Bolyai University June, 2000

submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL/BIOANALYTICAL CHEMISTRY

at the CLEVELAND STATE UNIVERSITY March, 2008 © Copyright by ALIETA CIOCEA 2008 This dissertation has been approved for the Department of CHEMISTRY and the College of Graduate Studies by

______Dissertation Committee Chairperson, Michael Kalafatis, Ph.D. ______Department & Date

______Tatiana V. Byzova, Ph.D. ______Department & Date

______Lily M. Ng, Ph.D. ______Department & Date

______Edward F. Plow, Ph.D. ______Department & Date

______Crystal M. Weyman, Ph.D. ______Department & Date

______Aimin Zhou, Ph.D. ______Department & Date to my dear son Alex ACKNOWLEDGMENTS

I have to acknowledge the patience and love of my four year old son, Alexander

Constantine Orban, to whom this thesis is dedicated. He was born and raised while both my husband and I were graduate students. A special thanks goes to my husband and colleague Dr. Tivadar Orban for being there for me and for sustaining me throughout all the hardships. I am grateful for all the help from my parents: Zorica and Constantin

Ciocea and my in-laws: Alexandru and Rozalia Orban. A very special place in my heart will always have my grandmother, Mos Veronica, who supported me to come to USA to start a new life and who was next to me for my first 17 years of my life. Unfortunately now she cannot enjoy my achievements, but I want to remember her here even though she cannot remember us anymore.

I would like first of all to acknowledge the support from my advisor, Dr. Michael

Kalafatis. Dr. Kalafatis gave me the gift of independence so I could follow my own research ideas. He supported financially this project and it was very hard for him because it was his “orphan” project like he likes to call it. My biology and biochemistry first insights are due to him. He introduced me to all this great people that supported me.

I am greatly indebted to all my committee members, Dr. Tatiana Byzova, Dr.

Crystal Weyman, Dr. Aimin Zhou, Dr. Lily Ng and Dr. Edward Plow for their helpful comments throughout all the stages for the completion of this thesis and all the moral support.

I would also like to acknowledge the help of Dr. Daniel J Lindner and his research technologist Rebecca M Haney for offering me training and guidance with the animal work performed for this project. Dr. Byzova offered her laboratory equipment and expertise throughout all the research steps done and I learned a lot from her and from her laboratory members (I would like to acknowledge Dr. Olga Razorenova, Dr. Natalya Narizhneva, Dr. Maria

Madjka for help with the platelets assays and blood collection and HUVEC cells).

Whenever I needed help and guidance Dr. Byzova was always available and friendly.

Dr. Crystal Weyman offered her expertise in apoptosis and she was always there for me with helpful advice and provided us with all her available instrumentation. Dr.

Weyman students Atossa Shaltouki and Terri Harford were very helpful and always nice.

Dr. Aimin Zhou is a very good experimentalist and I learned a lot from him about cell culture and various assays. Dr. Barbara Chase which was at the time in Dr Zhou’s laboratory also provided moral and scientific support.

Without Dr. Lily Ng I would have not started and neither finished this strenuous and long Ph.D. program, she offered me support and advice both moral and intellectual, she helped me with all my problems and I do not have words to thank her more.

Dr. Plow supported me throughout my entire work, he revised my first article and had permitted me to come to his department and seminars thus opening up for me the high level research world performed at Cleveland Clinic.

I developed an expertise in flow cytometry due to the access that I had to a flow cytometer, from Dr. Byzova and Dr. Plow. Bunny Cotleur and Sage O’Bryant from the

Flow Cytometry Core, Cleveland Clinic Foundation helped me at the beginning of my work and a lot of specialists from Purdue Cytometry Forum helped with advice.

Dr. Judith Drazba helped with all the microscopy experiments and she was very nice and teach me a lot of things. I would like to acknowledge Mei Yin and Linda Vargo from the Imaging Core Facility for all the help.

I would also like to thank to all of the graduate students from the chemistry department and from my laboratory especially to Dr. Evrim Erdogan which worked previously on this project and to Dr. Beatrix Budy who supported me at the beginning of my Ph.D. and who encouraged me to apply to CSU.

All the classes that I took here at CSU were very useful to me, because as I changed fields so radically from engineering to chemistry and biology I needed to learn a lot of things. Thus I would like to thank to all my professors.

This work would have not been possible without the financial support from my advisor Dr. Michael Kalafatis (who was supported by Dr. Saunders, the CSU Provost and

Dr. Schwartz, the CSU President), form the Department of Chemistry at Cleveland State

University (teaching assistantship and support for travel expenses), from the Biomedical and Health Institute at Cleveland State University (for travel expenses), from the

University Research Council at Cleveland State University (Doctoral Dissertation

Research Expense - 0300-0010-1722-10), from the College of Science at Cleveland State

University (support for travel expenses).

Alieta Ciocea

March, 2008 A CASEIN KINASE 2 INHIBITOR IS A POTENT ANTI-CANCER DRUG CANDIDATE

ALIETA CIOCEA

ABSTRACT

Cancer is so widespread and lethal that it can be considered the biggest health problem of

our century. Cancer drug development is a real challenge. Efforts outlined in this work

were directed towards showing solid evidence that casein kinase 2 inhibitors can be used

as a starting point for the development of potential cancer therapy. We first studied the

effect of a specific CK2 inhibitor, DMAT, in a malignant megakaryoblastic leukemia cell

line (MEG-01). Treated cells grew at a significantly lower rate than non-treated cells.

Apoptosis was induced by DMAT in MEG-01 cells, dose and time dependent. When

stimulated with DMAT, MEG-01 cells produced platelets in vitro and in vivo. These platelets were found to have a normal phenotype and normal function. MEG-01 tumor bearing mice had tumor growth inhibition due to DMAT. These mice had MEG-01 cells spleen infiltration, splenomegaly and high platelets counts. An in vivo pilot toxicity study showed that DMAT is not toxic to mice. Breast cancer, colon cancer and melanoma cells were treated with DMAT. We observed that in vitro DMAT induces significant proliferation arrest and apoptosis at a similar level in all the cancer cell lines tested. All the tumor bearing mice treated with DMAT showed tumor growth inhibition but this was

dependent oh hormone level for breast cancer and varied with the type of cancer

analyzed.

viii CITATIONS

Articles 1. Ciocea A, Byzova TV, Kalalatis M “Casein Kinase 2 Alpha Subunit Inhibition in MEG-01 Cell Line Results in Megakaryocytopoiesis, Apoptosis and Functional Platelets Release” (in resubmission at Blood)

2. Ciocea A, Lindner DJ, Byzova TV, Kalafatis M “A CK2 inhibitor, DMAT has a strong anti-cancer activity” (in progress)

Meeting publications 3. Ciocea A, Byzova TV, Kalalatis M, Casein kinase 2 inhibitors in hematologic cancers: biological effects and possible clinical application CLINICAL CHEMISTRY 53 (6): A134-A134 C-100 Suppl. S JUN 2007

4. Ciocea A, Byzova TV, Kalafatis M Role of casein kinase 2 in platelets release from megakaryocytes. BLOOD 108 (11): 441A-441A 1534 Part 1 NOV 16 2006

5.Ciocea A, Narizhneva NV, Erdogan E, Byzova TV, Kalafatis M Casein kinase II inhibition in MEG-01 cell line results in apoptosis, megakaryocytopoiesis and functional platelets release BLOOD 106 (11): 162B-162B 4328 Part 2 NOV 16 2005

ix TABLE OF CONTENTS Page

ABSTRACT…………………………………………………………………………..viii

CITATIONS …………………………………………………………………………...ix

TABLE OF CONTENTS……………………………………………………………….x

LIST OF TABLES……………………………………………………………………..xv

LIST OF FIGURES……………………………………………………………………xvi

ABBREVIATIONS………………………………………………………………….…xx

CHAPTERS

I. INTRODUCTION...... 1

1.1. COMPREHENSIVE LITERATURE REVIEW...... 1

1.1.1. Cancer and casein kinase 2 ...... 1

1.1.2 Casein kinase 2 inhibitors ...... 5

1.1.3. Cancer cells and apoptosis...... 6

1.1.5. Apoptosis and casein kinase 2 ...... 11

1.1.6. Bid apoptotic protein and casein kinase 2...... 11

1.1.7. Philadelphia chromosome positive disorders...... 14

1.1.8. Chronic myelogenous leukemia: actual treatments and problems...... 15

1.1.9. BCR-ABL oncoprotein and apoptosis ...... 16

1.1.10. Casein kinase 2 and BCR/ABL...... 16

1.1.11. Megakaryocytopoiesis and thrombocytopoiesis processes...... 16

1.1.12. Apoptosis and platelets production...... 18

1.1.13. Cell cycle and endoreplication in megakaryocytic cells...... 23

x 1.1.14 Casein kinase 2 and cell cycle ...... 26

1.1.15 Platelets...... 27

1.1.16 Casein kinase 2 and platelets ...... 29

1.2 GENERAL METHODS INTRODUCTION ...... 30

1.2.1 Principles of flow cytometry...... 30

1.2.2. Scanning Electron Microscopy (SEM) ...... 37

1.2.3. Transmission electron microscopy (TEM) ...... 39

1.2.4. Confocal microscopy ...... 41

1.3. REFERENCES ...... 44

II. TREATMENT OF MEGAKARYOBLASTIC LEUKEMIA CELLS WITH

CASEIN KINASE 2 INHIBITORS...... 68

2.1. ABSTRACT...... 68

2.2. INTRODUCTION ...... 70

2.3. EXPERIMENTAL PROCEDURES...... 73

2.3.1. Cell Culture...... 73

2.3.2. Cell Treatments...... 76

2.3.3. Platelets isolation from culture...... 76

2.3.4. Platelets isolation from whole blood (human)...... 77

2.3.5. Flow cytometric analysis ...... 81

2.3.6 MEG-01 samples gating of specific cell populations ...... 81

xi 2.3.7. Viability (proliferation) assay of MEG-01 cells, Trypan blue exclusion ...... 91

2.3.8. Anchorage independence in “soft agar” assay for MEG-01 cells...... 91

2.3.9. Light microscopy (phase contrast) and DAPI fluorescence microscopy...... 92

2.3.10. Scanning electron microscopy (SEM) ...... 92

2.3.11. Transmission Electron Microscopy (TEM) ...... 93

2.3.12. Confocal microscopy ...... 94

2.3.13. Statistical Analysis...... 95

2.4. RESULTS ...... 96

2.4.1. The effects of CK2 inhibitors on MEG-01 cells, as cancer cells...... 96

2.5. DISCUSSION...... 123

2.6. REFERENCES ...... 126

III. TREATMENT OF MEGAKARYOBLASTIC LEUKEMIA MURINE

XENOGRAFTS WITH DMAT ...... 137

3.1. ABSTRACT...... 137

3.2. INTRODUCTION ...... 138

3.3. EXPERIMENTAL PROCEDURES...... 140

3.3.1. Cell Culture...... 140

3.3.2. Creation of MEG-01 xenografts ...... 140

3.3.3. In vivo therapy of MEG-01 xenografts with DMAT...... 142

3.3.4. Pilot study for in vivo toxicity of DMAT ...... 143

xii 3.3.5. Hematoxylin & Eosin (H&E) staining of tumor tissue and organs ...... 143

3.3.6. Mice blood collection and blood counts ...... 145

3.3.7. Tail-bleeding assay ...... 145

3.3.8. Data Analysis...... 146

3.4. RESULTS ...... 147

3.5. DISCUSSION...... 171

3.6. REFERENCES ...... 175

IV. TREATMENT OF VARIOUS CANCER CELL LINES WITH DMAT.....181

4.1. ABSTRACT...... 181

4.2. INTRODUCTION ...... 182

4.3. EXPERIMENTAL DESIGN ...... 186

4.3.1. Cell Culture...... 186

4.3.2. Apoptosis Assays using Flow Cytometry with AnnexinV-FITC and propidium iodide (PI)...... 186

4.3.3. Viability (proliferation) assay...... 187

4.3.4. Anchorage independence in “soft agar” assay...... 188

4.3.5. Creation of the MCF-7, SW-480, and WM-164 xenografts ...... 188

4.3.6. In vivo therapy with DMAT of MCF-7 xenografts ...... 190

4.3.7. In vivo therapy with DMAT of SW-480 xenografts...... 190

xiii 4.3.8. In vivo therapy with DMAT of WM-164 xenografts ...... 190

4.3.9. Hematoxylin & eosin staining of tumor tissue and organs...... 190

4.3.10. Statistical analysis...... 191

4.4. RESULTS ...... 191

4.5. DISCUSSION...... 228

4.5. REFERENCES ...... 232

V. OVERALL CONCLUSIONS AND FUTURE DIRECTIONS ...... 239

5.1. OVERALL CONCLUSIONS...... 239

5.2. FUTURE DIRECTIONS ...... 242

5.3. REFERENCES ...... 246

xiv LIST OF TABLES

Table I. Most common CK2 inhibitors efficiency (Ki)…………………………………7

Table II. Emission and excitation spectra for most common fluorochromes…………..31

xv LIST OF FIGURES

Figure Page

Figure 1.1. Actions of CK2 depending on its localization...... 4

Figure 1.2. Apoptotic versus necrotic phenotypes...... 9

Figure 1.3. Main apoptotic and common pathways for CK2 and BCR/ABL proteins.13

Figure 1.4. Hematopoietic stem cell differentiation...... 21

Figure 1.5. The platelets production (thrombocytopoiesis) process...... 22

Figure 1.6. Cell cycle diagrams...... 25

Figure 1.7. Flow chamber and laser beam assembly of a flow cytometer...... 32

Figure 1.8. Optics of a flow cytometer...... 33

Figure 1.9. Scanning Electron Microscope (SEM)...... 38

Figure 1.10. Transmission electron microscope (TEM)...... 40

Figure 1.11. Confocal microscope...... 42

Figure 2.1. Schematics of platelets isolation from whole blood...... 78

Figure 2.2. Gating of the cells populations from MEG-01 culture...... 82

Figure 2.3. MEG-01 cells proliferation assays results...... 98

Figure 2.4. Anchorage independence assay in soft agar of MEG-01 cells...... 99

Figure 2.5. Anchorage independence assay in soft agar of MEG-01 cells...... 100

Figure 2.6. Maturation (differentiation) of MEG-01 cells due to DMAT...... 102

Figure 2.7. Fibrinogen binding to MEG-01 cells...... 103

Figure 2.8. DNA content analysis of MEG-01 cells treated with DMAT...... 104

Figure 2.9. Apoptotic assays...... 106

Figure 2.10. Phenotype change in MEG-01 cells treated with 10 µM DMAT...... 111

xvi Figure 2.11. Ultrastructural features of MEG-01 cells treated with DMAT...... 113

Figure 2.12. Platelets from MEG-01 cells obtained after DMAT treatment ...... 116

Figure 2.13. Platelets from MEG-01 cells (in culture) after DMAT treatment...... 117

Figure 2.14. Confocal microscopy analysis of platelets derived from MEG-01 cells.118

Figure 2.15. Ultrastructure of MEG-01 derived platelets (TEM)...... 119

Figure 2.16. HUVEC apoptosis versus MEG-01 cells apoptosis...... 122

Figure 3.1. MEG-01 xenograft treated with 2 mg DMAT/animal/day for 2 weeks. .149

Figure 3.2. MEG-01 xenograft treated with 3 mg DMAT/animal/day for 2 weeks. .151

Figure 3.3. Hematoxylin-eosin staining of MEG-01 tumor tissues...... 152

Figure 3.4. Whole blood counts for MEG-01 xenograft trial...... 154

Figure 3.5. Whole-blood cell counts histogram results...... 155

Figure 3.6. Hematoxylin-eosin staining of MEG-01 spleen...... 157

Figure 3.7. MEG-01 xenografts spleen sizes...... 158

Figure 3.8. Tail-bleeding times for MEG-01 xenograft...... 160

Figure 3.9. Hematoxylin-eosin staining of MEG-01 liver...... 161

Figure 3.10. DMAT toxicity level (10 mg/day/animal) treated male nude mice (2 weeks of treatment)...... 165

Figure 3.11. Normal healthy C57BL mouse (horizontal sections) H&E staining of organs

...... 167

Figure 3.12. Normal healthy C57BL mouse (transversal sections) H&E staining of organs

...... 169

Figure 3.13. Toxicity mouse whole-blood non-lysed flow cytometry...... 170

Figure 4.1. Proliferation assay of MCF-7 cells treated with DMAT 10 and 20 µM. 193

xvii Figure 4.2. Phase-contrast micrographs of MCF-7 cells after 24 hours of treatment with

DMAT 10 and 20 µM...... 195

Figure 4.3. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of

MCF-7 cells after 24 hours of treatment with DMAT 10 and 20 µM...... 197

Figure 4.4. Soft agar anchorage independence assay (one week treatment with DMAT 10

µM)...... 198

Figure 4.5. MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. 17-beta-estradiol (0.04 mg/day) was supplemented in water...... 199

Figure 4.6. MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. 17-beta-estradiol (0.02 mg/day) was supplemented in water...... 200

Figure 4.7. Hematoxylin and eosin staining of MCF-7 tumors...... 201

Figure 4.8. Hematoxylin and eosin staining of MCF-7 livers...... 202

Figure 4.9. Hematoxylin and eosin staining of MCF-7 spleens...... 203

Figure 4.10. Proliferation assay of SW-480 cells treated with DMAT 10 and 20 µM

(Trypan Blue exclusion)...... 206

Figure 4.11. Phase-contrast micrographs of SW-480 cells after 24 hours of treatment with

DMAT 10 and 20 µM...... 208

Figure 4.12. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of

SW-480 cells after 24 hours of treatment with DMAT 10 and 20 µM...... 209

Figure 4.13. Soft agar anchorage independence assay for SW-480 cells (one week treatment with DMAT 10 µM)...... 211

Figure 4.14. SW-480 xenograft treated with 1 mg DMAT/animal/day (40 mg/kg DMAT) for two weeks...... 212

xviii Figure 4.15. Hematoxylin and eosin staining of SW-480 tumors...... 213

Figure 4.16. Hematoxylin and eosin staining of SW-480 livers...... 214

Figure 4.17. Hematoxylin and eosin staining of SW-480 spleens...... 215

Figure 4.18. Proliferation assay of WM-164 cells treated with DMAT 10 and 20 µM

(Trypan Blue exclusion)...... 217

Figure 4.19. Phase-contrast micrographs of WM-164 cells after 24 hours of treatment with DMAT 10 and 20 µM...... 219

Figure 4.20. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of

WM-164 cells after 24 hours of treatment with DMAT 10 and 20 µM...... 221

Figure 4.21. Soft agar anchorage independence assay for WM-164 cells (one week treatment with DMAT 10 µM)...... 222

Figure 4.22. WM-164 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg

DMAT) for two weeks...... 224

Figure 4.23. Hematoxylin and eosin staining of WM-164 tumors...... 225

Figure 4.24. Hematoxylin and eosin staining of WM-164 livers...... 226

Figure 4.25. Hematoxylin and eosin staining of WM-164 spleens...... 227

xix ABBREVIATIONS

aa Amino acid residue

ACD Anticoagulant Citrate/EDTA

AChE Acetylcholinesterase

ADP Adenosine

AIF Apoptosis inducing factor

AIM-1 Apoptosis inhibitor of macrophage

A protein kinase named Akt, also known as PKB protein

AKT kinase B

ALL Acute lymphoblastic leukemia

AML Acute myeloid leukemia

AMN107 An BCR/ABL inhibitor, drug for CML

Anaphase A Anaphase A phase of cell cycle

Anaphase B Anaphase B phase of cell cycle

APAF apoptotic protease activating factor 1

APC Adenomatous ployposis coli

Apo1 A death receptor ligand, known as CD95 or FAS

Apo2L Apoptosis ligand 2

ATP Adenosine triphosphate

Aurora B Aurora B is a kinase

B23 Nucleophosmin

Bad A proapoptotic protein

Bax A name for a Bcl familiy protein

Bcl-2 B-cell lymphoma 2

xx Breakpoint cluster protein (BCR) fusion with Abelson kinase

BCR/ABL (ABL)

Bid BCL-2 interacting domain protein, pro-apoptotic

Bim A pro-apoptotic FoxO target

BK Blocking filter

BP Band pass

BSA Bovine serum albumine

CD Cluster of differentiation

CD34 Cellular display marker 34

CD41 Cellular display marker 41

CD61 Cellular display marker 61

CD95 Cellular display marker 95

CDBS Cell dissociation buffer cdc2 Cell divison cycle 2 phosphatase cdc25 Cell division cycle 25 phosphatse

CDK or cdk Cyclin dependent kinase

CEA Carcinoembrionic antigen

CK1 Casein kinase one

CK2 Casein kinase two

CML Chronic myelogenous leukemia

C-terminal The carboxylic end of a peptide sequence

C-terminus The carboxylic end of a peptide sequence

DAPI Diamidino-2-phenylindole dihydrochloride, a nuclear stain dATP 2’-deoxyadenosine 5’-triphosphate

DISC Death-Inducing Signalling Complex

DL Dichroic lens

DMAT 2-Dimethyl-amino-4,5,6,7-tetrabromo-benzimidazole

xxi DMSO Dimethylsulfoxide

DNA Deoxyribonucleic acid

DRB 5,6-dichloro-1-beta-ribofuranosyl benzimidazole

ECGS Endothelial growth factor suplement

EDTA Ethylene-diamino-tetraacetic acid

ENDO-G Endonuclease G

FAS A death receptor ligand, known as CD95 or Apo-1

FBS Fetal bovine serum

FITC Fluorescein isothiocyanate

FL Median fluorescence

FN Fibronectin

FS Forward side scatter

FSC Forward side scatter

FSC-H Forward side scatter height g Gravitational acceleration

G0 Phase G0 of cell cycle

G1 Phase G1 of cell cycle

G2 Phase G2 of cell cycle

GFP Green fluorescent protein

GTP Guanosine 5'-triphosphate h hours/hour

H1 Histone kinase

HBBS Hank's balanced salt solution

HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

Her-2/neu An oncogene name

HPLC High performance liquid chromatography

HSIX1 Homeobox gene from late S phase mammary carcinoma

xxii cells

HtrA2/OMI A proapoptotic factor

HUVEC Human umbilical vascular endothelial cells

IIa Thrombin

IL Interleukin

INF Interferon

JAK Jason protein kinase

Ki The dissociation constant for binding of inhibitor to enzyme kV kilo Volts

L-Glu L-Glutamine

M Molar concentration

M Mitotic phase of cell cycle

MDR Multidrug resistance

MMTV Mouse mammary tumor virus

MPF Maturation promoting factor mRNA Messenger Ribonucleic Acid

NF-kB Nuclear factor-kappa B nm Nanometers

NO Nitric oxide p53 A nuclear protein named p53

PAC-1 Activated platelets specific monoclonal antibody

PBS Phosphate saline buffer

PGE-1 Prostaglandin 1

Ph+ Philadelphia Chromosome positive

PI Propidium iodide

PI3K Phosphatydil-inositol 3 kinase

PKC Protein kinase C

xxiii PMA Phorbol-12-myristate-13-acetate

PS Penicilin/Streptomycin

An oncogene named from transforming genes Kirsten

Ras murine sarcoma viruses

RGD Platelets inhibiting peptide

RGDS Platelets inhibiting peptide

R-PE or RPE R-Phycoerythrin

RT Room temperature

S S phase of cell cycle

SCID Severe combined immunodeficient mouse

SEM Scanning electron microscopy

Ser Serine

SMAC Second mitochondria-derived activator of caspase

SMAD Signal transduction cascade protein

SS Side scatter

SSC Side scatter

SSC-H Side scatter height

STAT Signal transducer and activator of transcription

STI571 Imatinib Mesylate, a chronic myelogenous leukemia drug

TBB 4,5,6,7-Tetrabromobenzotriazole tBid Truncated Bid protein

TEM Transmission electron microscopy

TGF Transforming growth factor

Thr Threonine

TNF Tumor necrosis factor

TNS Trypsin neutralization solution

TPO Thrombopoietin

xxiv TRAIL Tumor necrosis factor related apoptosis inducing ligand

TRAP Platelets activating peptide

Tyr Tyrosine

U Units

U/G/O Unstained, green and orange fluorochrome stained cells

VEGF Vascular endothelial growth factor

VN Vitronectin vWf von Willebrand factor

xxv

CHAPTER I

INTRODUCTION

1.1. COMPREHENSIVE LITERATURE REVIEW

1.1.1. Cancer and casein kinase 2

Cancer is so widespread and lethal that it can be considered the biggest health problem of our century. Cancer rates could increase by 50%, to 15 million new cases, in the year 2020 worldwide according to the World Cancer Report. Various unknown causes, diverse genetic and protein abnormalities as well as very different and complex molecular mechanisms make cancer drug development a real challenge. Harmful side- effects are another problem that needs to be overcome in the search for possible cancer therapies.

Efforts outlined in this work were directed towards showing solid evidence that casein kinase 2 (CK2) inhibitors can be used as a starting point for the development of potential cancer therapy.

Why targeting casein kinase 2?

In different cancers, including leukemia, a kinase, named casein kinase 2 (CK2),

was found to be constitutively activated, over-expressed and to serve as an oncoprotein

[1-7]. CK2 is a pleiotropic, ubiquitous kinase that phosphorylates Serine, Threonine, and

Tyrosine amino acid residues. The protein is a heterotetramer with two catalytic subunits,

α and α’, and two regulatory β subunits [8]. Each subunit was shown to be able to

execute specific functions by itself or in the holoenzyme form, the αα’β2 (or α2β2) tetramer [9, 10]. CK2 was found to have both nuclear and cytoplasmic localization. It seems that CK2 is also characterized by the capability to undergo nucleo-cytoplasmic shuttling [3-5, 7, 11].

A combined effect from both nuclear and cytoplasmic CK2 will promote survival in cancer, see figure 1.1. In cancer cells, CK2 is deregulated (is hyperactive and elevated

3- to 7- folds) [7]. The up-regulation and hyperactivity of CK2 has an anti-apoptotic effect and is correlated with aggressive tumor behavior in different cancer types examined and results in abnormal platelet counts in leukemia [12].

Silencing of the CK2 alpha subunit was studied in vivo, in murine prostate cancer xenografts. A very interesting observation following this study showed that CK2 inhibition affects the tumors (by inducing apoptosis and tumor ablation), but does not affect the normal surrounding tissue [3, 7]. A differential response to CK2 inhibition

(with specific CK2 inhibitor, DRB and antisense nucleotides against CK2α mRNA) in different normal and benign cell lines (in vitro) was observed [6, 13]. Therefore it seems that normal and benign cells are less sensitive to the CK2 inhibition.

CK2 elevation in nuclear matrix in cancerous cells is related to cell growth, survival and even resistance to apoptosis [5, 7, 11]. Nuclear localization of CK2 in

malignant versus normal tissues was observed on histological analysis of normal tissues and neck squamos carcinoma tissues [3, 14]. The effects of nuclear CK2 are due to the action of CK2 on cell cycle and nuclear proteins like MPF (cdc2/cyclin B), cdc 25, topoisomerase II, surviving and p53 just to mention a few.

Inhibition of CK2 hinders angiogenesis in oxygen induced retinopathy, most probably due to the action of CK2 on AKT kinase [15]. CK2 is a positive regulator of

AKT kinase [16, 17]. PI3K-AKT pathway deregulation is important in tumor development and tumor response to anti-cancer treatment. CK2α inhibition with antisense nucleotides against CK2α mRNA, potentates the effect of specific inhibition of PI3K-

AKT signaling pathway. CK2 forms a complex with AKT, and also phosphorylates AKT

[16, 17]. This makes CK2 an angiogenic protein and thus by promoting angiogenesis will also promote tumor growth.

CK2 phosphorylates pro-apoptotic Bid protein making it less sensitive to caspase-

8 cleavage and by this action, CK2 promotes cell survival by inhibiting apoptosis [18-

23].

BCR/ABL chimeric abnormal tyrosine kinase enhances survival of leukemia cells through modulation of pro-apoptotic and anti-apoptotic molecules in chronic myelogenous leukemia. BCR/ABL is involved in Ras signaling, JAK/STAT kinase pathways, PI3K/AKT kinase pathway, just to mention the most important ones [24-27].

BCR/ABL was found responsible of decreased expression of pro-apoptotic protein Bim, a

Bcl-2 mediator [28-30]. BCR/ABL acts on PI3K/AKT pathway [31] that modulates activation of Bad and BCR/ABL prevents the translocation of Bad and Bax to the mitochondria [32-35].

Figure 1.1. Actions of CK2 depending on its localization. Cytoplasmic CK2 acts on major cellular pathways stimulating cellular survival and rescuing cells from apoptosis and nuclear CK2 regulates cell cycle and nuclear proteins.

Therefore BCR/ABL involvement in intricate mechanisms of cellular signaling is responsible for its oncogenic action. Interestingly, CK2α was found to be a substrate for the ABL domain of BCR/ABL [36] and to form a specific complex with the BCR domain of BCR/ABL [37]. It was hypothesized that CK2α impedes sterically the binding of the

ABL SH2 domain to BCR [37]. This results in proliferation abnormalities in Ph+ cells.

Therefore CK2α was shown to be a possible regulator of BCR/ABL function [36, 37].

These studies sustain the idea that CK2α is an attractive cancer treatment target even though it is a pleiotropic kinase. Other proteins like PI3K, AKT, PKA, PKC,

Topoisomerase II are targeted for cancer therapy, even though they are ubiquitous and pleiotropic. A general kinase inhibitor (also a CK2 inhibitor), heparin is used as a drug for thrombosis. Therefore, CK2 inhibitors can be exploited in order to develop new cancer drugs and also as an adjuvant of actual cancer therapies.

1.1.2 Casein kinase 2 inhibitors

CK2α protein kinase quite specific inhibitors have been developed. Such CK2α inhibitors belong to the classes of tetra-bromo-benzimidazole/triazole derivatives, indoloquinazolines and condensed polyphenolic compounds, see table I [38, 39]. TBB and DMAT inhibitors (ATP site directed) are cell permeable and are available for purchase at Calbiochem, USA. A hydrophobic pocket next to the ATP/GTP binding site

(which in the CK2 case is smaller than on most of the other kinases, due to the aminoacid residues side-chains that are less bulky) gives them the high selectivity [38, 39]. TBB and

DMAT are from the most selective (as screened from a panel of 38 kinases) and potent inhibitors of CK2α [40-42], see table I. DMAT has one of the lowest Ki value (40 nM) of

all the CK2α available chemically synthesized inhibitors and is one of the most specific inhibitors with respect to CK2, up to date. DMAT is ineffective on casein kinase 1 (CK1) up to 200 µM [40-42]. .The use of such inhibitors in various cancer cell lines in vitro produced apoptosis and proliferation arrest [1, 2, 6, 7, 10, 36, 43]. CK2 inhibitors use in human rhabdomyosarcoma cells increased the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL –Apo2L-) [20].

1.1.3. Cancer cells and apoptosis.

Cancer cells are characterized by increased proliferation and loss of the normal phenotype and function. Cancer is caused by defects in signaling pathways including deregulation of apoptosis process with the consequent imbalance in cell growth.

Apoptosis is a genetically programmed and evolutionary conserved mechanism through which normal development and tissue homeostasis are maintained [44-46].

Necrosis is cell death associated with an inflammatory process as a response to tissue damage. The programmed cell death, apoptosis, shows specific morphological features

(figure 1.2) such as: cell shrinkage, DNA fragmentation and nuclear breakdown, cell blebbing, phosphatidylserine redistribution at the cell surface [44, 46, 47]. These morphological features are different from the ones found in the necrotic process, i.e., loss of membrane integrity, swelling and rupture of the cells [48]. It is important to differentiate cell death originating through apoptosis or cell death originating through necrosis. It has been shown [49, 50] that malfunctioning of the apoptotic event, triggers autoimmunitary disorders, immunodeficiency, cancer, viral infections, neurodegenerative and hematological diseases.

CK2 INHIBITOR Ki (µM) Emodin (from the cactus Aloe vera) – related coumarinic 0.22 compound 8-hydroxy-4-methyl-9-nitrobenzo[g]chromen-2- one (NBC) Ellagic acid (from raspberry seeds) 0.02 Tetra-bromo-cinnamic acid (from cinnamon) 0.11 5,6-dichloro-1-ß-o-ribofuranosyl benzimidazole (DRB) 2 4,5,6,7-Tetrabromobenzotriazole (TBB) 0.56 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole 0.04 (DMAT) Indoloquinazoline derivative – 5-oxo-5,6- 0.17 dihydroindolo(1,2a)quinazolin-7-yl acetic acid (IQA) Heparin 0.14

Table I. Most common CK2 inhibitors efficiency (Ki)

1.1.4. The apoptotic pathways

Proliferation inducing oncoproteins malignantly transform cells in cooperation with anti-apoptotic processes [51]. For example studies of colon specimens harvested at diverse points along the adenoma-carcinoma conversion show that apoptosis is high in normal colonic epithelium, intermediate in adenomas and low in carcinomas [52].

Death signals, from inside and outside of the cell, activate signaling networks, and form the apoptosis mechanism, see figure 1.3. The most important pathways that lead to apoptosis are categorized as the extrinsic apoptosis pathway and the intrinsic apoptosis pathway. Once an apoptotic signal is received, the apoptotic process is carried out by the caspases cascade, and mitochondria pathways [50, 53, 54]. The caspases cascade includes the activation of caspase-3, 6 and 7 by caspase-8. Regarding the extrinsic apoptosis pathway cells were classified as type 1, from FAS death receptor (CD95/Apo-1) leading to an efficient formation of death inducing signaling complex DISC, and type 2, from

FAS death receptor (CD95/Apo-1) with slow DISC formation. In type 1 cells the caspases cascade is sufficient to induce cell death, whereas type 2 cells require an amplification of the death signal through the mitochondria pathway.

Figure 1.2. Apoptotic versus necrotic phenotypes. The phenotype of apoptotic cell (blebbing, nuclear shrinkage and condensation, nuclear fragmentation) is different from the necrotic cell phenotype (cellular swelling and cellular rupturing) [55]

The mitochondria pathway requires the proteolytic activation of the BH3-only protein

Bid by caspase-8 to a truncated form tBid (a C-terminal fragment) [56, 57] tBid translocates to the mitochondria and releases cytochrome c and other pro-apoptotic factors (SMAC/Diablo, AIF, ENDO-G, HtrA2/OMI) from mitochondrion. Release of the cytochrome c contributes to the formation of the apoptosome, i.e., cytochrome c, Apaf-1 and dATP. The apoptosome activates caspase-9, which in turn contributes to the caspase cascade by activating caspase-3 [48, 56, 58-63]. Bid was found to be involved in the intrinsic pathway too, in anoikis (Bmf activation through subcellular relocalization) and by being a p53 target. Bid is the most obvious connection between the extrinsic and intrinsic pathway [60, 64, 65]. Even more evidence shows that both type 1 and type 2 cells (FAS/CD95) activate mitochondria, releasing cytochrome c with different kinetics.

The cytochrome c release is slower for the type 1 cells. and the cascade is regulated and directed by the amount of caspase-8 [56]. Bid is also involved in the metabolic cycle of cardiolipin. Cardiolipin is the major component of the mitochondria membrane [22].

Patients with anti-cardiolipin antibodies have recurrent thrombotic events, like patients suffering from myeloproliferative disorders [66]. Bid and tBid were found to permeabilize the mithocondrial membrane and to oligomerize and interact with other proteins involved in apoptosis (anti and pro apoptotic Bcl-2 family) [19, 21-23, 50, 62,

67-69]. Therefore, Bid acts as a control in any event that results in loss of mithocondrial membrane potential and cytochrome c release and it may be said that is a very important link between the apoptotic pathways.

1.1.5. Apoptosis and casein kinase 2

CK2 protects prostate cancer cells from tumor necrosis factor-related ligand

(TRAIL/Apo2-L) mediated apoptosis, through prevention of caspase activation and blockage of mithocondrial apoptosis induced by TRAIL. TBB specific inhibition of CK2 in PC-3 cells enhances TRAIL induced apoptosis after 24 hours of treatment [70]. DRB, another CK2 specific inhibitor, enhances TRAIL induced apoptosis in rhabdomyosarcoma and human colon carcinoma cells, by enhancing activity of DISC and caspase-8 mediated cleavage of Bid [13, 20]. Inhibition of CK2 will prevent phosphorylation of Bid, and thus caspase-8 will cleave Bid to tBid (its truncated form) which is Bid’s active form (pro-apoptotic).

1.1.6. Bid apoptotic protein and casein kinase 2

It was found out that Bid is a phosphoprotein and the phosphorylated protein is less sensitive to cleavage by caspase-8 and caspase-3 [18, 57]. Murine Bid is phosphorylated by casein kinases CK1 and CK2 (CK1ε isoform and CK2α in vitro and in vivo). Inhibition with heparin, a specific CK2 inhibitor (it does not inhibit CK1) increased significantly the cleavage by caspase-8 [18]. CK2 alone phosphorylates murine recombinant Bid and murine Bid isolated from mouse kidney cytosol. By means of this phosphorylation, the rate of cleavage of Bid, by caspase-8, is significantly reduced [57].

In murine Bid the CK2 proposed phosphorylation site was Thr58. For CK1 the proposed phosphorylation sites where Ser61 and Ser64. Other phosphorylation sites of murine Bid, by CK2 were found [18]. A human Bid decapeptide phosphorylation in vitro at Thr59 (a

CK2 phosphorylation site) makes it less sensitive to cleavage by caspase-8 and caspase-3

[57].

Figure 1.3. Main apoptotic and common pathways for CK2 and BCR/ABL proteins.

Extrinsic apoptotic pathway starts from the outside of the cell from death receptors and it can go through the mitochondria or through the caspases cascade. The intrinsic apoptotic pathway gathers signaling from the inside of the cell and goes through the mitochondria. BCR/ABL chimeric oncoprotein as well as CK2 kinase are involved in important pathways like the Ras signaling, JAK/STAT kinase pathway, PI3K/AKT pathway and apoptotic pathways. [48, 56, 61]

Thr59 is more critical in human when compared with Ser61, Ser65 relative to the corresponding murine Thr58, Ser61 and Ser64 (in mouse, Ser61 and Ser64 count for the highest level of phosphorylation) [18, 57]. Therefore phosphorylation sites and their importance in the context, differ between species. This data suggests that for human Bid,

CK2 is the most important phosphorylating enzyme. Given this information we can conclude that CK2 is an important enzyme in the apoptotic cascade.

1.1.7. Philadelphia chromosome positive disorders

The reciprocal chromosomal translocation (9; 22), known as the Philadelphia positive chromosome (Ph+) [71] is associated with chronic myelogenous leukemia

(CML) [72], acute lymphocytic leukemia (ALL) [73, 74] and can appear in acute myelogenous leukemia (AML) [75]. This genetic abnormality results in a chimeric oncoprotein BCR/ABL tyrosine kinase, which is thought to be the main cause of the abnormal survival and over-proliferation of hematopoietic stem cells and their progeny

[76, 77]. In CML, the BCR/ABL tyrosine kinase is constitutively activated. Different intracellular pathways are transformed by the oncoprotein BCR/ABL, resulting in uncontrolled hematopoietic proliferation. The late phase of CML, named blast crisis (or blastic phase), is characterized by extreme overproliferation of stem cells in the bone marrow and their progeny. During blast crisis, the major complication is thrombosis due to high platelet counts [77]. Platelets (thrombocytes) are vital for maintaining normal hemostasis and for the response of the human body to trauma. Thrombosis is lethal because it leads to organ damage (stroke, hearth attack). In myeloproliferative disorders, like CML, the platelet count and function are abnormal due to malignant megakaryoblasts overproliferation, as well as impaired maturation of blastic cells.

1.1.8. Chronic myelogenous leukemia: actual treatments and problems

The treatment of choice for CML is Imatinib Mesylate (4-[(4-Methyl-1- piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-yrimidinyl]amino]-phenyl]- benzamide - methanesulfonate), a BCR/ABL tyrosine kinase inhibitor (Gleevec) formerly named STI571 [77]. Treatments with STI571 show good hematologic and cytogenetic remission in patients with early stage CML. However, patients in blast crisis are little or no responsive to the inhibitor. These patients also present additional and more aggressive adverse effects. The main cause of this problem is the development of STI571 resistance

[72, 77-79]. Point mutations, ATP phosphate-binding loop mutations [78], gene amplification and protein over-expression of BCR/ABL tyrosine kinase are the main cause of this resistance, besides other defects in intracellular signaling pathways [79, 80].

Other BCR/ABL tyrosine kinase inhibitors (Dasatinib and AMN107) show a better response than Imatinib in phase II trials, but acquired resistance still remains the major problem [79]. A point mutation in the BCR/ABL kinase (Thr315→Ile) was associated with resistance to all BCR/ABL pharmaceutical inhibitors. This specific mutation causes resistance, because of a hydrogen bond that impedes inhibitor binding and which does not affect the protein BCR/ABL carcinogenic function [81]. Altogether, the data demonstrated that the acquired mutations in BCR/ABL oncoprotein together with other molecular changes induce BCR/ABL tyrosine kinase inhibitors resistance. Activation of other kinases may contribute to the onset of the disease and also to the gained resistance.

Targeting such kinases with protein kinase inhibitors may offer a clinical benefit by providing drugs that could overcome the resistance to BCR/ABL tyrosine kinase inhibitors.

1.1.9. BCR-ABL oncoprotein and apoptosis

BCR/ABL tyrosine kinase enhances survival of leukemic cells through modulation of pro-apoptotic and anti-apoptotic molecules in chronic myelogenous leukemia.

BCR/ABL is involved in Ras signaling, JAK/STAT kinase pathways, PI3K/AKT kinase pathway, just to mention the most important ones [24-27]. BCR/ABL was found responsible of decreased expression of pro-apoptotic protein Bim, a Bcl-2 mediator [28-

30]. BCR/ABL acts on PI3K/AKT pathway [31] that modulates activation of Bad and

BCR/ABL prevents the translocation of Bad and Bax to the mitochondria [32-35].

Therefore BCR/ABL involvement in intricate mechanisms of cellular signaling is responsible for its oncogenic action. CK2 is an interacting partner and regulator of

BCR/ABL [2, 4-6, 13, 36, 82-86].

1.1.10. Casein kinase 2 and BCR/ABL

The up-regulation and hyperactivity of CK2 has an anti-apoptotic effect in leukemia and results in abnormal platelet counts [12]. Interestingly, CK2α was found to be a substrate for the ABL domain of BCR/ABL [36] and to form a specific complex with the BCR domain of BCR/ABL [37]. It was hypothesized that CK2α impedes sterically the binding of the ABL SH2 domain to BCR [37]. This results in proliferation abnormalities in Ph+ cells. Therefore CK2α was shown to be a possible regulator of

BCR/ABL function [36, 37]. Other functions of CK2α downstream of the BCR/ABL interaction may provide an overall cancer promoting response in Ph+ positive cells.

1.1.11. Megakaryocytopoiesis and thrombocytopoiesis processes

Megakaryocytes are polyploid cells, originating from hematopoietic stem cells in the bone marrow. All hematopoietic progenitor cells express surface antigens CD34 and

CD41 and characteristic to the megakaryocytic lineage is the expression of CD61 and increased expression of CD41 [87]. CD61 antigen recognizes a 110-kDalton protein, also known as GpIIIa, the common β subunit (integrin β3-chain) of the GpIIb/IIIa complex

(αIIbβ3) and the vitronectin receptor – αVβ3– [88]. The GpIIb/IIIa complex and αVβ3 are glycoproteins from the class of integrins. Integrins are α/β- heterodimeric glycoprotein complexes that are involved in cell adhesion. The CD41 antigen corresponds to the GpIIb

(αIIb) protein. The GpIIb/IIIa (αIIbβ3) complex is formed from the GpIIIa (antigen CD61) and GpIIb (antigen CD41) proteins. This GpIIb/IIIa (αIIbβ3) complex acts as a receptor for fibrinogen, von Willebrand factor (vWf), fibronectin and vitronectin on activated platelets. With the αVβ3, α-chain or αv (CD51 antigen), the GpIIIa (CD61 antigen) forms

αVβ3. αVβ3 mediates activation-independent cell adhesion to vitronectin, vWf, fibrinogen, and thrombospondin. The CD61 and CD41 antigens are found on all normal resting and activated platelets. The CD61 antigen is found on endothelial cells, megakaryocytes, and on some myeloid, erythroid, and T-lymphoid leukemic cell lines. CD41/CD61 complex is expressed on platelets, megakaryocytes, and early hematopoietic progenitors [89-93].

Megakaryocytes produce and release platelets into the circulation, through a complex process, normally regulated by cytokines (thrombopoietin –TPO– and interleukins –IL–). TPO and interleukins, which are modulating all the steps of megakaryocytic development (megakaryocytopoiesis) were proven not to be essential in the final stages of platelets production (pro-platelets formation and platelets release) [94].

More interesting, TPO was found to inhibit platelets formation in vitro [87, 95].

Megakaryocyte progenitors are usually identified the bone marrow by immunoperoxidase and AChE labeling. Megakaryoblasts undergo endomitosis (usually

to attain a DNA content of 16N, but higher DNA contents were noticed) and cytoplasmic maturation to the stage of megakaryocytes prior to platelets release, through a process called megakaryocytopoiesis (see figure 1.4). The first precursor in the megakaryocytic linage is the promegakaryoblast (2N ploidy level). The megakaryoblast comes from the promegakaryoblast and has two sets of chromosomes (4N) and is 10 to 50 µm diameter.

In Romanovski stain is intensely basophilic (this is an indication of high quantities of ribosomes). The platelet specific granules are not found in megakaryoblasts.

Promegakaryocyte stage is the last one before megakaryocyte stage is achieved.

Promegakaryocytes are bigger in diameter (20-80 µm) with the cytoplasm less basophilic and are positive for alpha granules. After the process of endomitosis is finished, megakaryocytes undergo cytoplasmic maturation. During this process, the cytoplasm fills with platelets-specific proteins, organelles and membrane systems (demarcation membrane system or “platelets maps”, the dense tubular system) and secretory granules.

Mature megakaryocytes reach the pro-platelets bearing stage as final level of this differentiation process. Proplatelets bearing megakaryocyte fragments give rise to the anucleated cells, platelets, through the process of thrombocytopoiesis, see figure 1.5 [87,

94, 96-106].

1.1.12. Apoptosis and platelets production

The thrombocytopoiesis process (platelets production from megakaryocytes) is not fully understood but is linked with constitutive apoptosis of megakaryocytic cells [99,

101-113]. It has been shown that pro-platelets bearing megakaryocytes contain nuclei with typical apoptotic chromosomal condensation and DNA fragmentation. The whole process of platelet formation from megakarycoytes consists of cytoskeletal

reorganization, membrane condensation, ruffling and specific apoptotic nuclear changes

[87]. Megakaryocytes with proplatelet extensions were stained positive for active caspases [99]. Caspase activation in megakaryocytes is connected with platelet production. Caspase 3 and 9 are active in mature megakaryocytes and their inhibition stops proplatelets formation [108]. Caspase 3 is overexpressed and caspase 9 is not present in platelets. Caspase 12 found in megakaryocytes is absent in platelets [87].

Apoptosis inducing cytokines like TNFα and INFα trigger the release of platelets [99,

109]. Megakaryoblasts from individuals with CML are capable of differentiating in vitro in response to Phorbol 12-myristate 13-acetate (PMA) [96, 114], nitric oxide (NO) [109], aphidicolin and nocodazole (which are apoptosis inducers, by inhibiting DNA synthesis)

[106, 111]. Megakaryocytopoiesis results in platelet formation as a consequence of mitochondrial cytochrome c release, either by intrinsic apoptotic pathway or by type 2 extrinsic apoptotic pathway [99, 108, 113]. Apoptosis inhibitory proteins such as Bcl-2 and Bcl-XL are expressed in early megakaryocytes but are not found in mature megakaryocytes and platelets [104, 112]. Nitric oxide is also expressed in mature megakaryocytes and has a pro-apoptotic function [109]. Transforming growth factor-β1 and SMAD induce apoptosis in megakaryocytes (pro-apoptotic) [115].

Figure 1.4. Hematopoietic stem cell differentiation. The megakaryocytopoiesis process starts with hematopoietic progenitor cells named pluripotent hematopoietic stem cells. Such cells become uncommitted stem cells. Further, due to specific stimuli (like cytokines) uncommitted stem cells will become committed stem cells. Myeloid stem cells are such committed stem cells, which can develop into megakaryocytic lineage. Committed myeloid stem cells will differentiate into megakaryoblasts, again due to specific stimuli. Megakaryoblasts will undergo a process of differentiation, going through the intermediary stage named pro-megakaryocyte to reach the final stage of megakaryocyte. [105]

Figure 1.5. The platelets production (thrombocytopoiesis) process. After the megakaryocytopoiesis process is accomplished, megakaryocytes A, increase their size and undergo nuclear endomitosis and cytoplasmic maturation B, microtubules move to the cell cortex and the cell starts to form pseudopods C, the megakaryocyte reaches the pro-platelets bearing stage D, proplatelets are released and finally platelets will rupture out and the megakaryocyte nucleus is extruded. [116]

Pro-apoptotic and pro-survival proteins are regulated towards apoptosis during megakaryocytopoiesis and thrombocytopoiesis [99, 103, 105, 107, 108, 112]. In megakaryoblasts, apoptosis inducers produce proliferation arrest, followed by maturation to megakaryocytes towards the pro-platelets bearing stage. Next, blebbing and compartmentalized fragmentation of megakaryocytes concludes with platelets shedding through an apoptotic process.

1.1.13. Cell cycle and endoreplication in megakaryocytic cells

DNA synthesis occurs independently of mitosis for endoreplication and for this to happen, several cell cycle checkpoints must be overcome (figure 1.6). The checkpoints that a cell needs to overcome to undergo endomitosis, are located in late S-phase (S/M, telophase, centrosome duplication checkpoints), in G2-phase (G2/M checkpoint), and in

M-phase (spindle and post-spindle checkpoints) [117-124]. In the endoreplication cycle, many of these checkpoints are naturally absent or are naturally overcome. Human megakaryocytes with 8N-128N giant nuclei express cyclin B1 correlated with H1 histone kinase activity. Endomitosis in megakaryocytes occurs because of abortion of mitosis in late anaphase and collapse of cytokinesis. Consequent reinitiation of G1 and S phases results in polyploidy [125-127]. 8N-ploidy represents a general limit, above which cells cannot execute mitosis. 16N-32N cells and cells of higher ploidy are usually unable to divide by mitosis, and they abrogate cytokinesis. Megakaryocytes belong to the so-called hyperploid class of polyploids [120, 121, 124, 127, 128].

The megakaryocytic progenitor cells start the cell cycle, undergoing the G1 phase, continues with the S phase for DNA synthesis and then the G2 phase (short) is continued with the beginning of the mitotic cycle. However, megakaryocytic cells go from prophase

to anaphase A, but do not enter anaphase B, neither telophase and therefore they do not undergo cytokinesis. After reaching anaphase A, megakaryocytes nuclear envelope breaks down and a specific spherical mitotic spindle forms. The spindle poles do not move apart and newly formed sister chromatides remain together. A new nuclear envelope emerges forming a lobed and big nucleus [129, 130].

As it can be seen in figure 1.6, G2 arrested cells can go directly from the G2/M damage checkpoint into the endocycle (the case of endomitotic cancer cells) or go on to undergo apoptosis (normal cells). If the G2/M checkpoint is adapted, cells can also enter into mitosis, and from there, they may arrest in the metaphase. In turn, M-arrest has three alternative exits: to return to the mitotic cycle, to undergo apoptosis (normal cells) or, through restitution, to enter the endocycle (cancer cells). Normally, mitosis restitution is unstable and can be aborted, therefore inducing apoptosis, however malignant cells cannot do this. Endocycling cells often rash apoptosis, but can also return through somatic reduction to the mitotic cycle. The checkpoint(s) for this last process are unknown [124, 131, 132].

Figure 1.6. Cell cycle diagrams. On the left of the figure is the cell cycle description. On the right of the diagram are the cell cycle checkpoints and connection with apoptosis and endoreplication. Left: G1 phase – synthesis of RNA and proteins, preparation for S phase, S phase – replication of DNA, G2 phase – preparation for M (mitosis), M phase – mitosis (prophase - chromosome condensation, nuclear envelope breakdown, mitotic spindle assembly; metaphase – kinetochore assembly at each centromere and association with microtubules, anaphase – separation of sister chromatids, telophase – mitotic spindle disassembly, chromosome decondensation); Right: The diagram shows the available switch-points from the mitotic pathway to the endocycle (endomitosis) in cancer cells and stimulation of this switch by DNA damage. [124]

1.1.14 Casein kinase 2 and cell cycle

A variety of signals regulate the endomitosis process. However Cdc2 and cyclin B form a protein complex named mitosis promoting factor (MPF) which seems to control the the entrance of cells into mitosis. MPF has a kinase activity and either inhibition of cdc2 or of cyclin B will result in endomitosis. However other signals play independent functions in the control of the mitosis process. Absence or inhibition of Aurora-B/AIM-1 and survivin are causing endomitosis [133, 134].

CK2 has been found to phosphorylate cell cycle proteins and by this to modulate their activity. The use of DRB (a CK2 inhibitor) as well as antisense depletion of CK2 resulted in an inhibition of MPF activation (cdc2/cyclin B) in response to nocodazole in epithelial cells. Interestingly, when the cell cycle characteristics of nocodazole treated cells were examined, it was found that depletion or inhibition of the catalytic subunit of

CK2, in the presence of microtubule inhibitors, resulted in a compromise of the G2 arrest

(spindle checkpoint). It was also found that CK2 increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription. Other cell cycle proteins that are regulated by CK2 are cdc25 and topoisomerase II [43, 135-138].

CK2 was found to regulate both apoptosis and cell cycle following DNA damage induced by 6-thioguanine (apoptosis inducer), by controlling caspase and cdc2 proteins activity. CK2α relocalize from nucleus to the endoplasmic reticulum after 6-thioguanine treatment. CK2 is essential for apoptosis inhibition due to 6-thioguanine [139]. Ionizing radiation induces CK2 to inhibit apoptosis and CK2α to move to perinuclear structures.

CK2α reduction affects cell cycle progression independent of ionizing radiation in the

transformed HeLa cells. CK2 negatively regulates caspase activity following ionizing radiation exposure of HeLa cells [140]. CK2 has a deregulated expression in tumors.

CK2 signaling activity related to cell growth is focused on sites like nuclear matrix and chromatin. A nuclear localization of CK2 is associated with the neoplastic transformation. Therefore a higher intracellular nuclear localization is observed in tumor cells compared with normal cells, where the cytosolic localization is dominant [3-5, 7,

141]. All these information related to nuclear localization of CK2 in malignant processes suggest that CK2 action on cell cycle progression protects cells from apoptosis first of all and induces abnormal proliferation.

BCR/ABL inhibits apoptosis and cause anchorage independent growth. BCR/ABL signaling acts on down-regulation of cyclin-dependent kinase activity and cell cycle arrest in serum and growth factor deprived cells, thus inhibiting both apoptosis and altering cellular proliferation [142]. As it was proposed [36, 37], CK2 maybe an arbitrator of the BCR/ABL function and a possible interacting protein. Therefore CK2 may play a very important role in the cell cycle regulation and cellular proliferation of the transformed CML cells.

Altogether, CK2 inhibition may provide a very useful tool in the study of megakaryocytic endomitosis and it points out again that CK2 deregulation endows cells with oncogenic properties.

1.1.15 Platelets

Platelets are very small blood cells, 2 to 5 µm and have a thickness of 0.5 µm and a mean cell volume of 10 femtoliters. The lifespan of platelets is 7 to 10 days. Platelets are also known as thrombocytes. Thrombocytes are anucleated cells and are characterized

by a very complex physiology [87]. They are capable to undergo shape-change in response to agonists and to release α-granules, various molecules (like coagulation factors), expose P-Selectin and phosphaditylserine and to aggregate and form the fibrin clot. In vivo animal studies with adenosine diphosphate (ADP), a platelet agonist, showed a link between platelets aggregate formation and myocardial infarction [143]. Platelet phospholipids present a very high content of arachidonic acid and have a transcellular eicosanoid metabolism between them, leukocytes and endothelial cells [144]. The blood coagulation cascade occurs on the lipid membrane of platelets [145]. This cascade culminates with the formation of fibrin from fibrinogen, which will “glue” the platelets, to form a “stable” clot. The platelets plug however presents the feature of “clot retraction” [146]. This is due to fibrin lysis as well as due to the large amounts of contractile proteins like actin and myosin, which are muscle proteins, present in platelets, which make platelets extremely mobile [87]. Surface platelets receptors like GpIIbIIIa or

αIIbβ3 (integrin complex) are at the molecular basis of Glanzmann thromboasthenia.

GpIIbIIIa acts as a receptor for fibrinogen. Integrins are involved in cell adhesion and aggregation [147]. Defects in platelets integrins cause various disorders, like Bernard-

Soulier syndrome [92]. Defects in platelets clot formation causes bleeding disorders like haemophilia (“A” due to factor VIII deficiency and “B” due to factor IX deficiency) and increased clotting like thrombosis (mainly due to factor V defects). Platelet counts are very important, low platelet counts (thrombocytopenia) causing bleeding and high platelet counts (thrombocytosis) causing thrombosis, ischemia, organ failure, stroke, myocardial infarction [87, 148].

Studies have shown that platelets can interact directly with tumors, and some tumors may induce or enhance platelet aggregation. Low platelets counts have been correlated with decreased tumor formation and inhibition of metastasis. Platelets are rich in vascular endothelial growth factor (VEGF) which is released when platelets are activating, makes platelets an important player in the tumor increased angiogenesis.

Platelets have been involved in the pathogenesis of psychiatric disorders (like anxiety disorders), Alzheimer, schizophrenia [87].

Therefore platelets are very important in maintaining normal hemostasis and play a vital function in the body.

1.1.16 Casein kinase 2 and platelets

CK2 was found to be constitutively active in platelets and appears to be a major messenger-independent protein. CK2 function in platelets is regulated by protein phosphatases or by accessibility of substrates [149]. Platelets CK2 phosphorylates coagulation factors [148]. CK2 also acts on adherence and blood coagulation pathways

[82, 148, 150-152]. Therefore in hematological malignancies like CML, CK2 high concentration and hyperactivity not only affects the hematopoietic stem cells but also platelet number and activation. This leads to thrombosis, due to both platelet counts and platelet abnormal function.

1.2 GENERAL METHODS INTRODUCTION

1.2.1 Principles of flow cytometry

Flow cytometry is a method that uses an instrument called flow cytometer (or cytofluorometer) to collect multi-parametric data from particles and cells (as small as 0.5

µm up to 200 µm).

Principles of light scattering, light excitation and emission of fluorochrome molecules.

Cells are hydro-dynamically focused in sheath fluid (PBS) and intercepted with a focused light source, a laser, see figure 1.7. The nozzle of the instrument suctions cells at variable speeds creating a single-cell flow. In this way the laser hits each cell separately and the scatter from each cell is collected, see figure 1.8. Each cell has its own scatter

(“auto-fluorescence”), but when labeled with fluorochromes they are scattered at a higher energy state and the fluorochrome emission signal can be identified. For commonly used fluorochromes emission and excitation spectra (table II). The most unique feature of flow cytometry is that measures fluorescence per cell or particle [153]. Sophisticated analysis

(statistics) and collection software provides high quality data that is used in advanced diagnosis (in clinical laboratory), providing accurate blood counts, and identifying diseases like hematological malignancies, as well as a powerful tool in medical research, molecular biology, marine biology.

Table II. Emission and excitation spectra for most common fluorochromes (dyes) used in flow cytometry.

FL1, FL2, FL3, FL4 correspond to each channel (laser). The wavelength is measured in nm. [153]

Figure 1.7. Flow chamber and laser beam assembly of a flow cytometer. Hydro-dynamic focusing of the cell suspension occurs in the flow chamber due to the sheath fluid. Single-cell suspension is hit by the laser light and the scattered light is collected. [153]

Figure 1.8. Optics of a flow cytometer. SS = side scatter, FS = forward side scatter, BP = band pass, BK = blocking filter, DL = dichroic long pass [153]

Flow cytometry applications.

Parameters that can be measured with flow cytometry are: volume and morphological complexity of cells, cell pigments such as chlorophyll or phycoerythrin,

DNA (cell cycle analysis, cell kinetics, proliferation etc.), RNA, chromosome analysis and sorting (library construction, chromosome paint), protein expression and localization, transgenic products in vivo, particularly the green fluorescent protein or related fluorescent proteins, cell surface antigens (Cluster of differentiation (CD) markers), intracellular antigens (various cytokines, secondary mediators etc.), nuclear antigens, enzymatic activity, pH, intracellular ionized calcium, magnesium, membrane potential, membrane fluidity, apoptosis (quantification, measurement of DNA degradation, mitochondrial membrane potential, permeability changes, caspase activity), cell viability, monitoring electro-permeabilization of cells, oxidative burst, characterising multidrug resistance (MDR) in cancer cells, glutathione, various combinations (DNA/surface antigens etc.) [153].

Basic flow cytometry operations.

Gating. Through the operation of gating we have to possibility to focus on a population of cells that is of interest. Gating is done through the software, is not a physical thing, but it allows the possibility to focus on what is important and some physical changes can be applied to the collection mode, like adjusting the best compensation for this specific population.

Compensation. Due to spectral overlap between the emission and excitation spectra of each dye (color), the optics of the instrument (see figure 1.8.), need the software, besides their factory setup in order to eliminate the “spillover”, and this process

is called compensation. Most software’s also allow the user to do compensation during analysis. Data is represented as histograms, dot plots isometric displays, contour plots, chromatic plots, 3D projections [153].

General compensation protocol for three dyes in tandem

To perform this work a FACS Calibur flow cytometers was used, which is an older generation instrument. Such instruments require compensation done by user, using specific flow cytometry software.

A mixture of approximately equal parts of unstained cells, green-only cells and orange-only cells (U/G/O mix) was used. The same mixtures were made for orange and red cells (U/O/R mix). Six samples were prepared for three-color compensation as follows: (1) unstained (no dyes) to remove background noise, (2) green-only that correspond to the FL1 channel (FL1-H), (3) orange-only that correspond to the FL2 channel (FL2-H) and (4) red-only that correspond to the FL3 channel (FL3-H), (5) the

U/G/O mix (equal parts of unstained, green and orange) and (6) the U/O/R mix (equal parts of unstained, orange, red). The samples are run on the instrument, in order. Sample

(1) was run with unstained cells to establish proper settings such as PMT and voltage.

This procedure enabled to visualize correctly the cells on the FSC-H, SSC-H dot plot.

Sample (2) was run with green cells and FL1-H was adjusted to get a good signal. In the same manner each of the following sample was run in order to adjust FL2H (orange) and

FL3H (red), respectively. After all the settings were done, sample (5) with U/G/O mix was run on the cytometer. FSC/SSC settings have to be adjusted more, in order to bring cells on scale, and then gating around the population of interest is required. Dot plots will be constructed for the pair FL2 (y) versus FL1 (x) and then for the pair FL3 (y) versus

FL2 (x). The plots are going to be set to display only events in the above gate. The FL1,

FL2 and FL3 amplifiers will be set to logarithmic mode. Now adjustment of the voltage on the FL1 detector to position the negative cell population at about FL1=100 will be performed, keeping the bright FL1 population on scale. The same procedure will be applied to FL2 channel. Sample (6) with the U/O/R mix has to be run next on the cytometer. FL3 versus FL2 plot will be referred. The voltage on the FL3 detector will be adjusted to position the negative cell population at about FL3=100, but keeping the bright

FL3 population on scale. Now, FL3-%FL2 compensation control is next adjusted, in order to bring the orange-only cell population downwards until the FL3 median of this population is approximately the same as the FL3 median of the negative cells. In the similar fashion, adjustment of the FL2-%FL3 compensation control to bring the red-only cell population leftwards until the FL2 median of this population is approximately the same as the FL2 median of the negative cells. Sample (6) the U/G/O mix is run on the cytometer. FL2 versus FL1 plot is referred. We need to adjust now the FL2-%FL1 compensation control in order to bring the green-only cell population downwards until the FL2 median of this population is approximately the same as the FL2 median of the negative cells. In the same way, we need to adjust the FL1-%FL2 compensation control to bring the orange-only cell population leftwards until the FL1 median of this population is approximately the same as the FL1 median of the negative cells. Some cytometers have an extra pair of compensation controls for FL1 and FL3. A dotplot of FL3 versus FL1 will be created and FL3-%FL1 and FL1-%FL3 adjusted, at this point. For most purposes it is adequate to set compensation "by eye" as in the two color example. If accuracy is required, then regions around the 3 cell populations will be draw, statistics will be

displayed and fine adjustments to the compensation controls will be made, in order to equalize the median fluorescence values. Compensation will be now set correctly. Newer generations of instruments have auto-compensation capabilities.

1.2.2. Scanning Electron Microscopy (SEM)

Principles of SEM

The Scanning Electron Microscope (SEM) provides an image of surfaces and is capable of both high magnification and good depth of field, so that even at low magnifications it is often more useful than an optical light microscope. SEM uses electrons instead of white light to view the specimen, see figure 1.9. The scanning electron microscope (SEM) provides a three-dimensional high-resolution image of cells and tissues. In SEM the surface of the tissue is studied. The electrons are generated from a thin tungsten wire in the "gun" of the SEM. Electricity is passed through the wire and then focused by magnets onto the sample. When the electrons from the gun strike the surface coating of gold, electrons are reflected back off the specimen to a detector, this is transmitted to a television screen where the image is viewed and photographed.

Sample Preparation

Tissues prepared for SEM are usually chemically fixed using glutaraldehyde buffer and then dehydrated in ethanol before being dried. Drying at critical point has become the preferred method. The tissue is introduced into liquid carbon dioxide which is then brought to its critical point. The critical point is the combination of pressure and temperature at which the fluid and gaseous phases exist together without an interface or meniscus. Thus there is no surface tension. The presence of surface tension during drying is disruptive to a tissue and causes visible distortions.

Figure 1.9. Scanning Electron Microscope (SEM). An SEM consists of an electron source (the electron gun), condenser lens, objective, scanning coil and sample chamber [154].

After drying, the surface of the tissue is sputter coated in a vacuum with an electrically conductive layer of gold. [109, 154, 155]. The coated dry sample is placed in a vacuum so that the electron beam can move without interference. SEM has a variety of applications including material surface studies (chemistry) and observation of detailed surface cell morphology.

1.2.3. Transmission electron microscopy (TEM)

Principles of TEM.

The TEM can resolve objects 0.001 µm apart. A TEM consists of a tall, evacuated column which has a heated filament that emits electrons at its top. The electrons are moved at uniform high velocity down the column by a high voltage (around 100kV) and pass through the slice of specimen at about half-way down. Because the density of the specimen varies, the “shadow” of the beam falls on a fluorescent screen near the bottom of the column and forms an image. A camera mounted beneath the screen records the image. The electron beam is controlled by magnetic fields produced by electric coils, called electron lenses. One electron lens, called the condenser, controls the beam size and brightness before it strikes the specimen. Another electron lens, called the objective, focuses the beam on the specimen and magnifies the image about 50 times. Other electron lenses below the specimen then further magnify the image. [100, 109, 110, 154,

155]

Figure 1.10. Transmission electron microscope (TEM). A TEM consists in an electron source, electron beam focusing on the sample, electromagentic lense and data collector device (camera) [154].

Sample Preparation

Uses of TEM are various like study of the internal structure of the cells as well as study molecular interactions, like protein complex formation.

Cells are fixed in 2.5% glutaraldehyde buffer When the sample is ready for TEM, the sample will be washed with PBS three to five times. Then cells are refixed in 1-4% osmium tetroxide buffer and then dehydrated in ethanol steps. Next samples are infiltrated with one part resin-epon/two parts solvent (overnight) and two parts resin- epon/one part solvent (overnight) and finally with 100% resin-epon- (one hour). The cells in 100% resin are placed in a suitable container (embedding). Polymerization is done at

60-700C overnight. Sections are cut through thin sectioning and then samples are analyzed using a Phillips CM12 TEM microscope.

TEM is a powerful imaging technique providing accurate intracellular details at very high magnifications making possible observation of intracellular ultrastructures like organelles.

1.2.4. Confocal microscopy

Confocal microscopy [155] is an optical imaging technique used to increase micrograph contrast. Confocal microscopy use includes reconstruction of three- dimensional images by using a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane, see figure 1.11. In a conventional fluorescence microscope, the entire specimen is flooded in light from a light source. Due to the conservation of light intensity transportation, all parts of the specimen throughout the optical path will be excited and the fluorescence detected by a photodetector or a camera.

Figure 1.11. Confocal microscope. A confocal microscope consists of a laser excitation source, dichromatic mirror which focuses the excitation light rays through the objective on the sample and the detector pinhole which eliminates out of focus light rays from the samples and the detector [155].

In contrast, a confocal microscope uses point illumination and a pinhole in an optically conjugate plane in front of the detector to eliminate out-of-focus information.

Only the light within the focal plane can be detected, so the image quality is much better than that of wide-field images. As only one point is illuminated at a time in confocal microscopy, 2D or 3D imaging requires scanning over a regular raster, which is a rectangular pattern of parallel scanning lines, in the specimen. The thickness of the focal plane is defined mostly by the square of thenumerical aperture of the objective lens, and also by the optical properties of the specimen and the ambient index of refraction.

Sample preparation for confocal microscopy.

Basically the protocol is the same as for the preparation of cells for regular fluorescence micrsocopy. Shortly cells are washed, then fixed (2% paraformaldehyde in

PBS), then permeabilized if necessary with Triton X-100, 0.1% solution in PBS. After permeabilization and fixation, cells are washed and stained with specific fluorochrome conjugated monoclonal antibody or protein (like phalloidin). After staining and washing to remove unbound stain, cells are mounted and then analyzed using the confocal microscope.

Confocal microscopy provides a wonderful imaging technique when various proteins from cells can be stained to observe their localization (nuclear or cytoplasmic), cellular membrane can be stained with „dyes” like Bodipy, Dil (Invitrogen, NY, USA), nucleus with DAPI. GFP expressing cells can also be observed in detail using this technique, like for example for imaging of embrionar stages in insects.

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CHAPTER II

TREATMENT OF MEGAKARYOBLASTIC LEUKEMIA CELLS

WITH CASEIN KINASE 2 INHIBITORS

2.1. ABSTRACT

Megakaryoblasts are hematopoietic progenitor stem cells that differentiate to the

stage of megakaryocytes. Mature megakaryocytes form pro-platelets and give rise to

platelets. We studied the effect of inhibition of casein kinase 2 alpha subunit (CK2α) with

specific inhibitors in a megakaryoblastic cell line (MEG-01) from a chronic myelogenous leukemia patient in blast crisis. We found that these inhibitors induce proliferation arrest while maintaining a steady cell number for a period of one week. Treated cells grew at a significantly lower rate than non-treated cells. Apoptosis was induced by CK2 inhibitors in MEG-01 cells, and this phenomenon was dose and time dependent. Necrosis was minimal in the presence of the inhibitors, suggesting that such compounds may not be cytotoxic. In the presence of CK2 inhibitors megakaryocytes matured to the pro-platelets bearing stage. Platelets were subsequently released through rupture, following

cytoplasmic fragmentation and nuclear extrusion. Thrombocytopoiesis due to the use of

CK2 inhibitors occurred both in suspension as well as with MEG-01 cells grown on a fibronectin matrix. The platelets release process has apoptotic features (blebbing, specific

DNA fragmentation). Platelets obtained following these treatments are functional. These platelets get activated in response to various agonists and form a fibrin clot. Such platelets adhere to collagen and fibronectin coated surfaces. The structure of such platelets was found to be comparable to normal blood platelets. These findings suggest that CK2 may be involved in MEG-01 cells differentiation and platelet production. Thus, the use of CK2 inhibitors as a tool for platelet generation in culture, provides the opportunity to study platelets production and to engineer MEG-01 cells to produce platelets that will express desired proteins.

2.2. INTRODUCTION

Normal cells are characterized by the capability to reproduce themselves exactly

and their proliferation rate corresponds to each type of cell (function and tissue) so the

body functions properly. Normal cells will self-destruct when they are damaged, or when

is necessary (through the apoptosis process) and they will become specialized or

“mature”. Normal cells signal with the surrounding cells and execute their specialized function accordingly. Cancer cells, on the other hand, are characterized by abnormal proliferation (never-ending reproducing with apparition and propagation of defects due to mutations) and loss of the normal phenotype and function. Such cancerous cells become unspecialized and can grow anywhere and manifest the feature of anchorage independence. Cancer cells do not signal with the neighboring cells and do not stick

together. Such cells do not mature and can move to other parts of the body (the metastasis

process) and due to their increased proliferation they form tumors. Cancer is caused by

defects in signaling pathways including deregulation of the apoptosis process.

Megakaryocytes are highly specialized hematopoietic precursor cells that release

into circulation the platelets. Megakaryocytes are polyploid cells, originating from

hematopoietic stem cells in the bone marrow. Megakaryoblasts undergo endomitosis

(DNA replication without cell division) and maturation to the stage of megakaryocytes,

through a process called megakaryocytopoiesis [1]. After the endomitosis is completed,

megakaryocytes undergo a cytoplasmic expansion characterized by the formation of the

demarcation membrane system (DMS). High quantities of proteins are expressed in

mature megakaryocytes and platelets specific proteins and granules are accumulating in

the cytoplasm. The demarcation membrane system reorganizes into beaded cytoplasmic

extensions called pro-platelets. Proplatelets bearing megakaryocytes fragment to give rise to platelets, through the process of thrombocytopoiesis [2]. Thrombocytopoiesis is the development of anucleated cells, platelets, from megakaryocytes [3].

Platelets (or thrombocytes) are vital for maintaining normal hemostasis and for the response of the body to trauma. The process of platelet formation is complex and not well understood [4]. The thrombocytopoiesis process was linked to the constitutive apoptosis of megakaryocytes. Caspase activation in megakaryocytes was connected with platelets production. Pro-apoptotic and pro-survival balance is shifted towards apoptosis during megakaryocytopoiesis and thrombocytopoiesis [3, 5-10].

The reciprocal chromosomal translocation t(9;22), known as the Philadelphia positive chromosome (Ph+) [11] is associated with chronic myelogenous leukemia

(CML). This genetic abnormality results in the chimeric oncoprotein BCR/ABL tyrosine kinase, which is thought to be the main cause of the abnormal survival and overproliferation of hematopoietic stem cells and their progeny [12-14]. In chronic myelogenous leukemia, the BCR/ABL tyrosine kinase is constitutively activated.

Different intracellular pathways are transformed by the oncoprotein BCR/ABL, resulting in uncontrolled hematopoietic proliferation. The late phase of chronic myelogenous leukemia, named blast crisis (or blastic phase), is characterized by extreme overproliferation of stem cells and their progeny in bone marrow. In myeloproliferative disorders, like chronic myelogenous leukemia, the platelet counts and function are abnormal due to malignant megakaryoblasts overproliferation [13, 15].

MEG-01 cell line. A megakaryoblastic leukemia cell line named MEG-01 was established in 1983. MEG-01 was isolated from a bone marrow aspirate of a patient with

chronic myelogenous leukemia in megakaryocytic blast crisis [16]. MEG-01

megakaryoblasts were found to release platelets that are morphologically

indistinguishable from normal blood platelets, viable and functional, when treated with

apoptosis inducers. These cells are of interest for our proposal because they are cytokine

independent [17-19], and therefore do not respond to cytokine treatments (interferons and

thrombopoietin), and can be induced to release platelets [6, 16, 20-23]. Cytokine

independence imposes a problem when using the cytokine treatment for the advanced

stage of CML (the blastic phase). MEG-01 cells were characterized as being

megakaryoblasts in the early stage of differentiation on the megakaryocytic lineage, and

they are committed progenitors towards platelet production. These cells have

Philadelphia positive chromosome, expressing BCR/ABL oncoprotein [16, 21, 22].

MEG-01 cells do not express c-mpl (the thrombopoietin receptor) and have a non-

functional p53 (due to a major deletion) [24, 25]. Due to the fact that c-mpl-/- knockdown mice survive and have functional megakaryocytes and platelets, even though they present a reduction in the number of megakaryocytes and do not present severe bleeding abnormalities and because it was shown that the thrombocytopoiesis process does not depend on thrombopoietin (TPO) only, it has been suggested that actually TPO is not required for the final stages of platelets release. Therefore other factors are responsible for megakaryocytic differentiation and platelets release [2, 26]. So such a cell line, like

MEG-01 (cytokine independent) can be used to study what other factors may contribute to the thrombocytopoiesis process.

The up regulation and hyperactivity of CK2 has an anti-apoptotic effect which is associated with abnormal platelet counts and function in leukemias [27]. Interestingly,

CK2α was found to be a substrate for the ABL domain of BCR/ABL [28] and to form a

specific complex with the BCR domain of BCR/ABL [29]. It was hypothesized that

CK2α impedes sterically the binding of the ABL SH2 domain to BCR [29]. This results

in proliferation abnormalities in Philadelphia positive cells. Therefore CK2α was shown

to be a possible arbitrator of BCR/ABL function [28, 29]. Other functions of CK2α

downstream of the BCR/ABL interaction give an overall oncogenic response in

Philadelphia positive cells.

CK2α protein kinase inhibitors have been developed and studied, like emodin, 5, 6-

dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), 4,5,6,7-Tetrabromobenzotriazole

(TBB), 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), ellagic acid

[30-36]. Inhibition of CK2 in various cancer cell lines produced apoptosis and proliferation arrest [30-32, 37-47].

Given these facts, it will be interesting to assess the effects of CK2 inhibitors on the

MEG-01 cell line which has an increased proliferation and survival rate.

2.3. EXPERIMENTAL PROCEDURES

2.3.1. Cell Culture

Materials

Cell Dissociation Buffer Solution (CDBS) - Gibco BRL (Gaithersburg, MD, USA)

DMEM F12 1X - Central Cell Services, Media Lab, Lerner Research Institute,

Cleveland Clinic (Cleveland, USA)

Endothelial Cell Growth Factor Supplement (ECGS) – BD Biosciences (Bedford,

MA, USA)

Fetal Bovine Serum (FBS) - Invitrogen (Carlsbad, CA, USA)

Fibronectin - BD Biosciences (Bedford, MA, USA)

L-Glutamine - Central Cell Services, Media Lab, Lerner Research Institute,

Cleveland Clinic (Cleveland, USA)

Hank’s Balanced Salt Solution (HBBS) – Gibco BRL (Gaithersburg, MD, USA)

HEPES Buffer - Invitrogen, (Carlsbad, CA, USA)

Heparin Sodium Salt- Calbiochem (San Diego, CA, USA)

Penicillin/Streptomycin - Invitrogen (Carlsbad, CA, USA)

Phosphate Saline Buffer (PBS) - Central Cell Services, Media Lab, Lerner

Research Institute, Cleveland Clinic (Cleveland, USA)

RPMI 1640 1X - Central Cell Services, Media Lab, Lerner Research Institute,

Cleveland Clinic (Cleveland, USA)

Sodium Pyruvate Solution - Invitrogen (Carlsbad, CA, USA)

Trypsin/EDTA Solution – Sigma Chemical Co (St. Louis, MO, USA)

Trypsin Neutralization Solution (TNS) – ScienCell (Carlsbad, CA, USA)

MEG-01 cells culture

MEG-01 megakaryoblastic cell line [16], was a generous gift from Dr. P. B. Tracy,

(Department of Biochemistry, University of Vermont, College of Medicine, Burlington,

VT, USA). MEG-01 cells were also purchased from American Tissue Culture Collection

(Manassas, VA). Cells were maintained in an incubator, with humidified atmosphere of

0 CO2 5%, and at 37 C. Cell culture media was RPMI 1640 1X with L-Glutamine (2mM),

was adjusted to contain 10 mM HEPES, and 1.0 mM Sodium Pyruvate and 10-20 % heat-

inactivated fetal bovine serum (FBS) and 5U Penicillin/Streptomycin. Cells were seeded

at 2 X 105 cells/ml, media was renewed and cell number was adjusted two times per

week. 20% of FBS was observed to improve the growth and endurance of MEG-01 cells.

Cells were washed by centrifugation (150 g for 5-10 minutes) and the washing buffer was

PBS. The mature MEG-01 cells become adherent to the culture flask wall and therefore

in order to provide a better surface area for them, flaks can kept on the side, this will also

help with the platelets production. For some experiments, MEG-01 cells were grown on

fibronectin coated plates ( 1 µg/cm2).

HUVEC cells culture

Human umbilical vascular endothelial cells (HUVEC) were purchased from Dr. P.

DiCorletto (LRI, Cleveland Clinic, Cleveland, OH, USA) by Dr. Byzova TV (LRI,

Cleveland Clinic, Cleveland, OH, USA). HUVEC cells were grown in DMEM F-12 cell

culture media supplemented with 15 % heat-inactivated fetal bovine serum (FBS) and

endothelial growth factor supplement (ECGS) 150 µg/ml, heparin sodium salt (90 µg/ml)

and 5U Penicillin/Streptomycin. HUVEC cells were grown on fibronectin coated

surfaces. Fibronectin was used at a concentration of 1 µg/cm2 in PBS buffer. Fibronectin

0 was incubated in PBS with the surface for at least 45 minutes at 37 C, in CO2 cell culture

incubator or overnight at 4 0C and after this coating procedure, the coated surface was washed twice with PBS buffer or HBBS buffer. The cell number was adjusted when confluence reached 70%, by splitting the cells. When cells needed to be detached, they were either detached with Trypsin/EDTA solution or with cell dissociation buffer (EDTA based, enzyme free) depending on application. When trypsin was used to detach cells, cells were immediately treated after trypsinization with trypsin neutralization solution

(TNS). Cells were washed by centrifugation (300 g for 5 minutes) and the washing buffer was either PBS or HBBS with 2 % FBS.

2.3.2. Cell Treatments

Materials

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) - Calbiochem

(San Diego, CA, USA)

Dimethylsulfoxide (DMSO) - Calbiochem (San Diego, CA, USA)

Phorbol 12-myristate 13-acetate (PMA) - Calbiochem (San Diego, CA, USA)

4,5,6,7-Tetrabromobenzotriazole (TBB) - Calbiochem (San Diego, CA, USA)

Method

Cells were treated with the CK2α inhibitors, TBB and DMAT [31, 32]. The vehicle for DMAT and TBB was DMSO. As a differentiation control, besides the untreated control (DMSO only), PMA was used [48]. Apoptosis and viability assays were performed with various inhibitors concentrations and time lengths of treatments in order to choose the non-cytotoxic concentrations of TBB and DMAT that have a significant

effect (titration assays). At the beginning of each treatment cells were counted with a

Neubauer hemacytometer (using Trypan blue dye at half-dilution) and were split to

~2X105 cells/ml. To assess the effect of CK2 inhibitors, the treatments length went up to

four days without splitting. Other treatments went up to one month with the required

splitting and re-treatments.

2.3.3. Platelets isolation from culture.

Platelets were separated from the megakaryocytic cells, by differential

centrifugation [9] and analyzed separately. Suspension cells (MEG-01 culture) were

centrifuged first at 400 g for 15 minutes, MEG-01 pellet was discharged. The platelets

rich supernatant was kept and centrifuged again at 2000 g for 15 minutes. The platelet

pellet was resuspended in the appropriate buffer (Tyrode’s buffer with CaCl2, pH 7.4), for

analysis. For impeding artefactual aggregation of platelets in the control sample, EDTA

and RGD or RGDS were added in the cell suspension from the beginning of the

centrifugations, and with each centrifugation step. 1 mg/ml RGD or RGDS and 10 mM

EDTA final concentrations, were used for negative control (inactivated) or resting

platelets. Platelets were activated with different agonists: 100 nM PMA, 1 µg/ml TRAP

and 0.5 U/ml human α-thrombin [49]. For most of assays TRAP 1 µg/ml was used. For scanning electron microscopy experiment (formation of the fibrin clot) we used human α- thrombin. For both sample sets, activated (with TRAP) and inactivated (EDTA, RGD or

RGDS), we used platelets collected from MEG-01 cultures incubated with 10 µM DMAT for 4 days.

2.3.4. Platelets isolation from whole blood (human)

Platelets isolation from whole blood was adapted as previously described [49-51].

Figure 2.1. Schematics of platelets isolation from whole blood. Whole blood treated with ACD and PGE-1 is centrifuged at 100 g, then the supernatant named platelets rich plasma is centrifuged again at 2400 g. After the second centrifugation step, platelets pellet is resuspended in plasma (1/3 volume) and the run over a column with Sepharose CL2B with Tyrode’s buffer 1X. Gel filtered platelets are collected from the void volume of the column (aproximative 4 ml for a 50 ml column).

Materials

Bovine Serum Albumine (BSA) – Sigma Chemical Co (St. Louis, MO, USA)

Sodium Chloride, NaCl - Sigma Chemical Co (St. Louis, MO, USA)

Sodium BiCarbonate, NaHCO3 - Sigma Chemical Co (St. Louis, MO, USA)

Potassium Chloride, KCl - Sigma Chemical Co (St. Louis, MO, USA)

Magnesium Chloride (hexahydrate), MgCl2*6H2O - Sigma Chemical Co (St. Louis,

MO, USA)

Calcium Chloride CaCl2 - Sigma Chemical Co (St. Louis, MO, USA)

Dextrose, HOCH2CH(CHOH)4O - Sigma Chemical Co (St. Louis, MO, USA)

Sodium Citrate (DiHydrate), C6H5Na3O7*2H2O - Sigma Chemical Co (St. Louis,

MO, USA)

Citric Acid (Monohydrate), C6H8O7*H2O - Sigma Chemical Co (St. Louis, MO,

USA)

Prostaglandin, PGE-1 - Sigma Chemical Co (St. Louis, MO, USA)

Sepharose CL-2B - Pharmacia, A.K.A. Amersham Biosciences (Uppsala, Sweden)

Net for column – Pharmacia A.K.A. Amersham Biosciences (Uppsala, Sweden)

Chelex 100 – BioRad (Hercules, CA, USA)

Instruments

Centrifuge: Beckmann Allegra 6, tabletop (rotor diameter is 39 cm)

Reagents

Tyrode’s stock buffer 10X (1.368 M Na Cl, 0.1199 M NaHCO3, 0.0255 M KCl

0.0206 M MgCl2*6H2O, MgCl2 is optional as well as 500 mM CaCl2)

Tyrode’s buffer 1X (diluted from 10X stock buffer 1:10 with miliq H2O), added 0.1

% BSA (A7638) and Dextrose 0.1 %, the final pH of the buffer is 7.2

ACD –anticoagulant– (84.8 mM C6H5Na3O7*2H2O, 64.8 mM C6H8O7*H2O, 111 mM Dextrose HOCH2CH(CHOH)4O)

Sepharose CL-2B (code 17-0140-01, 45-165 µm particle size)

Sepharose CL-2B column (40 to 50 ml bed volume, equilibrated with 1X

TYRODE’S buffer containing BSA and Dextrose at a pH of 7.2)

Net for column (code 19-0659-01, 45 µm, PTFE)

Chelex 100 (code 142-2832, biotechnology grade, 100 g, Na+, 100-200 dry mesh,

150-300 µm wet bead).

Observations: Plastic materials need to be used during all the procedure. Isolation of platelets requires room temperature.

Isolation of human platelets from whole blood using adapted Sepharose CL-2B

procedure, see figure 2.1.

Whole blood from healthy volunteers was collected (60-70 ml) and patients must be checked for aspirin usage. To the whole blood during collection was added an 1:6 ratio

ACD plus PGE-1 at 0.86 µM (final concentration, where molecular weight of PGE-1 is

354.5 g/mol. PGE-1 (Prostaglandin) has anticoagulant effects. Each blood tube was divided in half using new tubes. Samples were spun at 800 rpm (rotor 39 cm, 280 x g), without brake. Platelet rich plasma (PRP) was removed carefully with a plastic transfer pipet. Red cell pellet should not be touched. PRP may sit at RT (room temperature) for several hours before isolation of platelet. PRP was next spun at 2300 rpm (rotor 39 cm, is

2306 x g) for 15 minutes at room temperature, (Chronolog recommends 2400 g for 20

min at RT). Plasma was removed and platelet pellet was gently resuspended in 1 ml 1X

Tyrode’s buffer with 0.1% BSA and with 0.1% Dextrose, pH 7.2. Platelet suspension was

applied to the Sepharose Column. Washed platelets were recovered in void volume in a

final volume of about 4 ml. Platelets were counted using a Coulter counter or by

hemocytometer. At this point, the platelets isolated from human blood are ready to be

used in any experiment. Normal human platelets were used as normal control, when

appropiate.

2.3.5. Flow cytometric analysis

Cells were analyzed using a FACSCalibur flow cytometer (Becton-Dickinson), with CellQuestPro ver.3.3 software. The data was further analyzed with FlowJo ver. 6.2, and WinMDI 2.8 softwares.

2.3.6 MEG-01 samples gating of specific cell populations

Proper gating was performed to characterize each cell population (MEG-01 cells

and platelets). The population of cells high on forward side scatter (FSC-H) and side-

scatter (SSC-H), identified as large, non-granulated particles, corresponds to MEG-01

cells (figure 2.2).

Figure 2.2. Gating of the cells populations from MEG-01 culture. Proper gating was performed to characterize each cell population (MEG-01 cells and platelets). The population of cells high on forward side scatter (FSC-H) and side-scatter (SSC-H), identified as large, non-granulated particles, corresponds to MEG-01 cells (gate R1). Platelets are localized between 101-102 of the logarithmic scale (low on FSC-H and SSC-H (gate R2). A highly granulated and small population may consist in granules or debris (gate R3).

This population is involved in proplatelet formation. The population that corresponds to platelets (small, granulated particles), is PI negative because thrombocytes are anucleated cells (only the viable cells were considered). Platelets from MEG-01 cells were distinguished by their capacity to get activated, undergoing shape change in response to agonist and to show phosphaditylserine (PS) exposure when activated [6].

Platelets were further separated from the megakaryocytic cells, by differential centrifugation [9], considering the size difference between these cells populations (1-5

µm for platelets and 35-150 µm for megakaryocytic cell line MEG-01) and analyzed separately. Voltage and channels settings were adjusted accordingly. Analyzed values were obtained with WinMDI ver.2.8 or Flow Jo ver.6.2.

2.3.5.2. Flow Cytometric Apoptosis Assays with AnnexinV-FITC and propidium iodide (PI).

Materials

AnnexinV protein conjugated with Fluorescein Isothiocyanate (AnnexinV-FITC) is

a part of the kit AnnexinV-FITC and PI Apoptosis Kit I - BD Biosciences, (Bedford, MA,

USA)

AnnexinV-binding buffer 10X, is a part of the kit AnnexinV-FITC and PI

Apoptosis Kit I - BD Biosciences, (Bedford, MA, USA)

Propidium Iodide (PI) is a part of the kit AnnexinV-FITC and PI Apoptosis Kit I -

BD Biosciences, (Bedford, MA, USA)

Staurosporine - Sigma Chemical Co (St. Louis, MO, USA)

Tumor Necrosis Factor alpha (TNF α) – GenScript (Pistacaway, NJ, USA)

Ethanol, HPLC grade - Sigma Chemical Co (St. Louis, MO, USA)

Method

Apoptosis Assay Controls

For induction of typical apoptosis, cells were grown in the presence of 1µM staurosporine for 6 hours or for 24 hours with TNFα in the case of HUVEC cells (tumor necrosis factor is cytotoxic to HUVEC cells). The staurosporine treated cells were stained as follows: a control with PI, a control with AnnexinV-FITC and a control with both PI and AnnexinV-FITC in order to have the brightest controls for compensation and proper collection of the flow cytometry data. Cell necrosis was induced by heat shock (650C for

30 minutes) or ethanol treatment. Controls for necrosis were stained in the same way as for the Staurosporine treated cells. The unstained control signal was subtracted by gating from the stained control and the rest of stained samples, to get only the positive cells signal (stained). The stained control (PI and AnnexinV-FITC labeled), was then used as the reference in establishing the level of apoptosis induced by the treatments. Data was collected on logarithmic modes, two-colors. AnnexinV-FITC corresponds to FL1H channel, and PI to FL3H or FL2H channels. Proper compensation was established considering median values.

AnnexinV-FITC and PI apoptosis assay staining protocol

Freshly isolated from culture, 105 – 106 cells were stained with 50 µg/ml PI and with 0.5 µg/ml FITC-labeled Annexin V using the staining protocol provided by the supplier.

Briefly, cells were counted using a Neubauer hemacytometer, then washed (by

centrifugation with PBS buffer at 150 g for 5 minutes for MEG-01 cells and 300 g for 5

minutes with PBS buffer with 2 % FBS, for HUVEC cells). After washing, cells were

resuspended in the appropriate amount of AnnexinV-binding buffer 1X (provided in the kit), in order to have 106 cells in 100µl. Then PI and AnnexinV-FITC solutions were added to the cells. Incubation was performed for 15 minutes in the dark at room temperature. Cells were washed after incubation once as described previously, but with

Annexin V binding buffer 1X. Fresh samples were then analyzed immediately by flow cytometry. HUVEC cells when trypsinized need to be treated with trypsin neutralization solution, and the use of cell dissociation buffer is not recommended, because the high content of EDTA from this solution will inhibit AnnexinV binding to phosphaditylserine exposed on the cell membrane due to apoptosis (this binding is calcium dependent).

Treatment of cells for apoptosis assay. MEG-01 and HUVEC cells were treated with DMAT (10 and 20 µM) and TBB (25 and 50 µM) inhibitors for 24 h, 48 h, 72 h and

96 h (without splitting). Next cells were stained and analyzed as described above.

2.3.5.3. Flow cytometric DNA content assessment assay using PI/RNAse A.

Materials

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) - Calbiochem

(San Diego, CA, USA)

Dextrose - Sigma Chemical Co (St. Louis, MO, USA)

Ethanol, HPLC grade - Sigma Chemical Co (St. Louis, MO, USA)

Mesh 50 µm – BD Biosciences (Bedford, MA, USA)

PI – BD Biosciences (Bedford, MA, USA)

RNAse A I – Qiagen (Valencia, CA, USA)

Sodium Citrate – Sigma Chemical Co (St. Louis, MO, USA)

Sodium Chloride - Sigma Chemical Co (St. Louis, MO, USA)

Method

Cells were serum starved in order to synchronize them in the G0 phase of the cell cycle. Cells were fixed in 80% cold Ethanol/PBS added drop-wise and kept in the ethanol/PBS solution for at least 24 hours at 4 0C, until further processing. Before PI staining cells were washed with sterile, RNAse, DNAse free, PBS buffer. Fixed cells were incubated with 50 µg/ml PI and 100 µg/ml RNAse A-I in hypotonic citrate buffer

(0.37 g sodium citrate/L, 0.15 g sodium chloride/L, and 0.63 g dextrose/L, 0.1% Triton

X-100, pH adjusted to 6.2) for 30 minutes in dark, at room temperature. Cells were

passed through a 50 µm mesh (Falcon type) in order to eliminate clumps and further

analyzed by flow cytometry (logarithmic mode - because of the ploidy characteristic of

the cells -, one-color channel FL3H). All the protocol steps were performed at 4 0C.

Treatment of MEG-01 cells for DNA content assessment. Treatment with

DMAT 10 µM for a period of 4 days was performed and then cells were collected for

further processing. Control cells were also kept in culture like the DMAT treated without

splitting.

2.3.5.4. RPE-CD41a immunophenotyping of MEG-01 cells.

Materials

CD41a (anti human αIIbβ3 monoclonal antibody) conjugated with R-Phycoerythrin

(R-PE) - BD Biosciences (Bedford, MA, USA)

Sodium Azide (NaN3) – Sigma Chemical Co (St. Louis, MO, USA)

0.22 µm Filter – Millipore (Billerica, MA, USA)

Method

CD41a is the antigen for αIIbβ3 complex and it is found on platelets and platelet

precursors, including MEG-01 cell line [9, 16]. αIIbβ3 complex is a marker of differentiation for megakaryocytes. The staining protocol provided by BD Biosciences was used. Briefly, cells were washed (by centrifugation at 150 g) and resuspended in 1 X

PBS (with 0.1% FBS, 0.01% NaN3) 0.22 µm filtered buffer. Cells were counted and

adjusted to 106 cells/ml and 20 µl of RPE-CD41a was used for 180 µl cell suspension.

Cells were incubated with RPE-CD41a for 30 minutes, then washed with PBS buffer.

Usually cells were analyzed fresh, but they can also be fixed (2% paraformaldehyde in

PBS, overnight), when fixed, staining was done after washing thoroughly fixed samples.

RPE-CD41a stained cells were collected on FL2H channel and gating was performed on

FSC and SSC logarithmic modes. The unstained control signal was subtracted from the stained cell signal, in order to measure the staining of the cells without background noise.

2.3.5.5. Labeling of Fibrinogen with Alexa Fluor 488.

Human fibrinogen was conjugated with Alexa Fluor 488 fluorochrome (F-13191) following the recommended Molecular Probes procedure, using the Alexa Fluor 488 labeling kit. This labeling procedure was modified, in the way that the column step, was replaced with a dialysis step (performed overnight), and the bicarbonate buffer (from the

kit) was used at 1 M concentration. Before labeling, fibrinogen was also dialyzed in PBS

buffer (overnight) to remove any unwanted amines. Final concentration of fibrinogen

conjugated to Alexa Fluor 488 was determined using a spectrophotometer.

2.3.5.6. Fibrinogen-Alexa Fluor 488 staining of MEG-01 cells and analysis by

flow cytometry

The same protocol as for CD41a-FITC staining of MEG-01 cells was followed for labeling cells with Fibrinogen-Alexa Fluor 488, see point 2.3.5.4. Samples stained in this way were run on the flow cytometer and analyzed using WinMDI and GraphPad Prism software.

2.3.6. Platelets functional assays

Materials used in platelets functional assays

Human Fibrinogen - Sigma Chemical Co, (St Louis, MO, USA)

RGD peptide - Sigma Chemical Co, (St Louis, MO, USA)

RGDS peptide - Sigma Chemical Co, (St Louis, MO, USA)

TRAP peptide – Sigma Chemical Co, (St Louis, MO, USA)

CD62P (anti human P-Selectin monoclonal antibody) conjugated with FITC - BD

Biosciences (Bedford, MA, USA)

MicroDialysis Apparatus - Pierce Chemical Co (Rockford, IL, USA)

PAC-1 monoclonal antibody conjugated with FITC - BD Biosciences (Bedford,

MA, USA).

Human thrombin IIa (α)- Haematologic Technologies Inc (Essex Junction, VT,

USA)

Phalloidin conjugated with Alexa Fluor 488 - Molecular Probes (Eugene, OR,

USA)

Alexa-Fluor 488 protein labeling kit - Molecular Probes (Eugene, OR, USA).

Horm collagen - Hormon-Chemie (Munchen, Germany)

Material details.

Human Thrombin IIa (alpha) has a molecular weight of 36700 g/mol = 36700 Da.

Thrombin Unit (NIH) is defined as that amount of enzyme required to cleave 1 mg

of a test protein. Haematologic Technologies states that 3800 NIH Units of

1% Thrombin/mg and Thrombin extinction coefficient is E 1cm, 280 nM = 18.3 for

human alpha-thrombin.

TRAP peptide is an agonist, a platelet activator.

RGD and RGDS peptides are inhibitors of platelets activation.

Phalloidin is a toxin from the death cap (Amanita phalloides) and it binds actin (a cytoskeletal protein), preventing its depolymerization.

Alexa Fluor 488 is a fluorochrome commonly used in fluorescent staining of cells for microscopy or flow cytometry.

CD62P is a monoclonal antibody that recognizes a specific epitope on P-Selectin which is a protein exposed by platelets only when they are activated.

PAC-1 is a monoclonal antibody that recognizes an epitope specific only to an activated form of the protein αIIbβ3.

Fibrinogen is a zymogen, a soluble plasma glycoprotein that is synthesized in the

body, by the liver. Fibrinogen is very important in the coagulation cascade, because it makes the fibrin mesh (by polymerization when activated by thrombin) which forms together with platelets the hemostatic clot.

2.3.6.1. PAC-1-FITC binding due to platelet activation flow cytometric assay

The monoclonal antibody PAC-1 recognizes an epitope on the glycoprotein αIIbβ3 of activated platelets. PAC-1 binds only to the activated platelets. PAC-1 will not bind

EDTA and RGD or RGDS peptides treated platelets [50, 52, 53], therefore platelets

suspensions treated with any of these inhibitors were used as a negative control. Platelets

were collected from MEG-01 cells suspension, using two centrifugation steps. After

collection, platelets were activated with TRAP and then incubated with PAC-1-FITC

Activation of platelets with 1 µg/ml TRAP peptide was performed for 10 minutes. 20µl

PAC-1-FITC was used for 5 µl fresh platelets suspension, in Tyrode’s buffer with CaCl2.

The protocol provided by BD Biosciences for PAC-1-FITC staining was followed. PAC-

1-FITC antibody was incubated with the platelets for 30 minutes, in dark. After the incubation platelets were washed once by centrifugation. Samples prepared this way were run on the flow cytometer.

2.3.6.2. CD62P-FITC exposure due to platelet activation flow cytometric assay

Platelets were collected from MEG-01 cells suspension, using two centrifugation steps. After collection, platelets were activated with TRAP and then incubated with

CD62P-FITC. Activation of platelets with 1 µg/ml TRAP was performed for 10 minutes.

20µl CD62P-FITC were used for 5 µl fresh platelets suspension, in Tyrode’s buffer with

CaCl2. Incubation was performed in dark, at room temperature, for 30 minutes, as

recommended by BD Biosciences and then samples were washed by centrifugation with

Tyrode’s buffer. After washing platelets were analyzed by flow cytometry.

2.3.6.3. Fibrinogen-Alexa Fluor 488 binding to platelets flow cytometric assay

Platelets were collected from MEG-01 cells suspension, using two centrifugation

steps. After collection, platelets were activated with TRAP and then incubated with

Fibrinogen-Alexa Fluor 488. Activation of platelets with 1 µg/ml TRAP was performed

for 10 minutes. After activation, 300 nM (final concentration) labeled fibrinogen was

incubated with platelets suspension for 30 minutes, in dark, at room temperature. Further

platelets were washed at 2000 g, 15 minutes at room temperature then analyzed by flow

cytometry.

2.3.6.4. AnnexinV-FITC for phosphatidylserine (PS) exposure on platelets

Platelets were collected from MEG-01 cells suspension, using two centrifugation

steps. After collection, platelets were activated with TRAP and then incubated with

Annexin V-FITC. For this assay, AnnexinV-FITC (BD Biosciences) was used, as

recommended by the manufacturer. Following activation for 10 minutes with agonist (1

µg/ml TRAP), incubation of the platelets with Annexin V – FITC was performed in the

dark, at room temperature, for 30 minutes. Tyrode’s buffer with CaCl2 was used in all the steps.

2.3.7. Viability (proliferation) assay of MEG-01 cells, Trypan blue exclusion

Cells were counted using a Neubauer hemacytometer. Trypan blue dye was used

according to the manufacturer (Sigma-Aldrich). DMSO, which is the vehicle for TBB,

DMAT and PMA, was used as a mock control, considering the highest amount that was

used as a vehicle for TBB and DMAT.

2.3.8. Anchorage independence in “soft agar” assay for MEG-01 cells.

Agarose (Promega) was mixed with MEG-01 growth media RPMI1640 1X with

0 10% FBS. Cells were grown in 12 wells dishes at 37 C, 5% CO2, 90% humidity in a

VWR incubator. Colonies formation was observed and micrograph images were taken using an Olympus CK80 microscope. Cells were observed after one week. We compared the control (DMSO) samples with the samples grown in the presence of 25 µM DMAT.

Media was renewed on the top of the agar every 4 days and DMAT treatment was

performed every time. 85 colonies from 30 images were analyzed. Measurements of the

colonies areas were performed using the NIH Image software ver.1.63 for MacOS 9.

2.3.9. Light microscopy (phase contrast) and DAPI fluorescence microscopy

Cells were daily observed using phase-contrast inverted microscopes.

High quality pictures and live imaging were obtained at Cleveland Clinic Imaging

Core (Cleveland, OH). DAPI staining of cultured MEG-01 cells was performed with fresh cells. For live imaging (observing a single cell for a 24 hour period of time) of the thrombocytopoiesis process, MEG-01 suspension cells were made adherent by using

Fibronectin (FN) coated culture dishes and cells were kept in a special imaging chamber which reproduced the mammalian cell culture incubator conditions (CO2 5%, humidity

90%). FN was used, at 5 µg/cm2 and incubated at 37 0C for 1 h, as recommended by the supplier (BD Biosciences, Bedford, MA, USA) [54].

2.3.10. Scanning electron microscopy (SEM)

Cells treatments and prep before SEM

Cell preparation and pictures were performed at the Imaging Core Facility

(Cleveland Clinic). Cells were grown for four days in the presence of 10 µM DMAT and then collected by differential centrifugation. Cells were washed twice in phosphate buffer

(PBS) buffer by centrifugation. Further, cells were fixed in glutaraldehyde buffer

(provided by the Imaging Core). The clot was prepared with platelets collected from

MEG-01 cultures treated with 10 µM DMAT. Human thrombin 0.5 U/ml for 15 minutes was used to activate them.

SEM sample preparation protocol

The fixation buffer for SEM is 2.5% glutaraldehyde in 0.1M phosphate buffer

(PBS), pH 7.2 -7.4. Fixation needs to be performed for at least 24-48 hours. Tissue was washed with 0.1M phosphate buffer four times for 15 minutes. Tissue was next rinsed with distilled water three times for 5 minutes. Tissue was dehydrated with ethanol. Times for dehydration are dependant on specimen size and density. There is a succesion of dehydration steps: 50% ethanol 20 minutes, 70% ethanol 20 minutes. At this moment the tissue can be stored in 70% ethanol. When the final processing of the sample is required, then more dehydration steps are done: 80% ethanol 20 minutes, 90% ethanol 20 minutes,

95% ethanol 20 minutes and 100% ethanol three times for 20 minutes. At this point, samples were ready for critical point drying. After critical point drying samples were sputter-coated with gold. Finally, prepared samples were analyzed using a JEOL SEM microscope [23, 55].

2.3.11. Transmission Electron Microscopy (TEM)

Cells treatments and prep before TEM

Cell preparation and pictures were performed at the Microscopy Core Facility

(Cleveland Clinic). Cells were grown for 4 days in the presence of 10 µM DMAT and then collected by differential centrifugation. Cells were washed twice in phosphate buffer

(PBS) buffer by centrifugation. Further, cells were fixed in glutaraldehyde buffer and then further processed at the Core. The clot was prepared with platelets collected from

MEG-01 cultures treated with 10 µM DMAT. Human thrombin 0.5 U/ml for 15 minutes was used to activate them.

TEM sample preparation protocol

Cell were washed through centrifugation with PBS buffer two times. Next cells

were fixed in 2.5% glutaraldehyde buffer (glutaraldehyde in PBS, pH 7.2-7.4). Fixation is

for at least 2 hours at (40C) or rapid in microwave. The best fixation time is overnight.

Fixed cells can be kept for longer periods of time. When the sample was ready for TEM, the sample was washed with PBS three to five times. Then cells were refixed in 1-4% osmium tetroxide buffer and then dehydrated in ethanol steps. Dehydration steps: 25% ethanol, 50% ethanol, 70-75% ethanol, 90-95% ethanol and 100% ethanol. Next infiltration steps are required: 1 part resin-epon-/2 parts solvent (overnight) and 2 parts resin-epon-/1 part solvent (overnight) and 100% resin-epon- (one hour). The cells in

100% resin were placed in a suitable container (embedding). Polymerization was done at

60-700C overnight. Sections were cut through thin sectioning and then samples were analyzed using a Phillips CM12 TEM microscope [23, 55-57].

2.3.12. Confocal microscopy

Cells treatments and prep before confocal microscopy

Cell pictures were taken at the Microscopy Core Facility (Cleveland Clinic). MEG-

01 cells were grown for 4 days in the presence of 10 µM DMAT on fibronectin coated glass slides. Cells were washed twice in phosphate buffer (PBS) buffer. Platelets were isolated from the MEG-01 cell culture through differential centrifugation and whole- blood platelets were isolated through gel-filtration. Platelets were incubated at 370C with glass slides coated with collagen or fibronectin. Platelets adhesion to collagen was

performed as previously described by [49]. Coverslips were coated with 20 µg/mL Horm

collagen (Hormon-Chemie, Munchen, Germany) and platelets were added in the absence of calcium and in the presence of 2 U/mL apyrase. Attached platelets stained with Alexa

Fluor 488 - Phalloidin were viewed using a fluorescence microscope. Human thrombin

0.5 U/ml for 15 minutes was used to activate platelets samples that were already on the collagen coated slides. Platelets were washed and incubated in Tyrode’s 1X buffer.

Further, all cells were fixed in 2% paraformaldehyde buffer.

Sample preparation for confocal microscopy (for Phalloidin-Alexa Fluor 488 staining)

Paraformaldehyde 2% buffer was prepared freshly from a 10% stock solution

(prepared by heating paraformaldehyde powder in PBS buffer at 600C, plus one drop of

NaOH 1M, sterile filtered at 0.2 µm). Paraformaldehyde 2% buffer is the fixation

solution. Cells were fixed in 2% paraformaldehyde buffer for 15 minutes at room

temperature. After fixation samples were permeabilized in 0.1% Triton-X100 in PBS for

one minute. Cells were incubated in Phalloidin-Alexa Fluor or Rhdomanine-Phalloidin

(Molecular Probes) solution, diluted 1:100 in PBS, for 15 minutes. Washing three times

with PBS buffer was required in order to remove unbound Phalloidin-Alexa Fluor 488

from the sample. Cells on the glass slide were mounted for microscopy analysis using

DAPI-Vecta Shield mounting medium. Regular transparent nail polish was used on the

sides of the glass slide so the slide will not fall. Samples prepared in this way were

analyzed using a laser scanning confocal microscope. [58]

2.3.13. Statistical Analysis

Error bars are standard deviations (SD). Experiments were performed at least in

triplicates. Statistical analysis and graphing were performed using GraphPad, Prism

software ver.2.01. One-Way ANOVA Test-Repeated Measures followed by Dunnett’s

Multiple Comparison Test (which compares all treatment columns vs. the control

column) or student t-test were also performed. p < 0.05 (*) was considered significant, p

< 0.01 (**) very significant and p < 0.001 (***) extremely significant.

Briefly, for each sets of samples that need to be compared, the sample distribution

was plotted and the normality assumption was tested (to check whether or not we have

Gaussian distribution). If the normality was tested positive, then t test (one-sided two

sample) or One-Way Anova test (parametric) were performed. If the normality was not

tested, then non-parametric tests were performed, usually the Mann-Whitney-Wilcoxon

test.

2.4. RESULTS

2.4.1. The effects of CK2 inhibitors on MEG-01 cells, as cancer cells

The goal of this study was to determine the effects of CK2α inhibition with CK2

inhibitors on malignant megakaryoblasts. To accomplish our goal we selected the MEG-

01 cell line, which is characterized as early stage megakaryoblasts with Philadelphia

positive chromosome. The cells were initially isolated from a patient with CML, in blast

crisis [16]. These cells are extremely malignant with an increased proliferation rate.

MEG-01 cell proliferation arrest.

We first tested the effect of CK2α inhibitors (TBB and DMAT) on the proliferation rate of MEG-01 cells using Trypan blue proliferation assay (see figure 2.3). Because

DMAT and TBB were solubilized in DMSO, initial control experiments were undertaken to establish the effect of DMSO on cell growth. The data demonstrate that DMSO has no

effect on MEG-01 cells proliferation rate and apoptosis (figure 2.3). In the absence of

inhibitor there is a 6-fold increase in cell number (figure 2.3, open squares). In the

presence of 5 nM PMA, which is known to induce proliferation arrest and differentiation

in MEG-01 cells, a decrease in proliferation was observed (figure 2.3, open diamonds)

and the level of decrease was similar to that obtained in the presence of 25 µM DMAT

(figure 2.3, open circles) suggesting that DMAT may also induce differentiation. High

concentrations of CK2 inhibitors, (50 µM DMAT or 100 µM TBB) maintained a steady number of cells for the 4 days of treatment and induced significant reduction in the proliferation rate of MEG-01 cells (figure 2.3, filled triangles and filled circles). These data suggest that the inhibitors are capable of reducing the increased proliferation rate of

MEG-01 cells due to apoptosis, without inducing necrosis.

DMAT inhibits anchorage independence of MEG-01 cells.

We next tested the effect of DMAT on the malignant potential of MEG-01 cells, using anchorage independence assay in soft agar (figure 2.4, panels A and B). Anchorage independence of growth in soft agar assay is strongly connected with tumorogenicity and invasiveness. It has been well established that malignant cells form colonies when grown on soft agar, while non-transformed cells do not grow under similar experimental conditions. Our data show that the area and the number of the colonies formed by untreated MEG-01 cells are extensive (figure 2.4, panel A), whereas the DMAT-treated

MEG-01 cells do not form colonies (figure 2.4, panel B). The results in figure 2.5 summarize colony area determined from 30 representative images, 80 colonies.

Figure 2.3. MEG-01 cells proliferation assays results. Cell proliferation (viability) assay, with Trypan Blue exclusion. Five days of treatment of MEG-01 cells. Open squares represent the control (DMSO), open triangles 50 µM TBB, filled triangles 100 µM TBB, open circles 25 µM DMAT, filled circles 50 µM DMAT and open diamonds PMA 5 nM. Each day quadruplicate measurements were taken and triplicate sets of experiments were considered for the plot and analysis.

Figure 2.4. Anchorage independence assay in soft agar of MEG-01 cells. A. Control (DMSO) colony formation in soft agar by MEG-01 cells, magnification X20, phase-contrast micrograph. B. DMAT treated (25 µM) colony formation in soft agar by MEG-01 cells, magnification X20, phase-contrast micrograph.

Figure 2.5. Anchorage independence assay in soft agar of MEG-01 cells. Comparison of colonies areas (pixels) between control (DMSO) and DMAT treated MEG-01 cells. Data was collected from 30 representative images (80 colonies total).

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These data suggests that inhibition of CK2α by DMAT eliminates the anchorage

independence characteristic of MEG-01 cells.

Maturation of MEG-01 megakaryoblasts due to CK2 inhibitors (DMAT and

TBB)

Thrombocytopoiesis follows the maturation and differentiation of megakaryoblasts.

In order to assess the maturation process of MEG-01 cells in the presence of CK2

inhibitors, we used flow cytometric immunophenotyping method. The αIIbβ3 integrin complex is a megakaryocytic maturation marker (maturation correlates with increased levels of αIIbβ3). Following incubation with the inhibitor, the levels of expression of

αIIbβ3 increase. This increase is correlated with differentiation of megakaryoblasts, see

figure 2.6, panels A and B. figure 2.6. Panel A shows that 10 µM DMAT is sufficient to

obtain significant maturation levels compared to the control untreated cells. A further

increase in the concentration of DMAT (up to 20 µM) induces a slight increase in the

maturation level of MEG-01 cells. In figure 2.6, panel B, we show that 20 µM DMAT

induced similar maturation level as 1 nM PMA and 25 µM TBB. This was assessed based

on immunophenotyping for CD41 (αIIb). Increase in the fibrinogen binding to MEG-01 cells shows increased expression of αIIbβ3 complex (figure 2.7). This increase is due to maturation of the cells. Black line represents the Control (DMSO) collected after 4 days of culture, blue line represents DMAT 10 µM treatment (4 days) and gray line represents

DMAT 20 µM treatment (4 days) see figure 2.7. Finally, DNA content assay demonstrates that MEG-01 cells become polyploid (ploidy higher than 2N) in the presence of 10 µM DMAT (figure 2.8.) The increase in DNA content demonstrates that

MEG-01 undergo maturation in the presence of CK2α inhibitors treatments.

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Figure 2.6. Maturation (differentiation) of MEG-01 cells due to DMAT.

A. RPECD41a (αIIbβ3 integrin expression) flow cytometric immunophenotyping for DMAT, TBB and PMA treatments versus control (DMSO). Histogram shows results from one set of treatments (total relative fluorescence). Control (DMSO) unstained – red line, control (DMSO) stained –black line, 10 µM DMAT – green line, 25 µM TBB – blue line, 1nM PMA – purple line. B. Graph represents total relative fluorescence percentages for RPE-CD41a immunophenotyping, conform analysis of data in WinMDI ver 2.8, from triplicate experiments.

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Figure 2.7. Fibrinogen binding to MEG-01 cells.

Increase in the fibrinogen binding to MEG-01 cells shows increased expression of αIIbβ3 complex. This increase is due to maturation of the cells. Control (DMSO),4 days – black line, DMAT 10 µM treatment (4 days) –blue line and gray line represents DMAT 20 µM treatment (4 days)

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Figure 2.8. DNA content analysis of MEG-01 cells treated with DMAT.

MEG-01 cells were treated with 10 µM DMAT for 4 days, as assessed by PI and RNAase

A, flow cytometric assay. Gray filled line represents the Control (DMSO) and Black line

is DMAT 10 µM treatment.

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Figure 2.9. Apoptotic assays. Total apoptotic cells percentages were plotted for each treatment set daily. A. Quadrant gating of a control untreated MEG-01 cells after 24 hours (AnnexinV-FITC and PI flow cytometric assay). B. Quadrant gating of cells treated with 20 µM DMAT following 24 hours. C. The effect of TBB and DMAT treatment on MEG-01 cells apoptosis after 24 hours. D. The effect of TBB and DMAT treatment on MEG-01 cells apoptosis after 48 hours E. The effect of TBB and DMAT treatments on MEG-01 cells apoptosis after 72 hours. F. The effect of TBB and DMAT treatments on MEG-01 cells apoptosis after 96 hours. The results provided in C, D, E, F represent the average found in three independent experiments.

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If we compare the Control (DMSO) sample, represented by the gray filled line

(ploidy level achived 16 N) versus the DMAT 10 µM treated sample, black line (ploidy level achieved 64N) it is obvious that DMAT induces an increase in ploidy level which correlates with an increase in differentiation level. Collectively the data demonstrate that inhibition of CK2 in MEG-01 cells results in proliferation arrest followed by maturation of the cells.

Apoptosis of MEG-01 cells is induced by DMAT and TBB

In order to understand the effect of the inhibitors on MEG-01 cells and verify if the treatment is not cytotoxic, we assessed both apoptosis and necrotic levels, using an assay employing Annexin V and PI. Percentages of total apoptotic cells are the sum of (FL1H+,

FL3H-) lower right quadrant, corresponding to early apoptotic cells gate, with (FL1H+,

FL3H+) upper right quadrant, corresponding to late apoptotic cells. Necrotic cells correspond to the upper left quadrant gate (FL1H-, FL3H-). figure 2.9, panel A demonstrates that following 24 hours incubation in the absence of CK2 inhibitors 8.7% of the control untreated cells are apoptotic while 0.87% are necrotic. Following 24 hours treatment with 20 µM DMAT the level of apoptotic cells has significantly increased

(19%) while the level of necrotic cells remained low (figure 2.9, panel B). A comparative summary of the results obtained following 24 hours incubation with either TBB or

DMAT is provided in figure 2.9, panel C. The data shown in 2.9, panel D (48 hours of treatment). The effect is dose dependent and reaches a maximum after four days, see figure 2.9, panel E (72 hours of treatment) and figure 2.9, panel F (96 hours of treatment).

Following 96 hours incubation the results obtained with 10 µM DMAT are similar to the results obtained with 20 µM inhibitor. A direct comparison between control cells and

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DMAT-treated cells establish that treatment with DMAT induces significant apoptosis in

MEG-01.

Our findings also show that CK2 inhibitors may not be cytotoxic, due to necrotic

cells percentages which are not different between the DMSO treated sample versus

DMAT, TBB treated samples.

It has been well established that physiologically, platelets derive from

megakaryoblast following an apoptotic process [3, 5-10, 21, 22, 57].

Since DMAT and TBB induce apoptosis in MEG-01 cells, we next verified if

CK2α inhibitors could also be thrombocytopoiesis inducers. The data shown in figures

2.10 and 2.11 demonstrate that CK2α inhibition result in thrombocytopoiesis. As early as from the second day DMAT induced MEG-01 cells to form proplatelet extensions.

This process was dramatically enhanced following four days of treatment.

Thrombocytopoiesis phenotype.

CK2α inhibition with DMAT was found to result in thrombocytopoiesis. This is shown in figure 2.10 and figure 2.11. As early as from the second day DMAT induced

MEG-01 cells to form proplatelet extensions. This process was dramatically enhanced following four days of treatment (figure 2.10 panels A-E and figure 2.11). The megakaryocytes were found to undergo thrombocytopoiesis and showed apoptotic

features, DNA condensation and fragmentation. Comparing figure 2.10 A versus figure

2.10 B we notice bigger cells, blebbing, pro-platelets formation and particle release.

Phenotype change is induced in MEG-01 cells by DMAT treatment as seen in figure 2.10

D shows a scanning electron micrograph of a DMAT MEG-01 stimulated cell (blebbing

and pro-platelets in incipient phase) correlating with the previous results. MEG-01 cell

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(on fibronectin matrix) treated with DMAT 10 µM extended long beaded filaments.

These filaments will rupture releasing the platelets and in the mean time the same cell also undergoes extensive cytoplasmic fragmentation (figure 2.10 F left panel). In figure

2.10 E right panel we show the nucleus with DAPI stain of the MEG-01 cell from figure

2.10 F left panel. It is noticeable that the pro-platelets forming cell has specific nuclear apoptotic fragmentation. MEG-01 cell in suspension that undergo thrombocytopoiesis, following DMAT treatment, confirmed that platelets release from MEG-01 cells is not due to stimulation from the fibronectin matrix (see figure 2.10 C). The proplatelets and platelets do not stain positive with DAPI (figure 2.10, panel G, white arrow), showing that the beaded ends indeed will become anucleated cells.

Thrombocytopoiesis cellular structural insights

Cells were grown in suspension and incubated with DMAT 10 µM for 4 days.

Structural insights using transmission electron microscopy into this platelets production effect of DMAT on MEG-01 cells are shown in figure 2.11. In figure 2.11, panel A resting MEG-01 cells are shown where demarcation membrane system and nucleus can be observed. Progression of the thrombocytopoiesis process (figure 2.11 B) is characterized by extensive cytoplasmic fragmentation on the demarcation membrane system. The peripheral pro-platelets formation is shown in figure 2.11 panels C,D. The long filaments with bulbous ends (pro-platelets) are shown in figure 2.11 E. The proplatelets characteristic cytoskeleton can be observed in figure 2.11 G (a confocal laser scanning microscopy micrograph using 63 X magnification). Phalloidin-Alexa Fluor 488 is used for staining MEG-01 cells actin. Confocal microscopy shows specific nucleus that mature megakaryocytes have, big and lobulated.

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Figure 2.10. Phenotype change in MEG-01 cells treated with 10 µM DMAT. A. Control (DMSO) MEG-01 cells phase-contrast micrograph after 96 hours, magnification X 20, B. MEG-01 cells treated with 10 µM DMAT phase-contrast micrograph, following 96 hours of treatment X 20. C. Proplatelets formation, in suspension, phase-contrast micrograph, following DMAT treatment (10 µM) between 72 and 96 hours, magnification X 40. D. Scanning electron microscopy micrograph of MEG-01 cells, magnification X 7500, voltage 15 kV E. Proplatelets formation on fibronectin coat, phase-contrast micrograph, following DMAT treatment (10 µM) between 72 and 96 hours of treatment, magnification X 40. F. DAPI staining micrograph of proplatelets bearing MEG-01 megakaryocyte following DMAT treatment (10 µM) between 72 and 96 hours of treatment, magnification X 40. G. Platelets-like particles identified as anucleated cells with DAPI staining, magnification X 40 following DMAT (10 µM treatment) at 72 to 96 hours. Panels E and F represent the same cell.

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Figure 2.11. Ultrastructural features of MEG-01 cells treated with DMAT. Black arrows show nucleus, blue arrows show demarcation membrane system and red arrows show pro-platelets. A. MEG-01 cell in resting state (control), magnification 5600X B. MEG-01 cell that undergoes thrombocytopoiesis process (treated with DMAT 10 µM, 4 days), notice demarcation system formation, magnification 6300X, C. MEG-01 cell that undergoes thrombocytopoiesis process (treated with DMAT 10 µM, 4 days), notice pro-platelets formation, magnification 2000X, D. MEG-01 cell that undergoes thrombocytopoiesis process (treated with DMAT 10 µM, 4 days), notice pro-platelets formation, magnification 5600X, E. Pro-platelets with characteristic bulbous end formation in MEG-01 cells treated with DMAT 10 µM, 2000X and 4000X magnification, F. Confocal scanning electron micrograph of MEG-01 cells undergoing thrombocytopoiesis due to DMAT treatment, notice characteristic bulbous end formation at the tip of pro-platelets, 63X magnification.

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The staining for cytoskeleton shows pro-platelet and pseudopodia formation and this can

be better observed in the magnified image (figure 2.11. G right panel), 4X digital zoom,

over 63X physical zoom. Together the data demonstrate that CK2α inhibition induces apoptosis of MEG-01 cells, which in turn result in the release of platelet-like particles, following a maturation process (see figures 2.8-11).

DMAT induces functional platelets release from MEG-01 cells, in vitro

The next step was to verify if the platelet-like particles released following treatment with DMAT in culture, are indeed platelets and are functional. Several functional studies were performed and the results are reported in figure 2.12 panel A-D. The platelets are capable of undergoing shape change in response to agonists (human thrombin, TRAP,

ADP, and PMA). Activated platelets stain positive for PAC-1 (an antibody that recognizes a specific epitope on αIIbβ3 integrin, exposed only when platelets are

activated) (figure 2.12, panel A). Following activation the platelets were found to expose

P-Selectin (figure 2.12, panel B), phosphatidylserine (figure 2.12, panel C) and bind

fibrinogen (figure 2.12, panel D). Finally, following activation with 0.5 U/ml of human

α-thrombin (IIa) the platelets form a visible clot. SEM of this clot demonstrates that this

platelets develop spiked lamelapodia appearance, aggregation and formation of a fibrin

net (figure 2.13, panels A-C). Platelets were harvested from MEG-01 cells grown in the

presence of DMAT (10 (M, 72 hours). Human thrombin 0.5 U/ml (15 minutes exposure)

was used as agonist. A detail of the MEG-01 platelets, fibrin clot formation

magnification X 5,000, voltage 20 kV is depicted in figure 2.13 A. The platelets detail at

a X 7,500 magnification, voltage 20 kV is shown in figure 2.13 B.

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Figure 2.12. Platelets from MEG-01 cells obtained after DMAT treatment

MEG-01-derived platelets following treatment with DMAT (10 µM for 72) were collected and used. Platelets were activated with TRAP peptide. Controls were treated with EDTA and RGDS to prevent any artefactual activation. A. P-Selectin exposure (CD62P – FITC) by activated platelets. B. PAC-1 binding (PAC-1-FITC) by activated platelets. C. Fibrinogen-Alexa Fluor 488 binding to activated platelets. D. Annexin V- FITC binding to activated platelets (phosphatidylserine exposure). In all panels, the control platelets are represented by the red line, while the results obtained with activated platelets are represented by the green line. E. Resting MEG-01 derived platelets gate, F. Activated platelets gate.

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Figure 2.13. Platelets from MEG-01 cells (in culture) after DMAT treatment. Platelets were harvested from MEG-01 cells grown in the presence of DMAT (10 (M, 72 hours). Human thrombin 0.5 U/ml was used as agonist . A. Detail of the fibrin clot magnification X 5,000, voltage 20 kV. B. Platelets and fibrin net detail, magnification X 7,500, voltage 20 kV, C. fibrin net detail from the clot, magnification X 20,000 voltage 20 kV.

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Figure 2.14. Confocal microscopy analysis of platelets derived from MEG-01 cells. A. resting platelets from MEG-01 cells resulted during treatment with DMAT 10 µM for 4 days incubated with coverslips coated with 20 µg/ml collagen and for 15 minutes at room temperature. B. activated platelets with α-thrombin (IIa) 0.5 U/ml from MEG-01 cells incubated with coverslips coated with collagen, 63X zoom, C. activated platelets with α-thrombin (IIa) 0.5 U/ml from MEG-01 cells incubated with coverslips coated with collagen, 63X zoom, 8X digital zoom, D. activated platelets with α-thrombin (IIa) 0.5 U/ml from MEG-01 cells incubated with coverslips coated with collagen, 63X zoom, 4X digital zoom

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Figure 2.15. Ultrastructure of MEG-01 derived platelets (TEM). A. Resting MEG-01 derived platelet (with characteristic granules and lack of nucleus), magnification 10000 X, 60 kV B. Activated MEG-01 derived platelet (which releases its contents, undergoes shape change and still has some granules), 20000 X magnification, 60 kV. “g” represents the platelets specific granules.

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Finally the fibrin net detail from the clot at X 20,000 magnification and a voltage of 20

kV is shown in figure 2.13 C. Confocal microscopy experiments revealed specific platelet

cytoskeleton and lack of DAPI staining (anucleated cells) for MEG-01 derived platelets

adhered to collagen, see figure 2.14. MEG-01 derived platelets resting were found to

adhere to collagen coated slides (figure 2.14 panel A). These MEG-01 derived platelets

were found to become activated with α-thrombin (IIa) 0.5 U/ml (figure 2.14 panel B).

The cytoskeleton organization, see figure 2.14, panels C,D, is characteristic to platelets.

Such platelets extended cytoskeleton filaments when 0.5 U/ml human α-thrombin (IIa) was added to the adhered platelets on collagen slides, see figure 2.14, panels B-D. The ultrastructures of MEG-01 derived platelets due to DMAT 10 µM treatment (transmission electron microscopy) is depicted in figure 2.15. MEG-01 cells were treated with DMAT for 4 days and then platelets were collected through differential centrifugation. Resting

MEG-01 platelet is depicted in figure 2.15, panel A using a X 10,000 magnification and a voltage of 60kV. The activated MEG-01 platelets (0.5U/ml thrombin vortexed until they formed a platelet clot) are shown in figure 2.15, panel B using a X 20,000 magnification and a voltage of 60kV. These activated platelets show release of their contents. The lack of nucleus and α-granules can be observed, as well as the open canalicular systems

(figure 2.15).

HUVEC cells apoptosis versus MEG-01 cells apoptosis.

HUVEC apoptosis versus MEG-01 cells apoptosis show that after 24 hours the effect of DMAT is more pronounced in MEG-01 cells (malignant) versus primary

(normal) cells (see figure 2.16).

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Representative data from apoptosis assay (Annexin V and PI) for MEG-01 cells after 24 hours, treated with DMAT 20 µM are shown in the histogram shown in figure

2.16 A. Representative of apoptosis assay for HUVEC cells treated with DMAT 20 µM are shown in the histogram in figure 2.16 B. Lower right (FL1H+, FL3H-) quadrant which corresponds to early apoptotic cells has 13.7 % cells for MEG-01 sample versus

8.4 % cells for HUVEC sample. Late apoptotic cells quadrant (FL1H+, FL3H+), upper right, are 6.9 % for MEG-01 cells versus 4.9 % for HUVEC cells. Dead or necrotic cells quadrant (FL1H-, FL3H+), upper left, has 0.5% for MEG-01 cells versus 1.9 % HUVEC cells, which is not a significant difference for a flow cytometry experiment (error can be up to 2%). The positive control for induced apoptosis of HUVEC cells is shown in figure

2.16 C. For this, tumor necrosis factor (TNFα) which is very cytotoxic for HUVEC cells, was used. In figure 2.16 C, TNFα induces 34% total apoptosis (and most cells are dying already as noticed from the high percentage located in the late apoptotic gate). Three experiments data (as total apoptotic cell percentage: including late, early and dead cells gates) are represented in figure 2.16 D. In conclusion, it is suggested that apoptosis is induced different in malignant versus normal cells (figure 2.16). Mechanisms are unknown, but it may have something to do with the expression, localization and activity of CK2 that are different in MEG-01 cells versus normal primary cells. MEG-01 cells have various aberrations, deregulations, including the presence of BCR/ABL abnormal kinase, thus this result may not be as surprising as it seems at a first view.

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Figure 2.16. HUVEC apoptosis versus MEG-01 cells apoptosis. After 24 hours the effect of DMAT is more pronounced in MEG-01 cells (malignant) versus primary (normal) cells. A. HUVEC cells apoptosis, B. MEG-01 cells apoptosis, C. HUVEC cells apoptosis induced by treatment with TNFα, D. Comparation of averaged percentages of apoptotic cells after 24 h (HUVEC vs. MEG-01).

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2.5. DISCUSSION

Our data demonstrate for the first time that CK2α inhibition induces malignant

MEG-01 megakaryoblasts maturation and enhances functional platelet progeny release.

Interestingly, we found that CK2α inhibition with DMAT and TBB, induces proliferation arrest and apoptosis, without being cytotoxic. Proliferation arrest as well as apoptosis was correlated with length and amount of treatment. DMAT, which is a better inhibitor than

TBB, had effect at concentrations as little as 5 and 10 µM. Due to the importance of protein kinases in malignant processes, this study can be considered to have consequences for future therapeutic interest as previously described [27, 29, 37-39, 47,

59-63]

A striking observation that results from our studies is that CK2α inhibition with

DMAT and TBB induces thrombocytopoiesis. The megakaryocytes undergoing thrombocytopoiesis showed apoptotic features, as DNA condensation and fragmentation, blebbing and phosphatidylserine exposure. Mature megakaryocytes start to bleb and form pseudopodia. Following explosive fragmentation, long filaments with beaded ends

(proplatelets) are formed. The proplatelets do not stain positive in DAPI, demonstrating that the beaded ends indeed will become platelets. Platelets are expelled out from the proplatelets, and the fragmented nucleus slowly is extruded. Thrombocytopoiesis process occurred in cells bound to fibronectin matrix as well on cells in suspension.

The thrombocytopoiesis process observed in the present study follows the maturation and differentiation process of MEG-01 megakaryoblasts. This differentiation is similar to the effect observed with phorbol ester (PMA), however, CK2α inhibitors are not cytotoxic, whereas PMA is a potent tumorigenic substance as well as a powerful

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platelet activator [48, 64-66]. We speculate that maturation of MEG-01 cells is a result of proliferation arrest that makes incomplete repeated cell cycle to enter into endomitosis, probably due to the action of CK2 on the cell cycle. This hypothesis is strongly supported by the preponderant nuclear localization of CK2α in malignant cells as compared to its localization in normal cells [38, 40, 62, 67-69]. We assessed this maturation process using flow cytometry, by measuring the expression of αIIbβ3 integrin increase (which was shown to correlated with increase in size and differentiation of megakaryoblasts).

Increase in DNA content and cell size following incubation of the cells with the inhibitors; also adds further evidence that MEG-01 cells mature due to CK2α inhibition.

Anchorage independence assays in soft agar, is an experiment that identifies malignant cells versus normal cells due to their unique capability to be immature and

“live by themselves”. Cancerous cells grow in any conditions without a support or signaling with the neighboring cells. We found that MEG-01 cells treated with DMAT do not form colonies in soft agar and this maybe due to the fact that MEG-01 cells mature, regain their function to produce platelets and decrease their proliferation rate. Also, these results obtained from the soft agar experiments suggest that the adherence pathways controlled by CK2α maybe involved in the process of developing anchorage independence. BCR/ABL was found to be involved in the malignant transformation of

Ph+ cells, but its inhibition is not sufficient to suppress anchorage independence of such cells, suggesting involvement of other molecular mechanisms [70]. Our results connect

CK2 inhibition with apoptosis and the mechanism of thrombocytopoiesis that follow megakaryocytopoiesis. It has been previously shown that platelet shedding results from a constitutive form of apoptosis of megakaryocytes [3, 5-10, 21-23, 57]. Our study also

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shows for the first time that CK2 inhibition induces release of functional platelets from

malignant megakaryoblasts. In megakaryoblasts, CK2 inhibition first produces

proliferation arrest, followed by differentiation to megakaryocytes that culminate with

proplatelets formation, blebbing, and compartmentalized fragmentation of

megakaryocytes, finalized by thrombocytes release. Platelets obtained in culture,

following CK2α inhibition are functional. These platelets form a clot visible with the eye

when exposed to agonists. This clot was analyzed with SEM and fibrin net formation and

characteristic platelet phenotype were observed.

After 24 hours of treatment, the effect of DMAT is more pronounced in MEG-01 cells (malignant) versus primary (normal) cells. Overall, our data clearly demonstrate that treatment of MEG-01 cells with casein kinase 2 inhibitors results in proliferation arrest, maturation and release of functional platelets.

In conclusion, CK2α inhibition studies with TBB and DMAT, suggests a key role of CK2 in oncogenic development as well as in the megakaryocytopoiesis and thrombocytopoiesis processes and at least provides wonderful applicability of such compounds.

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CHAPTER III

TREATMENT OF MEGAKARYOBLASTIC LEUKEMIA MURINE

XENOGRAFTS WITH DMAT

3.1. ABSTRACT

Megakaryoblasts are hematopoietic progenitor stem cells that differentiate to the stage of megakaryocytes. MEG-01 cells are malignant megakaryoblasts isolated from a patient with chronic myelogenous leukemia in blast crisis. MEG-01 cells are presenting a malignant phenotype showing increased proliferation and capacity to produce solid tumors in vivo. MEG-01 cell line may provide a useful model for testing the in vivo efficacy of anti-tumor agents and immunotoxins, and for studying the pathophysiological mechanisms of human megakaryoblastic leukemia. Our findings following treatment of

MEG-01 murine xenografts with the CK2 inhibitor DMAT were striking. The MEG-01 tumors infiltrated the spleen, and leukemic megakaryoblastic cells seem to appear in the blood of mice with MEG-01 tumors (as high abnormal cell counts). All MEG-01 tumor bearing mice had splenomegaly (treated and untreated). The tumors released platelets in

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vivo and this phenomenon was increased due to casein kinase 2 inhibitor treatment

(DMAT). DMAT treatment induced significant proliferation arrest of tumors and necrosis and apoptosis in tumor tissue. Angiogenesis was reduced in tumor tissue due to

DMAT treatment. Liver, brain, kidney, muscle tissue seem to be normal in DMAT treated mice.

3.2. INTRODUCTION

Megakaryoblasts are hematopoietic progenitor stem cells that differentiate to the stage of megakaryocytes. MEG-01 cells are malignant megakaryoblasts isolated from a patient with chronic myelogenous leukemia in blast crisis [1]. MEG-01 megakaryoblasts were found to release platelets that are morphologically indistinguishable from normal blood platelets, viable and functional, when treated with apoptosis inducers. These cells are of interest for cancer research because they are cytokine independent [2-4], and therefore do not respond to cytokine treatments (interferons and thrombopoietin), and can be induced to release platelets [1, 5-9] and are very proliferative. Cytokine independence imposes a problem when using the cytokine treatment for the advanced stage of CML

(the blastic phase). MEG-01 cells were characterized as being megakaryoblasts in the early stage of differentiation on the megakaryocytic lineage, and they are committed progenitors towards platelet production. These cells have Philadelphia positive chromosome, expressing BCR/ABL oncoprotein [1, 6, 7].

MEG-01 cells are presenting a malignant phenotype showing increased proliferation and capacity to produce solid tumors in vivo [10, 11]. MEG-01 cell line may provide a useful model for testing the in vivo efficacy of anti-tumor agents and

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immunotoxins, and for studying the pathophysiological mechanisms of human megakaryoblastic leukemia.

Inhibition of CK2 in various cancer cell lines produced apoptosis and proliferation arrest [12-25].

Antisense nucleotides against CK2α were studied in mouse human prostate cancer xenografts, in vivo [14]. A very interesting observation was that antisense nucleotides treatments against CK2α in prostate cancer mouse xenografts, were affecting the tumors

(by inducing apoptosis and tumor ablation), but not the normal surrounding tissue. This was explained as being caused by different localizations of the alpha subunit of casein kinase 2 in normal versus malignant tissue. This conclusion was based on histological analysis of normal and neck squamos carcinoma tissues [14, 26-28]. These studies sustain the idea that CK2α is an attractive cancer treatment target even though is a pleiotropic kinase.

Our preliminary results suggest that apoptosis and proliferation arrest, followed by megakaryocytopoiesis and thrombocytopoiesis were induced by DMAT in CML Ph+ cell line MEG-01. MEG-01 platelets obtained after DMAT treatments were found to be functional.

The last step before assessing the DMAT effects into a murine model of CML

Ph+ (that mimics the disease) is to create first a murine xenografts model with MEG-01 tumors. In this murine model we can analyze the platelets levels and function following

DMAT treatments.

The formulation of the drug is very important considering the solubility of DMAT

(it is not in a polar solvent soluble form). DMAT is soluble in DMSO.

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This model will also provide the means to design a targeted drug delivery of

DMAT as an ultimate goal, in order to avoid as much as possible any side effects that

may occur due to the localization of CK2 in both malignant and normal tissue.

3.3. EXPERIMENTAL PROCEDURES

3.3.1. Cell Culture

Materials, see Chapter II (Cell Culture protocol)

Method

MEG-01 megakaryoblastic cell line [1], was a generous gift from Dr. P. B. Tracy,

(Department of Biochemistry, University of Vermont, College of Medicine, Burlington,

VT, USA). MEG-01 cells were also purchased from American Tissue Culture Collection

(Manassas, VA). Cells were maintained in an incubator, with humidified atmosphere of

CO2 5%, and at 37 0C. Cell culture media, RPMI 1640 1X with L-Glutamine (2mM), was adjusted to contain 10 mM HEPES, 1.0 mM Sodium Pyruvate and 10-20 % heat- inactivated fetal bovine serum (FBS) and 5U Penicillin/Streptomycin. Cells were seeded at 2 X 105 cells/ml, media was renewed and cell number was adjusted two times per week. 20% of FBS was observed to improve the growth and endurance of MEG-01 cells.

More mature MEG-01 cells become adherent to the culture flask wall and therefore in order to provide a better surface area for them, flaks were kept on the side.

3.3.2. Creation of MEG-01 xenografts

Animals.

Immunodeficient male athymic nude nu/nu mice were used. These animals were provided and housed by Dr. Lindner, DJ from Taussig Cancer Center, Cleveland Clinic and Case Western Reserve University. Mice were checked every day. Mice were housed

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in filtered air flow cabinets with autoclaved bedding at a density of 5 mice/cage. They

were fed autoclaved Purina Lab Rodent Chow 5010 and HCl-acidified distilled water ad

libitum and were placed in rooms with controlled temperature, humidity and 12-hr light-

dark cycles. Procedures involving animals and their care were conducted in conformity

with the institutional guidelines that are in compliance with national and international

laws and policies (EEC Council Directive 86/609, OJL 358, December 1, 1987, and the

National Institutes of Health Guide for the Care and Use of Laboratory), Animals, NIH

Publication 85-23, 1985).

The average weight of the mice used in these sets of experiments was 30-35 g.

Animals were observed for distress or dehydration and their weight was measured

every two days.

Engraftment of MEG-01 cells in nude mice

For engraftment, washed cells, resuspended in cell culture media were injected.

MEG-01 myeloid blast crisis cells (10×106 cells/100 µl in cell culture media (RPMI 1640

1X, 10 % FBS) were injected subcutaneously into the lower flanks of mice (left and right) using 0.5 ml tuberculin syringes with 25G needles. Cells were counted with a

Neubauer hemacytometer using Trypan blue, before innoculation. Only live cells were counted.

After about ten days all the animals developed tumors big enough to start the treatment. Tumors were visible after 6-7 days from inoculation. Treatment with DMAT was started when tumors were at least 100-200 mm3 volumes (prolate spheroid).

Tumor volumes were calculated as prolate spheroid using the formula described

bellow:

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V=(4/3)*π*(a)2*(b)

Were „a” is half the minor axis and „b” is half the major axis of the prolate spheroid.

“2a” and “2b” were measured with a caliper (mm).

Animals were treated with DMAT for approximately two weeks (when tumor

volumes reached an unacceptable size with the IACUC protocols, animals were sacrificed

in a CO2 euthanasia chamber). Tumors were collected following necropsy for further

histological analysis.

3.3.3. In vivo therapy of MEG-01 xenografts with DMAT

Therapy with DMAT was done in two trials. DMAT in DMSO, as well as just

DMSO as a control, were administered by injection subcutaneous in the fat pad tissue of

the neck (exogenous from the tumor). Tumor measurements were used to assess the

effects of DMAT on MEG-01 xenografts. Tumor diameters were measured using a

caliper and tumor volume was calculated using the prolate-spheroid formula.

Treatment of MEG-01 murine xenografts with DMAT

First trial with n = 4 (2 tumors on left and right flanks) was started when tumor

volumes were quite big (500-650 mm3). DMAT in DMSO was administered daily at 2

mg/ml per animal (an average of 61.5 mg/kg DMAT considering a male mouse weight

between 30-35 g). DMAT injection was performed subcutaneous, into the neck (in the

neck fat pad), exogenous from the tumors. Injection volume was 50 µl (DMAT 2 mg/animal per day). The trial length was two weeks. Tail-bleeding and blood collection were performed on these animals before sacrifice, under anesthesia.

Second trial with n = 6 (2 tumors on left and right flanks) was started when tumor

volumes were smaller, at 100-200 mm3. DMAT in DMSO was administered daily as 3

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mg/animal per day (an average of 92.3 mg/kg DMAT considering a male mouse weight

between 30-35 g). DMAT injection was performed subcutaneous, into the neck (in the

neck fat pad), exogenous from the tumors Injection volume was 50 µl. The trial length

was two weeks.

At the end of the trial, tail-bleeding times assay was performed and blood was

collected for blood counts from live and anesthetized mice. After these experiments were

completed mice were sacrificed and tumors and organs were collected. Samples were

fixed (at least overnight at room temperature) in formalin fixative for further processing.

Two batches of control mice (with MEG-01 tumors), n = 4 and respectively n = 6,

were treated with 50 µl DMSO, which is the vehicle for DMAT. The control batch was

analyzed and compared with the DMAT treated mice (as tumor volumes, tissue histology,

tail-bleeding and blood counts).

3.3.4. Pilot study for in vivo toxicity of DMAT

A male nude mouse (without tumors) was treated with DMAT to determine a

toxicity level. A saturated solution of DMAT in DMSO was inoculated daily in the neck,

subcutaneous (100 µl of 100 mg/ml DMAT in DMSO). Therefore this animal received 10

mg DMAT per day (308 mg/kg/day). After two weeks this animal was sacrificed and

organs were collected for further analysis.

3.3.5. Hematoxylin & Eosin (H&E) staining of tumor tissue and organs

After euthanasia with CO2, mice were suposed to necropsy. Tumors were collected from under the skin from both flanks and were measured for the last time and then fixed in formalin fixative. Spleen, liver, kidney, brain, lungs and legs were collected and fixed.

Spleens were also measured (as lenght, mm). Fresh tissue was immersed immediatly into

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liquid nitrogen and kept frozen at -800C. Fixed tissue was embedded in parafin and next

processed for H&E (the basic dye hematoxylin, and the alcohol-based synthetic material,

eosin) by the Cleveland Clinic Histology Core facility. Sections (4-µm thick) were stained with hematoxylin and eosin and evaluated for pathologic changes in a blinded fashion. H&E staining gives morphological information (vascularization, normal proliferating tissue, necrosis and apoptosis of the tissue).

Hematoxylin & Eosin Staining Protocol

Materials

Hematoxylin – Richard-Allan Scientific (Kalamazoo, MI, USA)

Eosin - Richard-Allan Scientific (Kalamazoo, MI, USA)

Ethanol – Sigma Chemical Co. (Carlsbad, CA, USA)

Paraformaldehyde - Sigma Chemical Co. (Carlsbad, CA, USA)

Xylene - Sigma Chemical Co. (Carlsbad, CA, USA)

Permount solution – Vector Laboratories (Burlingame, CA, USA)

Microscope glass slides – Fisher Scientific (Pittsburgh, PA, USA)

Method

Paraformaldehyde fixed tissue was washed thouroughly with running water (1-2 h). Then tissue was washed for 2 minutes with 100% ethanol. After alcohol washing, staining was performed with hematoxylin (8 minutes), then washed with running tap water for 30 minutes, then tissue was incubated with eosin for 3 minutes. After eosin staining, tissue underwent a series of alcohol incubation steps. First alcohol step was done overnight with 70% ethanol, next two times (15 minutes) with 80% ethanol, then three times (30 minutes) with 95% ethanol and finally three times (45 minutes) with

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100% ethanol. An incubation with xylene, three times (10 minutes) on shaker was performed. This processed tissue was embeded in parafin and then thin sliced. The slices were mounted with Permount solution on microscope glass slides and visualized with an

Olympus CK40 inverted microscope and pictures were taken for each slide, usually at 10

X and 20 X magnification.

3.3.6. Mice blood collection and blood counts

Materials, see Chapter II (human platelets isolation protocol).

Method

Blood counts are dependent upon the method and time of blood collection [29].

Whole blood was collected from the retro-orbital sinus (under the eye) of anesthesiated mice (both DMSO treated and DMAT treated batch). EDTA – citrate buffer (ACD) and prostaglandin E1 (PGE1) were used at collection to prevent clotting during blood collection. 500 µl mouse whole blood with 100 µl anticoagulant (EDTA 25 mM, as a 1:5 dilution) was used for counting.

A hematological analyzer (a clinical flow cytometer located at Cleveland Clinic

Stem Cell Research Department) was used for getting whole blood counts from the mice blood. Samples were compared (gated) with normal mice (C57BL strain).

3.3.7. Tail-bleeding assay

Materials

Ketamine – Fort Dodge Animal Health (Fort Dodge, IA, USA)

Xylasine – Vedco (St. Joseph, MO, USA)

Saline solution – Baxter Healthcare (Deerfield, IL, USA)

Scalpel - Roboz Surgical Instrument Co. (Gaithersburg, MD, USA)

Method

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Tail-bleeding times are important to investigate whether the platelets could

establish hemostasis in vivo. Platelet aggregation and clot retraction in response to physiologic agonists adenosine diphosphate (ADP), epinephrine, and thrombin will affect

tail-bleeding times. Normal tail-bleeding times are between 1.5-3 minutes in C57BL

mouse strain [30].

We used pre-warmed tubes of saline solution at 37ºC and these tubes were

maintained at this temperature during the measurements. Inhalation of isoflurane vapors

or, alternatively, intraperitoneal injection of (ketamine, xylasine and saline buffer

cocktail) was used to induce general anesthesia. Using a sharp new razor or a scalpel

blade, tails were cut exactly 0.5 cm of the distal tip of the tail of the adult mouse and

immediately inserted into the pre-warmed tube of saline. A stopclock was started at this

time. The tail was hold gently, near its base, to avoid a "torniquet effect." Venous blood

flowing into the tube can be observed. In this way it can be easily detected when the

bleeding stops. The stopclock provides an accurate bleeding time.

3.3.8. Data Analysis

To assess the statistical significance of the difference between pairs of means of

tumor volumes, student’s two-tailed t test was usually used. p < 0.05 (*) was considered significant, very signficant, p < 0.01 (**),extremly significant, p < 0.001 (***) , for each set of data, daily. Data was ploted in order to establish whether or not the population follows a normal distribution and then depending on the type of data, parametric or non- parametric tests were performed, to establish a proper „p” value. When was necesary due to the data type, unpaired student t-test was used to examine statistical differrences.

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Data was plotted as average tumor volume (mm3) from all tumors (n = 8 for trial 1 and n = 12 for trial 2) or averaged blood counts, spleen sizes, necrotic areas, respectively vascularized areas.

3.4. RESULTS

Our in vitro studies of MEG-01 cells treated with the casein kinase 2 inhibitor,

DMAT, revealed that DMAT has anti-cancerous activity (see Chapter II). MEG-01 cells are malignant megakaryoblasts with high proliferation rate. DMAT inhibited malignant proliferation and induced apoptosis dose and time dependent, in vitro, in MEG-01 cells.

And more interesting is that the primary cell line (normal) HUVEC is not so sensitive to

DMAT as MEG-01 cells are, as observed from apoptosis assays.

MEG-01 xenografts were treated with DMAT for a period of two weeks. Thus, based on these results, it is necessary to test DMAT in vivo in order to be able to acknowledge

DMAT as a possible anti-cancer drug candidate. The in vivo model chosen for these preliminary pre-clinical studies was the MEG-01 murine xenograft.

Two experimental sets for these in vivo studies were considered. First set consisted of animals with bigger tumors (n = 4, with tumors on both flanks left and right) were studied, see figure 3.1. This trial was started when tumor volumes were quite big (500-

650 mm3). DMAT in DMSO was administered daily as 2 mg/animal, subcutaneous, into the neck (in the neck fat pad), exogenous from the tumors. Tumors were measured daily and tumor volume was calculated for each tumor using the prolate-spheroid formula

(mm3). Control mice received a 50 µl DMSO injection daily. The treatment length was

two weeks. From the 7th day of treatment with DMAT, tumors start to stop their

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development maintaining a constant size, slightly decreasing until the end of the

treatment. From the 7th day of treatment the difference between treated (filled triangles) and untreated tumors (filled squares) becomes statistically significant different, as seen on figure 3.1.

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Figure 3.1. MEG-01 xenograft treated with 2 mg DMAT/animal/day for 2 weeks.

Black filled squares represent an average of tumor volumes of 8 tumors for Control batch (DMSO only) represented versus tumor volumes from DMAT treated batch (8 tumors) represented with filled triangles. * represent p < 0.01 as assessed daily with student t-test.

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Figure 3.2. shows the results from a MEG-01 xenograft treatment with DMAT second

trial. This second trial (n = 6, 12 tumors on both flanks left and right) was started when

tumors were smaller (100-200 mm3), see figure 3.2. DMAT in DMSO was administered

daily at 3 mg/animal exogenous from the tumors. Tumors were measured daily and tumor

volume was calculated for each tumor using the prolate-spheroid formula (mm3). Control mice received a 50 µl DMSO injection daily. The treatment length was two weeks.

Because of a dose increase in the DMAT treatment and smaller starting tumor size, the effect of DMAT was more pronounced, see figure 3.2 versus figure 3.1. Significant effect was observed earlier than with the first trial, starting from the 4th day (3 days earlier).

From this day up to the end of the trial, tumor development inhibition and tumor ablation

(filled triangles) were observed. In contrast, the control tumors (just DMSO, filled

squares) were growing exponentially for both experimental sets.

Thus we can conclude that the treatment of MEG-01 xenografts induces

proliferation arrest in vivo. This tumor proliferation arrest is due to apoptosis-necrosis of

tumor tissue and reduced angiogenesis, as observed from the histological analysis of the

tumor tissue (H&E staining), see figure 3.3. Figure 3.3, panel A and B shows the

hematoxylin-eosin staining of MEG-01 tumors (Control-DMSO versus Treated with

DMAT). Figure 3.3, panel C shows a plot of necrotic area, apoptotic area versus normal

areas (pixels) from hematoxylin and eosin images from all the MEG-01 tumors. Figure

3.3, panel D shows a plot of angiogenesis area in untreated (DMSO) MEG-01 tumors

(pixels) versus angiogenesis areas in DMAT treated MEG-01 tumors.

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Figure 3.2. MEG-01 xenograft treated with 3 mg DMAT/animal/day for 2 weeks.

Black filled squares represent an average of tumor volumes of 12 tumors for Control batch (DMSO only) represented versus tumor volumes from DMAT treated batch (12 tumors) represented with filled triangles. * represent p < 0.01

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Figure 3.3. Hematoxylin-eosin staining of MEG-01 tumor tissues.

A. Control – DMSO MEG-01 tumor, magnification X10 B. Treated with DMAT tumor, magnification X10, C. MEG-01 H&E apoptotic-necrotic areas, D. MEG-01 H&E angiogenesis areas

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Based on our in vitro observations that MEG-01 cells treated with DMAT produce platelets, we tested whether or not this phenomena occurs in vivo also. Thus platelets counting (figure 3.4 and figure 3.5) and tail-bleeding experiments (figure 3.6) were performed. Figure 3.4. shows the processed results of blood counts for the second set (n =

6) of MEG-01 xenograft. Figure 3.4, panel A shows platelets counts from MEG-01 xenograft mice (Control, DMAT treated) versus normal murine platelet counts (cells/µl).

Figure 3.4, panel B shows abnormal cell counts from MEG-01 xenograft mice

(Control, DMAT treated) versus normal murine abnormal cell counts (cells/µl). It is interesting to observe that the mice bearing MEG-01 tumors have increased platelets counts, as well as high abnormal cell counts. Details of such blood counting experiment can be observed in the figure 3.5. Figure 3.5 shows a MEG-01 Control (whole blood) sample versus a MEG-01 treated sample, when analyzed with the hematological analyzer to determine blood counts. Figure 3.5. panel A, is a representative of a Control-MEG-01 xenograft (DMSO only), with very high abnormal cells percentage. In contrast, in figure

3.5, panel B, is a representative DMAT treated sample from MEG-01 xenograft with very high platelets counts, but low abnormal cell counts. These results are very interesting because it seems that MEG-01 tumors in mice will produce circulating blast cells or that this tumors may produce stimulating factors that will induce the release of such abnormal cells from mice bone marrow. Mice treated with DMAT had higher platelets counts than the ones treated just with the vehicle DMSO, showing that DMAT induces platelets release in vivo from MEG-01 tumors.

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Figure 3.4. Whole blood counts for MEG-01 xenograft trial.

A. Platelet counts (counts*106 cells/µl) for Control-DMSO MEG-01 xenograft, DMAT- treated MEG-01 xenograft versus Normal mice blood counts (no tumor), B. Abnormal cell percentages for Control-DMSO MEG-01 xenograft, DMAT-treated MEG-01 xenograft versus Normal mice blood counts (no tumor). Unpaired student t-test was used to see statistical differrences (* significant, p<0.05, ** very signficant, p<0.01, *** extremly significant, p<0.001)

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A B

Figure 3.5. Whole-blood cell counts histogram results.

A. Control-MEG-01 xenograft DMSO only, with high abnormal cells percentage, B. DMAT treated MEG-01 xenograft with very high platelets counts.

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The mice treated with DMAT had lower abnormal cell counts which may be correlated with the maturation process from blast to megakaryocytes induced by DMAT in MEG-01 cells, thus less blasts are available to be release in the blood stream. It can also be speculated that, because DMAT decreases tumor size, smaller tumors will produce less stimulating factors and thus we will have less abnormal cells.

However, the examination of spleen tissue sections (H&E, figure 3.6) revealed megakaryocytic cell infiltration in spleen of both treated and non-treated MEG-01 tumor bearing mice (cells with huge nucleus are highlighted with white squares). Figure 3.6 shows comparatively, the hematoxylin-eosin staining of spleens from MEG-01 tumor bearing mice (Control-DMSO, figure 3.6 panel A versus DMAT treated, figure 3.6 panel

B) versus normal spleen from normal healthy mice (figure 3.6 panel C –horizontal section- and figure 3.6 panel D – transversal section).

Next we measured spleeen sizes for all sets of mice, as it can be observed in figure

3.7 (length, cm). Normal mice, MEG-01 xenograft Control (DMSO) and MEG-01 xenograft treated with DMAT (2 and 3 mg/animal/day) averaged spleen lengths were compared with normal spleens sizes from normal mice. This analysis showed very significant statistical differences between the normal mice and MEG-01 tumor bearing mice. The tumor bearing mice treated with DMAT had smaller spleens than the non- treated mice with tumors. Thus, we can conclude that the observed abnormal cell counts are due to MEG-01 blasts released into circulation from MEG-01 tumor bearing mice.

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Figure 3.6. Hematoxylin-eosin staining of MEG-01 spleen.

A. Control –DMSO MEG-01 xenograft spleen, B. Treated with DMAT xenograft spleen, C. Control no tumors, no treatment, horizontal section (normal), D. Control no tumors, no treatment, transversal section (normal). Magnification X10

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Figure 3.7. MEG-01 xenografts spleen sizes.

Spleens were measured (length, cm). Student unpaired t-test was used to analyse data, MEG-01 Control (DMSO only) spleen sizes were compared with MEG-01 DMAT treated (2 and 3 mg/animal/day) DMAT, (*, p<0.05) and compared each of them through student unpaired t-test with Normal spleen sizes (***, p<0.001) showed very significant statistical differences.

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Because MEG-01 mice had high platelets counts, it was interesting to examine if this thrombocytosis induces any diferrence versus normal and DMAT treated tail- bleeding times. Figure 3.8. shows the results of tail-bleeding times from both MEG-01 trials versus normal tail-bleeding times for both sets of mice (higher tumors at the beginning of treatment and 2 mg/animal/day DMAT dose and smaller tumor at the beginning of treatment and 3 mg/animal/day). This data show that there is no statistically significant diference between the tail-bleeding times of this set of mice, for the second set

(3 mg/ml) there is some effect. It seems that MEG-01 mice have a slightly decreased tail- bleeding time than the normal untreated mice, whereas the DMAT treated mice had a slightly increased tail-bleeding time versus the the MEG-01 tumor bearing mice left untreated.

However for this type of experiments like tail-bleeding, a much bigger number of samples (an averge optimal n = 100) should be considered, in order to be able to draw any conclusions. Thus considering these results we can speculate that DMAT may have a mild platelet aggregation inhibitory effect, like heparin, which is also a CK2 inhibitor.

These DMAT treated mice seem to be fine (they do not present symptoms of blood clotting problems like severe bleeding or thrombosis) which sustains these tail-bleeding results.

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Figure 3.8. Tail-bleeding times for MEG-01 xenograft.

Squares represent tail-bleeding times (minutes) for MEG-01 Control (DMSO) mice, triangles represent tail-bleeding times (minutes) for MEG-01 Treated (2 mg/animal/day DMAT) and circles represent normal values. A. Tail-bleeding times for trial 1 (2 mg/ml), B. Tail-bleeding times for trial 2 (3 mg/ml)

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Figure 3.9. Hematoxylin-eosin staining of MEG-01 liver.

A. Control MEG-01 – liver from MEG-01 xenograft mouse (DMSO treated) B. Liver from a MEG-01 mouse xenograft treated with DMAT, C. Normal liver from a healthy normal mouse (horizontal section), D. Normal liver from a healthy normal mouse (transversal section). Magnification X10.

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In order to see if there is any cytotoxic effect of DMAT on MEG-01 xenografts, liver sections stained with H&E were examined (figure 3.9). MEG-01 tumor bearing mice show some abnormal liver features, like increased spaces (liver fibrosis) see figure

3.9, panel A. The liver from MEG-01 tumor bearing mouse treated with DMAT (figure

3.9 panel B) looks normal like a healthy liver from C57BL (figure 3.9, panel C and D).

This data suggests first of all that DMAT is not toxic and that the MEG-01 tumors may induce stress on the liver.

A toxicity pilot study was performed. A normal healthy nu/nu male mouse was treated with a high quantity of DMAT (10 mg/animal/day, which is 308 mg/kg/day) in the same way as the other treatments were executed. The animal was observed for three weeks. The comportament of the animal was relatively normal, he ate, slept, drinked water. The animal presented increased soreness and swollen tissue at the injection site.

After two weeks, blood was collected from this animal and then he was euthanized and tissues were collected for histology. Tissues from various parts of the body were collected and stained with H&E. Results are summarized in figure 3.10 (H&E of brain, liver, kidney, spleen, injection site and muscle). If we compare these histological results with normal healthy mouse tissues (from a C57BL animal), see figure 3.11 and figure

3.12, we can definetly say that the organs of the DMAT treated mouse are normal. Figure

3.11 shows H&E staining of tissues (horizontal sections) from organs isolated from a healthy normal C57BL mouse (panel A brain, B. Liver, C hearth, D kidney, E lung, F aorta). Figure 3.12 shows shows H&E staining of tissues (transversal sections) from organs from a C57BL mouse that was normal and healthy.

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From this pilot toxicity study we can withdraw the conclusion that DMAT is not toxic to the animal even at this high dose. But it may also be possible that DMAT was wasted and not metabolized because it is not soluble. DMAT deposits were observed like calcifications in the tissue were the injections were performed.

Blood from this toxicity study mouse was also collected and runned on the flow cytometer whithout any staining just to observe wether or not the blood presents normal cell populations, see figure 3.13 (panel A logarithmic scale, FSCH-SSCH histogram, panel B linear scale, FSCH-SSCH histogram). From this figure we can tell that there were no changes in the whole blood cells populations due to DMAT treatment. Cell percentages for gated populations are in normal ranges, see figure 3.13, panel B. Gate R1

(monocytes) is 2%, gate R2 (lymphocytes) is between 20-40%, gate R3 (granulocytes) is about 6 % and platelets gate R4 is about 40%. These values are normal [31, 32].

DMAT induced tumor proliferation arrest and ablation. Tumors from mice treated with DMAT had high necrotic and apoptotic areas. Vascularization areas of such tumors were significantly reduced versus the non-treated tumors, suggesting a possible anti- angiogenic effect. DMAT enhanced platelets release from MEG-01 cells in vivo. Mice bearing MEG-01 tumors had high abnormal cell counts and highly polyploid cells infiltrates the spleens of such mice. MEG-01 xenografts also had splenomegaly and

DMAT treated mice had smaller spleens and lower abnormal cell counts. DMAT administered at high concentration did not have toxic effects. Thus we can propose

DMAT as a candidate for developing new therapies for chronic myelogenous leukemia.

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Figure 3.10. DMAT toxicity level (10 mg/day/animal) treated male nude mice (2 weeks of treatment).

A. brain H&E, B. Liver H&E, C. Kidney H&E, D. Spleen H&E, E. Injection site, F. Muscle from leg

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Figure 3.11. Normal healthy C57BL mouse (horizontal sections) H&E staining of organs

A. brain H&E, B. Liver H&E, C. Hearth H&E, D Kidney H&E, E. Spleen H&E, F. Lung, H&E F. Aorta H&E

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Figure 3.12. Normal healthy C57BL mouse (transversal sections) H&E staining of organs

A. brain H&E, B. Liver H&E, C. Hearth H&E, D Kidney H&E, E. Spleen H&E, F. Lung, H&E F. Aorta H&E

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Figure 3.13. Toxicity mouse whole-blood non-lysed flow cytometry

A. logarithmic scale (FSCH and SSCH histogram), gate R1 – RBC cells, gate R2 – platelets, gate R3 - debris B. Linear scale (FSCH and SSCH histogram), gate R1 – monocytes (2%), gate R2 – lymphocytes (25%), gate R3 – granulocytes (6%), gate R4 – platelets (40%).

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3.5. DISCUSSION

MEG-01 cells are malignant megakaryoblasts isolated from a patient with chronic

myelogenous leukemia in blast crisis [1]. MEG-01 cells are presenting a malignant

phenotype showing increased proliferation and capacity to produce solid tumors in vivo.

MEG-01 cell line may provide a useful model for testing the in vivo efficacy of anti-

tumor agents and immunotoxins, and for studying the pathophysiological mechanisms of

human megakaryoblastic leukemia [11]. Thus we created MEG-01 murine xenografts to

study the effects of DMAT in vivo.

First thing that we noticed with these studies is that DMAT induces tumor

proliferation arrest in vivo, in MEG-01 xenografts. DMAT has a better effect on smaller

tumors, which is normal. Also if the concentration of DMAT is increased, the response is

better. This proliferation arrest of the tumors is due to apoptosis and necrosis induced.

These effects were expected based on the fact that DMAT induces apoptosis and

proliferation arrest of MEG-01 cells, as observed from in vitro experiments previously

described in Chapter II. DMAT through its action on CK2 may modulate the AKT kinase

[33, 34] and therefore this could be a possible explanation of reduced angiogenesis in

treated tumor tissue versus non-treated, see figure 3.3 panel D and this reduction in

angiogenesis may result in tumor growth arrest. Tumors are highly vascularized because

they need a lot of nutriments to sustain their accelerated growth, which are brought to

them by blood.

The MEG-01 tumors released platelets in vivo due to casein kinase 2 inhibitor treatment (DMAT). We observed very high platelets counts for both MEG-01 treated and untreated xenografts. Tail-bleeding times for MEG-01 xenograft mice were close to

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normal (slightly decreased for Control-DMSO untreated and slightly increased for MEG-

01 DMAT treated mice). Based on the functional platelets assays performed in vitro with

MEG-01 platelets, we know that these MEG-01 platelets (released due to DMAT

treatment) are functional and can aggregate. These tail-bleeding results maybe due to

several possibilites: first thing, murine platelets respond to a different thrombin (IIa is not

evolutionary conserved). IIa in humans activates PAR-1 and PAR-4 receptors on human

platelets whereas on murine platelets PAR-3 and PAR-4 receptors are involved [35]. IIa

in human has a Na+ binding site, whereas due to a mutation (Asp 222 to Lys) in murine

IIa, this Na+ binding site does not exist [36]. Thus considering these facts, it is possible that murine platelets may respond to human thrombin, but the reverse is less likely, because the mechanism of platelet activation by IIa is more complex in human than in mouse. Therefore if MEG-01 tumors produce platelets in vivo due to DMAT action, these human platelets will not be able to form the stable clot, because murine thrombin will not act properly on them, so the only stable clots will occur with mouse’s own platelets.

Another possibility is that DMAT being a CK2 inhibitor, like heparin and as any other kinase inhibitors is not 100% specific to CK2, therefore it may inhibit other required phosphorylations so it will induce a short delay in the in vivo clotting. Anyway the differences versus normal tail-bleeding times are not as big to create a problem in the survival of these animals. More tail-bleeding experiments (n = 100) are required in order to be able to indeed have enough data points to be certain of a specific effect.

It is certain though that the MEG-01 tumors may infiltrate the spleen of MEG-01 xenografts, see figure 3.6 (both control and treated) and leukemic megakaryoblastic cells appeared in the blood of mice with MEG-01 tumors (as high abnormal cell counts) see

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figure 3.4, and figure 3.5. MEG-01 mice had all splenomegaly (treated and untreated),

see figure 3.7. This splenomegaly is correlated directly with high abnormal cell counts

(found in both treated and non-treated mice), see figure 3.4. Control mice with bigger

tumors, have bigger spleen sizes and also have higher percentages of abnormal cell

counts, which maybe correlated with release of MEG-01 undifferrentiated blasts in the blood stream. Also, high platelets counts maybe responsible for increased spleen sizes.

Now what it is interesting is that chronic myelogenous leukemia is characterized by circulating blasts (high abnormal cell number) and high platelets counts [37-39]. Various models of murine chronic myelogenous leukemia were created, like murine CML models made by transferring human stem cells that express BCR-ABL from retroviral vectors

[40, 41]. An alternative approach to developing an in vivo model of CML was developed based on the fact that normal human haemopoietic cells will engraft relatively efficiently

in immunodeficient mice. Transplantation of cell suspensions from patients with chronic phase CML into irradiated SCID or NOD/SCID mice has required the use of relatively large cell doses in order to detect cells with engrafting potential. Similarly, although some Ph+ cells can be produced in the immunodeficient murine hosts, a greater

engraftment of the mice with normal (Ph−) cells is typically seen [40, 42]. Thus MEG-01

engrafted mice makes an inexpensive, easy to maintain and produce model of chronic

myelogenous leukemia which manifests itself by releasing circulating blasts and

thrombocytosis, which are characteristic symptoms of CML.

Thus having a murine model and the potential drug candidate DMAT which works

in vivo in the same manner as in vitro (see Chapter II), we wanted to see if DMAT has

toxic effects. Livers from DMAT treated mice were stained with H&E and examined.

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Liver tissues from MEG-01 mice treated with DMAT had a normal histology. This data

suggests first of all that DMAT is not toxic. In order to see if indeed DMAT is not toxic,

then a pilot toxicity study was done. A nude healthy mouse was injected daily with high

amounts of DMAT (10 mg/animal). Liver, brain, kidney, muscle tissue seem to be normal in DMAT toxicity level treated mice, when H&E sections from this animal were examined and compared with normal tissues from C57BL healthy mice. The injection site shows necrosis. This is due to insoluble DMAT deposits and DMSO (the vehicle for

DMAT). The lack of toxicity at a such high concentration of DMAT maybe also due to the insolubility in body buffers of the CK2 inhibitor. Accumulation at the injection site was observed in all the treated mice (even with very low concentrations), but with such a high concentration, crystal-like solid deposits formed. A novel formulation of the substance maybe required if it presents interest as a drug.

Overall this data shows that DMAT may behave as a megakaryocytic maturation inducer in vivo and this is correlated with in vitro studies. DMAT also has potent anti- cancerous activity and does not seem to be toxic. This may have very important clinical value.

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CHAPTER IV

TREATMENT OF VARIOUS CANCER CELL LINES WITH DMAT

4.1. ABSTRACT

Casein kinase 2 was found to be up-regulated and over-expressed in tumor tissue and maybe responsible for the cancer apparition and sustainability. Studies with various

CK2 inhibitors (antisense nucleotides) and ATP site inhibitors in malignant cell lines as well as in murine xenografts revealed that such inhibitors maybe considered as possible tools in developing new cancer therapies. Such inhibitors induced apoptosis and proliferation arrest in vitro and in vivo in different cancer cell lines. MCF-7 cells are breast cancer cells, estrogen dependent, with a highly abnormal proliferation rate. SW-

480 are malignant colon cells (ephitelial cancer cells). WM-164 are very agressive melanoma cells. Such cells are all resistant to apoptosis and manifest anchorage independence by growing colonies in soft agar (a sign of malignicity). All these cell lines

were not tested before us in vitro and in vivo for effects due to the CK2 inhibitor DMAT.

Based on our preliminary results with MEG-01 megakaryoblastic leukemia cell line, and

based on literature data, we decided to test this inhibitor in a few very differrent cancer

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cell lines to see if it can be a general cancer drug. Results were as expected. DMAT induced proliferation arrest in vitro, due to apoptosis and inhibited growth in soft agar. In vivo results were very good for the MCF-7 xenografts (at very low concentrations of

DMAT). This may suggest involvement of CK2 in the female hormone metabolism.

MCF-7 showed proliferation arrest in tumors, even small tumor ablation due to DMAT treatment. Similar results were obtained for colon cancer and skin cancer (but at much higher concentrations of inhibitor). Tumors treated showed necrosis, apoptosis and reduced angiogenesis versus untreated (DMSO only) tumors. All these data together suggest a common mechanism were CK2 acts that exists in all the cancer cells.

4.2. INTRODUCTION

Casein kinase 2 (CK2), was found to be constitutively activated, over-expressed and to serve as an oncoprotein in various cancers [1-7]. In cancer cells, CK2 localization is nuclear and also CK2 is deregulated, being elevated 3- to 7- folds. The up-regulation and hyperactivity of CK2 has an anti-apoptotic effect and is correlated with aggressive tumor behavior in different cancer types examined and results in abnormal platelet counts in leukemia [8]. Antisense nucleotides treatments against CK2α mRNA were studied in mouse human prostate cancer xenografts. A very interesting observation following these studies showed that CK2 inhibition affects the tumors (by inducing apoptosis and tumor ablation), but does not affect the normal surrounding tissue [3, 7]. This was explained as being caused by different localizations of the alpha subunit of casein kinase 2 in normal

(preponderant cytoplasmic) versus malignant tissue (preponderant in the nuclear matrix).

CK2 elevation in nuclear matrix in cancerous cells is related to cell growth, survival and

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resistance to apoptosis mainly due to cell cycle control [5, 7, 9]. The disparity effect of

CK2 inhibition in malignant versus normal cells (as well as tissues) was observed on histological analysis of normal and neck squamos carcinoma tissues [3, 10]. Also differential response to CK2 inhibition (with antisense nucleotides against CK2α mRNA, as well as with specific CK2 inhibitor, DRB) in different normal and benign cell lines was observed [6, 11]. Therefore it seems that normal and benign cells are less sensitive to the CK2 inhibition. Mechanisms in which CK2 plays a role are very complex and occur both in the nuclear matrix as well as in the cytoplasm. What succession of events occurs first (the cytoplasmic or nuclear) was not determined, thus to speculate that it can be either one it would not be realistic.

MCF-7 breast cancer cell line.

MCF-7 is a breast cancer cell line established from the mammary gland of a 69 years old woman, it is an adenocarcinoma derived from pleural effusion (metastatic site)

[12]. MCF-7 are differentiated mammary epithelium cells that express estrogen receptor.

MCF-7 cells express oncogenes (WNT7B and Tx-4) and are sensitive to TNF alpha, which inhibits their growth [13]. MCF-7 growth in vivo is hormone dependent (estradiol)

[14]. MCF-7 produce insulin-like growth factor binding proteins (IGFBP). IGFBP secretion from MCF-7 can be modulated by treatment with anti-estrogens [15].

Clinical studies have shown that therapeutic agents preventing the synthesis and actions of estrogens are highly successful in the treatment of breast cancer. The mechanisms by which estrogens stimulate cell proliferation, however, are still not clear.

The epithelial breast cancer derived MCF-7 cell line is one of the most frequently used model systems. Casein kinase 2 was found to promote aberrant activation of nuclear

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factor- B, transformed phenotype, and survival of breast cancer cells [16]. The Her-2/neu

oncogene is a member of the epidermal growth factor (EGF) receptor family, encodes a transmembrane tyrosine kinase receptor. Her-2/neu activates nuclear factor (NF)- B via a

phosphatidylinositol 3 kinase (PI3-K)-Akt kinase signaling pathway in mouse mammary tumor virus (MMTV)-Her-2/neu NF639 mouse breast cancer cells. Because ectopic CK2

activity appears sufficient to induce NF- B, the elevated CK2 activity observed in many primary human breast cancers likely plays a role in aberrant activation of NF- B and,

suggesting that CK2 represents a potential therapeutic target, in breast cancer [16].

Human SIX1 (HSIX1) is a protein implicated in muscle, eye, head, and brain

development. CK2 and HSIX1 have both been implicated in cancer and in cell cycle

control, it was proposed that HSIX1, whose activity is regulated by CK2, is an important

target of CK2 in G(2)/M checkpoint control and that both molecules participate in the

same pathway whose dysregulation leads to cancer, in MCF-7 cells [17]. Ultimately

TNF-α was found to induce apoptosis in breast cancer cells. CK2 amplifies the effect of

TNF-α [18].

SW480 colon cancer cell line.

SW480 is a colorectal adenocarcinoma from Duke’s type B tumor stage. This cell

ine secretes TGFβ, keratin and carcinoembrionic antigen (CEA).[19]

To identify mechanisms by which CK2 promotes survival, specific CK2 inhibitors

TBB and DMAT were used to treat colon cancer cells. TBB and DMAT significantly

decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. These

authors concluded that CK2 kinase activity promotes survival by increasing survivin

expression via β-catenin–Tcf/Lef-mediated transcription [20]. Mutations in the

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adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and colorectal cancer development. The tumor suppressor protein APC and

CK2 interaction discovery suggests that growth-inhibitory effects of APC may be regulated by inhibition of CK2 [21].

WM164 melanoma cell line.

WM164 is a nodular melanoma in vertical growth phase cell line. The WM164 line was established from a metastatic melanoma from a lymph node from a 21 year old male.

This cell line is highly agressive and develops tumors in nude mice.[22, 23]

A proteomics study performed in several melanoma cell lines, found that a nucleolar and centrosome associated protein named nucleophosmin B23 is responsible for the malignant phenotype in melanoma. B23 is a target for cyclin E/CDK2 thus affecting cell cycle control. B23 is up-regulated and shows enhanced phosphorylation in tumor cells [24]. B23 was found to be mainly phosphorylated by CK2 and this phosphorylation was inhibited by heparin, apigenin and DRB in vitro and in vivo.[25-29]

p53 protein is a tumor suppressor and its gene disruption is associated with

approximately 55% of human cancers. The p53 protein is a regulatory cell cycle protein,

either preventing or initiating apoptosis. Phosphorylation by CK2 activates the specific

DNA binding of p53 and stimulates its ability to suppress cellular growth. [30-33]

The PTEN tumor suppressor gene encodes a phosphatidylinositol 3'-phosphatase

that is inactivated in human tumors, like glioblastoma, melanoma, and prostate and endometrial carcinoma. PTEN is a seryl phosphoprotein and a substrate of the kinase

CK2. CK2 inhibits the PTEN kinase, and this plays a role in oncogenesis [34].

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Considering all the possibilities of action of CK2 on various cancers, all finalizing

with the same effect, inhibition of CK2 proves to be of clinical importance.

4.3. EXPERIMENTAL DESIGN

Materials used in these experiments were previously described in the Chapters II and III.

4.3.1. Cell Culture

MCF-7 breast cancer, SW-480 colon cancer and WM-164 cell lines were a

generous gift from Dr. D.J. Lindner (Taussig Cancer Center, Cleveland Clinic). Cells

0 were maintained in an incubator, with humidified atmosphere of CO2 5%, and at 37 C.

Cell culture media was DMEM F12 with L-Glutamine (2mM), containing 10 mM

HEPES, and 1.0 mM Sodium Pyruvate and 10% heat-inactivated fetal bovine serum

(FBS) and 5U Penicillin/Streptomycin were used to grow all these cell lines.

When cells reached 70-80 % confluence were splitted accordingly.

4.3.2. Apoptosis Assays using Flow Cytometry with AnnexinV-FITC and propidium iodide (PI).

For induction of typical apoptosis, cells were grown in the presence of 1µM staurosporine for 6 hours. The staurosporine treated cells were stained as follows: control

1 with PI, control 2 with AnnexinV-FITC and control 3 with both PI and AnnexinV-

FITC, in order to have the brightest controls for compensation and proper collection of the flow cytometry data. Cell necrosis was induced by heat shock (650C for 30 minutes).

Controls for necrosis were stained in the same way as for the Staurosporine treated cells.

The unstained control signal was subtracted by gating from the stained control and the

rest of stained samples, to get only the positive cells signal (stained). The stained control

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(PI and AnnexinV-FITC labeled), was then used as the reference in establishing the level

of apoptosis induced by the treatments. Data was collected on logarithmic modes, two- colors. AnnexinV-FITC corresponds to FL1H channel, and PI to FL3H or FL2H channels. Cells were gated out from debris on FSCH and SSCH histogram using appropriate flow cytometry software (WinMDI 2.8 or Flow Jo ver 6.2).

AnnexinV-FITC and PI staining protocol

Apoptosis AnnexinV-FITC and PI kit from BD Biosciences was used. 105 – 106

cells were stained with 50 µg/ml PI and with 0.5 µg/ml FITC-labeled Annexin V using

the staining protocol provided by the supplier. Briefly, after trypsinization, cells were

counted using a Neubauer hemacytometer, then washed by centrifugation (400g, 5

minutes) and resuspended in the appropriate amount of AnnexinV-binding buffer 1X

(provided in the kit), in order to have 106 cells in 100 µl buffer. Then PI and AnnexinV-

FITC were added to the cells (5 µl each). Incubation was performed for 15 minutes in the dark at room temperature. Samples were then analyzed immediately by flow cytometry.

4.3.3. Viability (proliferation) assay

Cells were counted using a Neubauer hemacytometer. Trypan blue dye was used according to the manufacturer (Sigma-Aldrich). DMSO, which is the vehicle for DMAT was used as a mock control, considering the highest amount that was used as a vehicle for

DMAT. Phase-contrast micrographs of cells were taken using an inverted microscope

Olympus CK40, using monochromatic settings, in order to correlate the effect of DMAT on proliferation with pictures.

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4.3.4. Anchorage independence in “soft agar” assay

Agarose was mixed with MCF-7, SW-480 and WM-164 growth media DMEM F12

0 with 10% FBS. Cells were grown in 12 wells dishes at 37 C, 5% CO2, 90% humidity in a VWR incubator. Colonies formation was observed and micrograph images were taken using an Olympus CK40 microscope. Cells were observed after one week. We compared the control (DMSO) samples with the samples grown in the presence of 10 µM DMAT.

Media was renewed on the top of the agar every 4 days and DMAT treatment was performed every time. 50 colonies from 20 images were analyzed. Measurements of the colonies areas were performed using the NIH Image software ver.1.63 for MacOS 9.

4.3.5. Creation of the MCF-7, SW-480, and WM-164 xenografts

Materials

17-beta-estradiol - 0.36 mg E2/90-day release pellet- Innovative Research of

America (Sarasota, FL, USA)

Method

Immunodeficient male and female athymic nude nu/nu mice were used, provided

and housed by Dr. Lindner, DJ from Taussig Cancer Center, Cleveland Clinic and Case

Western Reserve University. Mice were checked every day and were housed in filtered

air flow cabinets with autoclaved bedding at a density of 5 mice/cage. They were fed

autoclaved Purina Lab Rodent Chow 5010 and HCl-acidified distilled water ad libitum

and were placed in rooms with controlled temperature, humidity and 12-hr light-dark

cycles. Procedures involving animals and their care were conducted in conformity with

the institutional guidelines that are in compliance with national and international laws and

policies (EEC Council Directive 86/609, OJL 358, December 1, 1987, and the National

188

Institutes of Health Guide for the Care and Use of Laboratory), Animals, NIH Publication

85-23, 1985).

For engraftment, cells were resuspended in cell culture media. A specific number of cells (depending on the cell line) were injected in each flank of the mouse (left and right) subcutaneous. Colon cancer cells SW-480 were injected at 2 X 106 cells/100 µl.

Melanoma WM-164 and breast cancer cells MCF-7 were injected at 3 X 106 cells/100 µl.

Cells were counted with a hemacytometer using Trypan blue (only live cells were

counted).

The average weight of the mice used in these sets of experiments was 30-35 g for

male mice and 20-25 g for female mice. Female mice weree used for MCF-7 xenografts,

because MCF-7 is female hormone depended, thus these mice require hormone

supplementation (17β-estradiol). This hormone was provided in drinking water with glucose to be more paleatable.

Treatment with DMAT was started when tumors were at least 100-200 mm3 volumes (prolate spheroid). Tumor volumes were calculated as prolate spheroid shown

bellow:

V = (4/3*π*(a)2*(b)

were „a” is half of the minor axis and „b” is half of the major axis of the prolate

spheroid. The prolate sheroid axes were measured with a caliper (mm). Animals were

treated with DMAT for approximately two weeks (when tumor volumes reached a size

unacceptable with the IACUC protocols, animals were sacrificed in a CO2 euthanasia chamber). Tumors were collected for further histological analysis.

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4.3.6. In vivo therapy with DMAT of MCF-7 xenografts

Therapy with DMAT was done in two trials (n = 4 both, with 2 tumors per mice).

The drinking water for mice was supplemented with 17-beta-estradiol. Tumor diameters were measured using a caliper and tumor volume was calculated using the prolate- spheroid formula. DMAT in DMSO as well as just DMSO as a control were administered by injection subcutaneous in the neck (exogenous from the tumor).

4.3.7. In vivo therapy with DMAT of SW-480 xenografts

Therapy with DMAT was done in one trial (n = 4 both, with 2 tumors per mice).

Tumor diameters were measured using a caliper and tumor volume was calculated using the prolate-spheroid formula. DMAT in DMSO as well as just DMSO as a control were administered by injection subcutaneous in the neck (exogenous from the tumor).

4.3.8. In vivo therapy with DMAT of WM-164 xenografts

Therapy with DMAT was done in one trial (n = 4 both, with 2 tumors per mice).

Tumor measurements will show if DMAT induces tumor ablation in the previously described murine xenografts.

4.3.9. Hematoxylin & eosin staining of tumor tissue and organs

After euthanasia with CO2, mice were supposed to necropsy. Tumors were collected from both flanks from under the skin and were measured for the last time and then fixed in formalin fixative. Various organs (spleen, liver, kidney, brain, lungs and legs) were collected and fixed. Spleens were also measured (as lenght, mm). Fresh tissue

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was immersed immediatly into liquid nitrogen and kept frozen at -800C. Fixed tissue was embedded in parafin. Sections (4-µm thick) were stained with hematoxylin and eosin and evaluated for pathologic changes in a blinded fashion. The protocol for processing and staining tissue with hematoxylin and eosin was described in detail in Chapter III.

4.3.10. Statistical analysis.

Statistical analysis was performed as described at Chapters II and III. Briefly student t-test and Anova One-Way test were performed depending on the sample type

(parametric or non-parametric). p < 0.05 (*) represents significantly diferent values.

4.4. RESULTS

CK2 is implicated in cellular transformation and development of tumorigenesis and aggressive tumor behavior. CK2 is over-expressed in cancers of mammary gland, lung, kidney, colon, head and neck, just to mention a few more studied. Various mechanisms are responsible for the effects of CK2 in these cancers. Based on these facts and our preliminary data with chronic myelogenous leukemia cells, we decided to test the effects of the CK2 inhibitor DMAT on hormone dependent human breast cancer cells (MCF-7), colorectal adenocarcinoma cells (SW-480) and melanoma cells (WM-164). Our first finding was that DMAT induces proliferation arrest in vitro in MCF-7 cells, dose and time dependent. This proliferation arrest can be observed in figure 4.1 (proliferation assay with Trypan blue) and figure 4.2 (phase-contrast micrographs of MCF-7 cells). The figure 4.1 shows that the effects of DMAT on MCF-7 cells are dose and time dependent, and these results are sustained by the results from figure 4.2. In the figure 4.2, panels A and B show control MCF-7 cells (DMSO), 5X magnification and 10X magnification

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respectively. Difference in confluence (showing inhibition) is observed in figure 4.2 panel C and D, were cells were treated with DMAT 10 µM. The effect of DMAT is more pronounced with the doubling of the dose of DMAT (20 µM), figure 4.2 panels E and F.

Figure 4.3 shows that DMAT induces apoptosis dose dependent in MCF-7 cells in culture. Figure 4.3 represents results from flow cytometric apoptosis assay (AnnexinV-

FITC and PI) of MCF-7 cells after 24 hours of treatment with DMAT 10 and 20 µM.

Control (DMSO), has 4.1 % apoptotic cells, whereas DMAT 10 µM treatment of MCF-7 cells for 24 hours induced 28% apoptosis and DMAT 20 µM treatment of MCF-7 cells for 24 hours induced 49% apoptosis. From these results we can tell by comparison with

MEG-01 cells analyzed in Chapter II, that MCF-7 are much more sensitive to DMAT than the chronic myelogenous leukemia cells. This maybe due to different mechanisms of

CK2 action specific to MCF-7 cells, like hormone related involved pathways. MCF-7 cells are known to present the anchorage independence feature, due to activation of

MAPK through phosphatidylinositol-3 kinase by insulin-like growth factor I [35]. Based on the fact that CK2 is involved in this pathway [36], we decided to test the effects of

CK2 inhibition with DMAT on anchorage independence in soft agar assay of MCF-7 cells.

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MCF-7 cells, proliferation assay

6 9.0×10 Control DM AT 10 µM 8.0×10 6 DM AT 20 µM

7.0×10 6

6.0×10 6

l 5.0×10 6 /m lls

e 4.0×10 6 c

3.0×10 6

2.0×10 6

1.0×10 6

0 1 2 3 4 5 days

Figure 4.1. Proliferation assay of MCF-7 cells treated with DMAT 10 and 20 µM.

Squares - control (DMSO), triangles - DMAT 10 µM, stars - DMAT 20 µM. Treatments length was four days.

193

194

Figure 4.2. Phase-contrast micrographs of MCF-7 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. Control treated only with the vehicle DMSO, 5X magnification, B. Control treated only with the vehicle DMSO, 10X magnification, C. DMAT 10 µM treatment of MCF-7 cells, 5X magnification D. DMAT 10 µM treatment of MCF-7 cells, 10X magnification, E. DMAT 20 µM treatment of MCF-7 cells, 5X magnification, F. DMAT 20 µM treatment of MCF-7 cells, 10X magnification.

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The results are summarized in the figure 4.4, for one week treatment with DMAT

10 µM. From this figure we can conclude that DMAT inhibits anchorage independence in

MCF-7 cell line. This is most probably due to the inhibition of action of CK2 on the

MAPK and PI3K pathway. Having these results in MCF-7 cells in culture, next step was to test DMAT in a hormone-dependent in vivo model, to see if female hormone mechanisms makes a difference. So DMAT was administered to MCF-7 xenografts

(which received 17-beta-estradiol supplementation daily). Figure 4.5 shows the effects of

DMAT in vivo in the first trial that we performed. A low amount of DMAT induces proliferation arrest in vivo in MCF-7 xenografts, as seen in the figure 4.5. Figure 4.5 shows MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. In the figures 4.5 and figure 4.5, black filled squares are averaged values of tumor volumes for control batch (DMSO, 8 tumors) versus tumor volumes from DMAT treated batch (8 tumors) represented with filled triangles. Figure 4.6 shows that very low amount of DMAT is necessary to induce proliferation arrest in vivo in MCF-7 xenografts and tumor ablation if the tumors growth rate is decreased (versus figure 4.5). Figure 4.6 shows MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. These two trials had different growth of tumors due to different 17-beta- estradiol amount administered to the animals (lower dosage).

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Figure 4.3. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of MCF-7 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. Control( DMSO) B. DMAT 10 µM C. DMAT 20 µM, D. Quantification of apoptosis induced by DMAT after one day (triplicates).

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Figure 4.4. Soft agar anchorage independence assay (one week treatment with DMAT 10 µM).

A. Control (DMSO) MCF-7 colonies, B. DMAT treated, MCF-7 colonies, C. Colonies areas from various images were measured and quantified.

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MCF-7 xenograft treated with DMAT 10 mg/kg (in DMSO) per animal, daily, animal weight was averaged at 25 g at the start of treatment

2500 Control MCF-7 cells Treated MCF-7 cells n = 8 2250 * * 2000 3 m 1750 ) m d i * o r e

h 1500 p s

te a l 1250

o * r (p

e *

m 1000 u l o v r

o 750 m tu 500

250

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 days

Figure 4.5. MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. 17-beta-estradiol (0.04 mg/day) was supplemented in water.

Tumors were 200 mm3 in volume at start. Black filled squares - Control batch (DMSO) versus tumor volumes from DMAT treated batch represented with filled triangles. * represent p < 0.01 as assessed daily with student t-test.

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Figure 4.6. MCF-7 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks. 17-beta-estradiol (0.02 mg/day) was supplemented in water.

Tumors were 200 mm3 in volume at start. Black filled squares - average of tumor volumes for Control batch (DMSO) versus tumor volumes from DMAT treated batch represented with filled triangles. * represent p < 0.01 as assessed daily with student t-test.

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Figure 4.7. Hematoxylin and eosin staining of MCF-7 tumors.

A. Control (DMSO), MCF-7 tumor, B. Tumor from MCF-7 DMAT treated mice, C. MCF-7 H&E apoptotic-necrotic areas, D. MCF-7 H&E angiogenesis areas. Magnification 10X.

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Figure 4.8. Hematoxylin and eosin staining of MCF-7 livers.

A. Control (DMSO), liver from MCF-7 tumor bearing mouse B. Liver from MCF-7, DMAT treated mice, C. Liver from normal healthy wild type mouse C57BL (horizontal section), D. Liver from normal healthy wild type mouse C57BL (transversal section). Magnification 10X.

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Figure 4.9. Hematoxylin and eosin staining of MCF-7 spleens.

A. Control (DMSO), spleen from MCF-7 tumor bearing mouse B. Spleen from MCF-7 DMAT treated mice, C. Spleen from normal healthy wild type mouse C57BL (horizontal section), D. Spleen from normal healthy wild type mouse C57BL (transversal section). Magnification 10X.

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Observing such effects in vitro and in vivo, we analyzed the tissues from MCF-7 xenografts in order to see if there is apoptosis or necrosis in the tumor tissues from

DMAT treated animals. Organs were also examined (H&E) in order to see if they are affected in any way by these treatments. Thus histological analysis was performed. The figure 4.7 shows hematoxylin and eosin staining of MCF-7 tumors. The control (DMSO) tumor (figure 4.7, panel A) was compared with a tumor from MCF-7 DMAT treated mouse (figure 4.7, panel B). Necrosis and apoptosis are induced by DMAT in MCF-7 tumors as observed and this areas were quantified as averaged data from all the tumor samples, see figure 4.7 panel C. Organs samples did not show tumor infiltration or other abnormalities in MCF-7 tumor bearing mice (both treated and untreated). Normal liver and spleen phenotypes were observed in comparison with wild type C57BL healthy tissues. Figure 4.8 and figure 4.9 show that DMAT does not affect these organs in MCF-

7 xenografts and that MCF-7 cells does not infiltrate the spleen and the liver of the MCF-

7 xenograft.

In colon cancer cells, increased expression of casein kinase 2 (CK2) is associated with hyper-proliferation and suppression of apoptosis. One such mechanism is due to the fact that CK2 increases survivin expression, which causes the malignant transformation of colon cells. CK2 inhibitor TBB was found to inhibit this signaling [20]. If TBB affects this surviving expression, it is normal to presume that DMAT which is a better CK2 inhibitor than DMAT will have similar or better effects and thus DMAT will inhibit SW-

480 colon cancer cells growth. Figure 4.10 shows the effects of CK2α inhibitor (DMAT) on the proliferation rate of SW-480 cells using Trypan blue proliferation assay. DMAT

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was found to induce proliferation arrest in vitro in SW-480 cells dose and time dependent.

205

SW-480 cells, proliferation assay 1.2×10 7 Control 1.1×10 7 DM AT 10 µM DM AT 20 µM 1.1×10 7 1.0×10 7 9.5×10 6 9.0×10 6 8.5×10 6 8.0×10 6 7.5×10 6 7.0×10 6 6 l 6.5×10

m 6

/ 6.0×10 s l 5.5×10 6 6 cel 5.0×10 4.5×10 6 4.0×10 6 3.5×10 6 3.0×10 6 2.5×10 6 2.0×10 6 1.5×10 6 1.0×10 6 5.0×10 5 0 0 1 2 3 4 days

Figure 4.10. Proliferation assay of SW-480 cells treated with DMAT 10 and 20 µM (Trypan Blue exclusion).

Squares - control (DMSO), triangles - DMAT 10 µM, stars - DMAT 20 µM Treatment of SW-480 cells was for four days.

206

207

Figure 4.11. Phase-contrast micrographs of SW-480 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. Control treated only with the vehicle DMSO, 5X magnification, B. Control (DMSO), 10X magnification, C. DMAT 10 µM treatment of SW-480 cells, 5X magnification D. DMAT 10 µM treatment of SW-480 cells, 10X magnification, E. DMAT 20 µM treatment of SW-480 cells, 5X magnification, F. DMAT 20 µM treatment of SW-480 cells, 10X magnification.

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Figure 4.12. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of SW-480 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. DMAT 10 µM, B. DMAT 20 µM, C. Control (DMSO), D. Treatment of SW-480 cells with DMAT for one day, quantification of apoptosis (triplicates).

209

This proliferation arrest can be observed better in the figure 4.11, in the phase-contrast

micrographs of SW-480 cells. Figure 4.11, panel A and B shows control (DMSO) SW-

480 versus panels C and D, which shows SW-480 cells incubated with DMAT 10 µM

for 24 hours. Figure 4.11 panels E and F show DMAT 20 µM treatment of SW-480 cells

for 24 hours. Corresponding to the proliferation arrest we observed that DMAT induces

apoptosis dose dependent in SW-480 cells in culture. Figure 4.12 summarizes results

from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of SW-480 cells after 24

hours of treatment with DMAT 10 and 20 µM. Control (DMSO) has 3 % apoptotic cells,

whereas DMAT 10 µM treatment of SW-480 cells for 24 hours induced 19% apoptosis

and DMAT 20 µM treatment of SW-480 cells for 24 hours induced 48% apoptosis. These

results are comparable with the ones obtained with MCF-7 cells. Over-expression of

PKC beta1 and adenomatous polyposis coli (APC) signaling in the SW-480 colon cancer

cell line are responsible for the anchorage independence growth in soft agar. CK2 is

involved in these pathways and thus DMAT which is a potent CK2 inhibitor should inhibit the anchorage independence phenotype of SW-480 cells. Figure 4.13, soft agar anchorage independence assay (one week treatment with DMAT 10 µM) shows that indeed DMAT inhibits anchorage independence in SW-480 cell line. If DMAT represses malignant proliferation in vitro it should have similar effects in vivo. Therefore DMAT was used as a treatment of SW-480 tumor bearing mice. As expected we found that

DMAT induces proliferation arrest in vivo in SW-480 xenografts. Figure 4.14 shows

SW-480 xenograft treated with 1 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks.

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Figure 4.13. Soft agar anchorage independence assay for SW-480 cells (one week treatment with DMAT 10 µM).

A. Control (DMSO) SW-480 colonies, B. DMAT treated, SW-480 colonies, C. Colonies areas measured and quantified from several pictures. Magnification 10X.

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SW-480 xenograft treated with 40 mg/kg DMAT (in DMSO) per animal, daily animal weight was averaged at 35 g at the start of treatment

2500 SW-480 Control DMAT Treated SW-480 n = 8 2250 *

2000

3 * m 1750 ) m * d i o r * e

h 1500 p s

te * a l 1250

o * r * (p e *

m 1000 u l * vo r o 750 * m tu 500

250

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 days

Figure 4.14. SW-480 xenograft treated with 1 mg DMAT/animal/day (40 mg/kg DMAT) for two weeks.

Tumors were 200 mm3 in volume at start. Filled squares - average of tumor volumes for Control batch (DMSO) versus tumor volumes from DMAT treated batch represented with filled triangles. * represent p < 0.01 as assessed daily with student t-test.

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Figure 4.15. Hematoxylin and eosin staining of SW-480 tumors.

A. Control (DMSO) tumor SW-480, B. Tumor from SW-480 DMAT treated mice, C. SW-480 H&E apoptotic-necrotic areas, D. SW-480 H&E angiogenesis areas Magnification 10X.

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Figure 4.16. Hematoxylin and eosin staining of SW-480 livers.

A. Control (DMSO), SW-480 xenograft, liver B. Liver from SW-480 DMAT treated mice, C. Liver from normal healthy wild type mouse C57BL (horizontal section), D. Liver from normal healthy wild type mouse C57BL (transversal section). Magnification 10X.

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Figure 4.17. Hematoxylin and eosin staining of SW-480 spleens.

A. Control (DMSO), SW-480 tumor bearing, spleen B. Spleen from SW-480 DMAT treated mice C. Spleen from normal healthy wild type mouse C57BL (horizontal section), D. Spleen from normal healthy wild type mouse C57BL (transversal section). Magnification 10X.

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The effect less stronger than in the MCF-7 xenografts (we used four times more DMAT), suggesting potential involvement of different mechanisms of action, especially female hormone pathways (due to CK2 signaling). Tumor sections were analyzed (histology) for necrosis and apoptosis. Figure 4.15 show hematoxylin and eosin staining of SW-480 tumors. Figure 4.15, panel A, Control tumor (DMSO) is compared with panel B (tumor from SW-480 DMAT treated mice). This figure shows that necrosis and apoptosis are induced by DMAT in SW-480 tumors to an even higher extent than in MCF-7 xenografts. Organs were checked (H&E) and as seen in the figure 4.16 (spleen) and figure 4.17 (liver) show that DMAT does not affect these organs in SW-480 xenografts.

SW-480 cells did not infiltrate the spleen and liver of SW-480 xenografts.

CK2 maybe involved in the development of melanoma cells due to its action on

B23 nuclear protein, as well as due to its effects on AKT/PI3K pathways. First things that were tested were effects of DMAT on proliferation and apoptosis in vitro in WM-164 melanoma cells. Results were like expected, DMAT induced proliferation arrest in melanoma cells, dose and time dependent, maybe due to a dominant mechanism different than in breast cancer cells, for example the VEGF signaling. In the figure 4.18 we can observe that DMAT induces proliferation arrest in melanoma cells dose and time dependent. Figure 4.19 show that DMAT induces apoptosis dose dependent in WM-164 cells in culture. In the figure 4.19 are results from flow cytometric apoptosis assay

(AnnexinV-FITC and PI) of WM-164 cells after 24 hours of treatment with DMAT 10 and 20 µM. Control (DMSO) has 4.2 % apoptotic cells, whereas DMAT 10 µM treatment of WM-164 cells for 24 hours induced 30% apoptosis and DMAT 20 µM for 24 hours induced 50% apoptosis.

216

WM -164 cells, proliferation assay 1.0×10 7 Control DM AT 10 µM DM AT 20 µM 9.0×10 6

8.0×10 6

7.0×10 6

6.0×10 6 l

/m 5.0×10 6 ls l e c 4.0×10 6

3.0×10 6

2.0×10 6

1.0×10 6

0 0 1 2 3 4 days

Figure 4.18. Proliferation assay of WM-164 cells treated with DMAT 10 and 20 µM (Trypan Blue exclusion).

Squares – Control (DMSO), triangles - DMAT 10 µM, Stars - DMAT 20 µM Treatment length was four days.

217

218

Figure 4.19. Phase-contrast micrographs of WM-164 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. Control (DMSO), 5X magnification, B. Control (DMSO), 10X magnification, C. DMAT 10 µM treatment of WM-164 cells, 5X magnification D. DMAT 10 µM treatment of WM-164 cells, 10X magnification, E. DMAT 20 µM treatment of WM-164 cells, 5X magnification, F. DMAT 20 µM treatment of WM-164 cells, 10X magnification.

219

220

Figure 4.20. Results from flow cytometric apoptosis assay (AnnexinV-FITC and PI) of WM-164 cells after 24 hours of treatment with DMAT 10 and 20 µM.

A. Control (DMSO) B. DMAT 10 µM treatment of WM-164 cells, C. DMAT 20 µM, D. Quantification of the results from diferent apoptosis assays performed after one day of treatment with DMAT (triplicates).

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Figure 4.21. Soft agar anchorage independence assay for WM-164 cells (one week treatment with DMAT 10 µM).

A. Control (DMSO) MCF-7 colonies, B. DMAT treated, WM-164 colonies, C. Colonies areas were measured from several images.

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These results are comparable with the ones obtained with MCF-7 cells and SW-

480 cells (about 20% induced apoptosis after 24 hours and 50% apoptosis after 48 hours).

WM-164 cells also present the characteristic feature of malignant cells, which is growth independence. In melanoma, deregulations of growth regulatory pathways have been described and are responsible for the anchorage independence of these cells [37, 38]. We analyzed the effects of DMAT (and thus CK2) in an anchorage independence soft agar assay, see figure 4.20. The soft agar anchorage independence assay (one week treatment with DMAT 10 µM) shows that DMAT inhibits anchorage independence in WM-164 cell line. We found that DMAT induces proliferation arrest in vivo in WM-164 xenografts, this effect was not as strong as observed in the MCF-7 cells, but still significant at the end of the two weeks of treatment. Figure 4.21 shows WM-164 xenograft treated with 1 mg

DMAT/animal/day (10 mg/kg DMAT) for two weeks. Tumors and organs from theses mice were analyzed histologically (H&E). Figure 4.22 shows hematoxylin and eosin staining of WM-164 tumors. Figure 4.23, panel A. Control (DMSO only treated), magnification 10X can be compared with panel B, tumor from WM-164 DMAT treated mice. This figure shows that necrosis and apoptosis are induced by DMAT in WM-164 tumors extensively. Organs examined in the same manner show that DMAT does not affect organs in the WM-164 xenografts and that WM-164 cells do not infiltrate the spleen and the liver. Figure 4.23 and 4.24 are H&E micrographs of staining for WM-164 liver and spleen tissues (control-DMSO versus DMAT treated).

We can conclude that DMAT at this amount is not toxic to mice and it has real potential to be used as a starting point in developing an anti-cancer therapy.

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WM-164 xenograft treated with DMAT 10 mg/kg (in DMSO) per animal, daily, animal weight was averaged at 35 g

1500 Control WM-164 Both Treated WM-164 Both n = 8

1250 * 3 m m

) id

o 1000 r e h p s e t

la 750 o r p (

e m lu

o 500 v r o m u t 250

0 4 5 6 7 8 9 10 11 12 13 14 days

Figure 4.22. WM-164 xenograft treated with 0.25 mg DMAT/animal/day (10 mg/kg DMAT) for two weeks.

Tumors were 200 mm3 in volume at start. Filled squares - average of tumor volumes for Control batch (DMSO) versus tumor volumes from DMAT treated batch (8 tumors) represented with filled triangles. * represent p < 0.01 as assessed daily with student t-test.

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Figure 4.23. Hematoxylin and eosin staining of WM-164 tumors.

A. Control WM-164 tumor (DMSO), B. Tumor from WM-164 DMAT treated mice, C. WM-164 H&E apoptotic-necrotic areas, D. WM-164 H&E angiogenesis areas. Magnification 10X.

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Figure 4.24. Hematoxylin and eosin staining of WM-164 livers.

A. Control (DMSO) liver, WM-164 tumor bearing mouse, B. Liver from WM-164 DMAT treated mice, C. Liver from healthy normal wild type mouse C57BL (horizontal section), D. Liver from healthy normal wild type mouse C57BL (transversal section). Magnification 10X.

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Figure 4.25. Hematoxylin and eosin staining of WM-164 spleens.

A. Control (DMSO) spleen, WM-164 tumor bearing mouse, B. Spleen from WM-164 DMAT treated mice, C. Spleen from healthy normal wild type mouse C57BL (horizontal section), D. Spleen from healthy normal wild type mouse C57BL (transversal section). Magnification 10X.

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4.5. DISCUSSION

CK2 is involved in many human cancers causing cellular transformation and development of tumorigenesis and aggressive tumor behavior. CK2 is up-regulated in cancers of mammary gland, lung, kidney, colon, head and neck and serves as an oncogene in lymphocytes. Various mechanisms and intricate signaling are responsible for the effects of CK2 in these cancers [39]. Based on all the evidence from literature and our preliminary data with chronic myelogenous leukemia cells, we decided to test the effects of the CK2 inhibitor DMAT on hormone dependent human breast cancer cells (MCF-7), colorectal adenocarcinoma cells (SW-480) and melanoma cells (WM-164).

CK2 enhances Wnt-β-catenin signaling in mammary epithelial cells. NFκ-B activation and up-regulation of c-myc are another mechanism through which CK2 induces malignant transformation of breast cells [1, 40]. MCF-7 cells are known to present the anchorage independence feature, due to activation of MAPK through phosphatidylinositol-3 kinase by insulin-like growth factor I [35]. Based on the fact that

CK2 is involved in this pathway [36], we decided to test the effects of CK2 inhibition with DMAT on MCF-7 cells first on proliferation and anchorage independence assay in soft agar. Results were astonishing. Proliferation and colony formation were strongly inhibited, much better than of the megakaryoblastic leukemia cells, that we tested previously, see Chapter II and Chapter III. We also found that DMAT induces apoptosis in MCF-7 after 24 hours and that the level of apoptotic cells doubles in 48 hours. The percentage of apoptotic cells produced by DMAT treatment is much higher than in the megakaryoblastic cells. If we have such a strong apoptotic response, most probably this is due also to caspase activation combined with the previously described mechanisms.

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MCF-7 cells form tumors in vivo only if they are stimulated with feminine hormone estrogen. We created MCF-7 xenografts which were treated with the hormone precursor

17-β-estradiol. Tumor growth was monitored and histological analysis of the tissue samples from tumors (treated and non-treated with DMAT) was performed. From these experiments we found that DMAT induced tumor growth arrest, ablation in smaller tumors and these were due to apoptosis and necrosis in the tumor. Tumor growth varies with the amounts of hormone administered and we observed that effects of DMAT were more pronounced in smaller tumors (less hormone) sustaining the idea that CK2 inhibitors maybe developed to create adjuvant therapy for breast cancer. Organ tissue samples were analyzed and were normal from both tumor bearing mice treated and non- treated suggesting that DMAT does not have toxic effects at the amounts administered.

For variety, other completely different cancer cell lines were treated with DMAT and analyzed in the same manner.

In colon cancer cells, increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis. Mutations in the tumor suppressor

APC (adenomatous polyposis coli) are frequent in colon cancer and often increase levels of β-catenin–T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 increases survivin expression via these signaling mechanisms, which provokes in this way the malignant transformation of colon cells. CK2 inhibitor TBB was found to inhibit this signaling [20]. If TBB affects this survivin expression, it is normal to presume that DMAT which is a better CK2 inhibitor than DMAT will have similar or better effects and thus DMAT will inhibit SW-

480 colon cancer cells growth. Over-expression of PKC beta1 and adenomatous

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polyposis coli (APC) signaling in the SW-480 colon cancer cell line are also responsible for the anchorage independence growth in soft agar. CK2 is involved in these pathways and thus DMAT which is a potent CK2 inhibitor should inhibit the anchorage independence phenotype of SW-480 cells. Therefore, we tested effects of DMAT on apoptosis, proliferation and anchorage independence in soft agar and we found that

DMAT inhibits all these processes almost as at the same level as in MCF-7 cells, in vitro.

We had to use a much higher amount of DMAT in vivo, to observe tumor proliferation arrest and necrosis/apoptosis in the SW-480 tumors treated with DMAT. One reason could be that the animals that we used for these experiments were males whereas the

MCF-7 xenografts were females treated with feminine hormone. These difference suggests that CK2 maybe involved in the hormone metabolism or defective function of receptors for estrogen, or at least that DMAT acts on such pathways.

CK2 maybe involved in the development of melanoma cells due to its action on

B23 nuclear protein, as well as due to its effects on AKT/PI3K pathways. WM164 melanoma growth is modulated by VEGF and BFGF factors [37]. These factors induce angiogenic and tumorogenicity characteristics to melanoma cells. VEGF and BFGF activate PI3K/AKT signaling [38]. Thus it was interesting to see whether DMAT affects angiogenesis and tumor development in melanoma. Deregulation of growth regulatory pathways has been described on four levels and are responsible for the anchorage independence of melanoma cells: aberrant production of autocrine growth factors, alterations in the response to negative autocrine growth factors, interleukine-6 (IL-6) and transforming growth factor (TGF-beta), over-expression of epidermal growth factor receptors (EGF-R) and alterations of cellular oncogenes involved in signal transduction

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(RAS, MYB) and growth suppression (p53) [41]. In in vitro cellular assays (apoptosis, proliferation, anchorage independence in soft agar) DMAT had similar effects on WM-

164 cells as on MCF-7 and SW-480 suggesting that just in the cellular environment, defective mechanisms in which CK2 is involved maybe common. We speculate that the mechanism that involves cell cycle and the defective extrinsic apoptotic pathway will cooperate to have similar results in all cancer cell lines tested. The melanoma xenografts, treated with the same amount of DMAT, as MCF-7 cells, responded to these treatment only at the end of the study (after two weeks). These results suggest that in body, where various stimuli and signals modulate tumor growth, there are other mechanisms involved which create the unique characteristics and variety of pathways of each cancer type.

To conclude, all tumors from treated mice showed high necrotic and apoptotic areas versus untreated (DMSO) tumors and tumor growth was arrested. All organs seem to look normal based on the hematoxylin-eosin pictures. Therefore, DMAT and in general CK2 inhibition prove of utmost importance as tools in developing new cancer therapies.

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18. Izeradjene, K., L. Douglas, A. Delaney, and J.A. Houghton, Influence of casein kinase II in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human rhabdomyosarcoma cells. Clin. Cancer Res., 2004. 10: p. 6650-6660.

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26. Louvet, E., H.R. Junera, I. Berthuy, and D. Hernandez-Verdun,

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27. Lawson, K., L. Larentowicz, L. Laury-Kleintop, and S.K. Gilmour, B23 is a downstream target of polyamine-modulated CK2. Mol Cell Biochem, 2005. 274(1-2): p. 103-14.

28. Szebeni, A., K. Hingorani, S. Negi, and M.O. Olson, Role of protein kinase CK2 phosphorylation in the molecular chaperone activity of nucleolar protein b23. J Biol Chem, 2003. 278(11): p. 9107-15.

29. Siemer, S., S. Kriener, J. Konig, K. Remberger, and O.G. Issinger,

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34. Miller, S.J., D.Y. Lou, D.C. Seldin, W.S. Lane, and B.G. Neel, Direct identification of PTEN phosphorylation sites. FEBS Lett, 2002. 528(1-3): p. 145-53.

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CHAPTER V

OVERALL CONCLUSIONS AND FUTURE DIRECTIONS

5.1. OVERALL CONCLUSIONS

Cancer is one of the major health problems of this century. The variety of cancer types and complex mechanisms and various molecular and genetic defects that cause cancer makes drug development a real challenge for researchers today. Different strategies were developed by focusing on specific molecules involved in cancer development. It is extremely hard to find specific molecules that are entirely the cause of the cancer development, but there are a lot that are deregulated, therefore they are targeted for drug design.

A protein named casein kinase 2 (CK2) was found deregulated in several types of cancer. CK2 controls both cell cycle [1-3] and apoptotic pathways [4-6]. This kinase has dual localization: cytoplasmic and nuclear. In different cancer types examined, CK2 was found to be up-regulated and constitutively activated. CK2 started to become an interesting cancer target, especially after various inhibitors were developed and studies in

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cells and animals (mice) were done [4, 6]. Knockout CK2 alpha [7-11] and beta mice and

silencing of CK2 alpha in mice were studied [12, 13]. CK2 has a highly conserved

aminoacid sequence between the species suggesting that may have a fundamental role in

cell function. Our studies sustain these data and bring novel observations that promote the

idea of using CK2 inhibitors as a potential drug for various types of cancer.

We observed for the first time that the use of the CK2 inhibitor DMAT in the

human malignant megakaryoblastic cell line (MEG-01) induces proliferation arrest and apoptosis. A very unique observation was that DMAT enhances platelets production from these cells in vitro (we characterized this process in detail) and in vivo. I consider this observation the most important of my entire work because it opens up various possibilities, just for basic research. We have a model of platelets production in vitro, easy to obtain and fast. These platelets were characterized as normal and have a normal function. Other models of platelets production in vitro were developed, like the one using

CD34+ stem cells. In this model, CD34+ stem cells are stimulated to become megakaryocytes with specific cytokine cocktails and then these megakaryocytes are stimulated to release platelets. These procedures are time (months) and money consuming. The CD34+ cells are primary cells and thus they need to be isolated each time from bone marrow aspirates or they need to be purchased. We can use MEG-01 platelets for studying various proteins by using genetic engineering of megakaryocytes so that the platelets will express the engineered protein. Platelets do not have nucleus and thus they cannot produce their own proteins, they need them to be produced in megakaryocytes. Such models were created using CD34+ cells and their platelets.

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An amazing observation that I made is that MEG-01 cells form tumors in immunodeficient mice and these tumors release platelets in vivo. This release seem to be stimulated by DMAT. These animals show spleen infiltration with megakaryocytic cells and have splenomegaly, as well as high platelets counts. This creates an easy to obtain model of chronic myelogenous leukemia, in which anti-leukemia drugs can be tested.

Another possibility, on the long run, for the applicability of this present work is the use of such inhibitors as developing adjuvant therapy for chronic myelogenous leukemia patients. We are targeting specifically the patients with resistance to the actual therapies (imatinib, and imatinib –like drugs).

DMAT was tested in various cellular and murine xenograft models. This work brings a few more cell lines that were not studied previously into the cohort of others that were tested up to date with other CK2 inhibitors. We found (like expected) that DMAT inhibits malignant cell growth in vitro and in vivo by inducing apoptosis in these cells

(breast cancer, colon cancer and melanoma). Effects of DMAT on cancer cells, in vitro, were similar with all the cell lines tested, but in vivo results were different (tumor growth was inhibited with different amounts of DMAT), suggesting that the organism milieu creates signaling mechanisms that may stimulate more the malignant cells growth. Such stimuli are hormones. MCF-7 (breast cancer) xenografts were treated with 17-beta- estradiol. When hormone level was decreased, tumor growth was slower and thus DMAT had a better effect than in the exponentially growing tumors. DMAT had the best effect on breast cancer, very good on colon cancer and good on melanoma.

A pilot toxicity study was performed with DMAT. A male immunodeficient mouse received a high quantity of DMAT for a period of two weeks. The animal behavior was

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observed and after two weeks was euthanized and histological analysis was performed on its organs. The animal did not present any abnormal behavior and he ate and drunk water regularly. He presented soreness and a big cyst formation at the injection site, which was due to deposits of DMAT. DMAT is not soluble in polar solvents (only DMSO) and that was the reason for this cyst formation. This maybe the reason that even at this high amount it was not lethal. However it seems that somehow DMAT maybe absorbable up to a point that it has effect from an exogenous tumor site. DMAT was injected into the neck fat pad subcutaneous, far from the tumors. From this study, the first conclusion to draw would be that DMAT is not toxic at the level tested.

Overall from all the studies performed in this work we can conclude that a CK2 inhibitor, DMAT is a potent candidate for anti-cancer drug development.

5.2. FUTURE DIRECTIONS

The first thing that I would do to continue this work, would be to administer

DMAT to animals by gavage. This can be done by using oil-emulsion (sesame-seed oil).

Insoluble substances can be administered in this way on the oral route. I will repeat the toxicity study with more animals, considering that one animal is not enough. More animals are also needed for the tail-bleeding study, in order to withdraw any clear conclusions from it. For the tail-bleeding study, I would also perform a clot retraction tail-bleeding time. The xenografts studies performed until now need to be repeated with a higher animal number.

As a more tangible and immediate direction of this research, I would analyze more in detail the platelets production from MEG-01 tumor bearing mice. First thing that

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needs to be done is to assess if we have circulating MEG-01 blasts. This can be easily done by blood smear. Blood smears will show all the blood cells and the abnormal cells can be easily spotted. With blood smears, accurate cell counts can be performed based on cell density per field. This is a very common practice in clinical laboratories. I would also examine lungs for megakaryocytic infiltration, because it was proposed that platelets production may not take only in the bone marrow but also in the lungs from migrated megakaryocytes.

Another way, more complicated but more elegant, to study blast and platelets circulation would be to transfect MEG-01 cells with green fluorescent protein (GFP) or luciferase and to examine if platelets from these cells are “green”, in vitro (fluorescence microscopy and flow cytometry). If these platelets are green, then GFP MEG-01 cells could be used to create xenografts with green tumors. In this way we can quantify exactly how many platelets are human and how many are murine, how they get activated and where they go. We can also monitor tumor infiltration and have a possible metastasis model. This model can prove useful in studies like platelets involvement in angiogenic response in ischemia conditions.

It would be interesting to see what effects has DMAT on CD34+ megakaryocytes

(normal and from patients with chronic myelogenous leukemia). A murine model of chronic myelogenous leukemia [14] is available from the Jackson Laboratory and it can be used to study DMAT in the context of the real disease in animal.

A mechanistic study is also required. One direction would be to consider the cytoplasmic localization of CK2 and another to consider the nuclear site. For the cytoplasmic study it seems that CK2 controls apoptotic pathways preponderantly through

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Bid protein as a common mechanism in all cancers. CK2 phosphorylates Bid and phosphorylated Bid is less sensitive to cleavage by caspase-8 and caspase-3. The active form of Bid is the truncated one, tBid. tBid will relocate to mithocondria and start the mitochondrial apoptotic pathway [5, 15-18]. I would use western blot analysis of cell lysates (cancer cells treated and not-treated with DMAT and as a control cell lysates where silencing of CK2α was performed) to quantify amounts of various apoptotic proteins and first ones to quantify would be tBid and Bid. Mitochondrial membrane potential changes could be assess using flow cytometry (JC-1, Mitotrack). Cytochrome c release can be quantified by western blot and also caspase-3, caspase-9 and caspase-7 and other apoptotic proteins levels can be observed. A second direction in an mechanistic approach would be to understand the events that involve CK2 and control cell cycle [1, 3,

19-22]. For this normal, cancer cells (diploid and polyploid) will be used. Cyclins, cdk’s and B23 protein levels can be assess by western blotting and cell cycle can be analyzed for both diploid and polyploid cells by flow cytometry.

There are a few strategies that can be followed up in order to obtain a cancer drug starting with DMAT.

One possibility (the least desirable) is to cling to DMAT like it is. In order to become a drug this substance needs to be soluble and absorbable, thus it needs a drug delivery strategy. This can be done by sonication with a delivery vehicle (nano-particles) or by using liposome drug delivery [23].

As a second possibility, other CK2 inhibitors are continuously developed [24-28] and therefore options are endless. As a chemist I would change the structure of DMAT to make it first of all soluble, even more specific and also original.

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The third possibility would be to test other potent CK2 inhibitors, that are naturally

occurring like ellagic acid and cinnamic acid. Such acids can be purchased from Sigma.

Ellagic acid, for example, is a polysaccharide and thus is soluble in polar solvents.

Ellagic acid is a polyphenol antioxidant found in berries, various nuts, pomegranates and

interestingly was found to act on factor XII and stimulate blood clotting. These natural

occurring substances are sold as natural extracts (remedies), not approved by FDA, in

natural stores and can be also purchased from internet stores. Cancer patients that used

them claim very good results. If we as scientists would test more this compounds and push for clinical studies, maybe FDA would approve their use and standardize it so they can be prescribed by doctors to all patients and this would be of great benefit to so many people. Unfortunately patent and financial issues may hinder such developments.

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5.3. REFERENCES

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3. Negi, S.S. and M.O. Olson, Effects of interphase and mitotic phosphorylation on the mobility and location of nucleolar protein B23. J Cell Sci, 2006.

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