EFFICACY AND MECHANISTIC STUDIES OF A NOVEL SMALL MOLECULE
THERAPEUTIC AGENT “XB05” IN ACUTE MYELOID LEUKEMIA (AML)
By
Jaspreet Singh Grewal

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Clinical Laboratory Sciences—Master of Science
2014

ABSTRACT
EFFICACY AND MECHANISTIC STUDIES OF A NOVEL SMALL MOLECULE
THERAPEUTIC AGENT “XB05” IN ACUTE MYELOID LEUKEMIA (AML)
By
Jaspreet Singh Grewal
Acute myeloid leukemia (AML) is the most common acute leukemia in adults
characterized by a clonal proliferation of myeloid precursors with a reduced capacity to
differentiate into more mature cellular elements resulting in pancytopenia. Current
treatment options include induction chemotherapy followed by consolidation and
hematopoietic stem cell transplantation (HSCT). DNA hypomethylating agents such as
decytabine have been used to treat AML, especially in older patients. The drug has
shown significant clinical benefit in a recent Phase III trial in older patients
demonstrating the proof of concept of inhibiting DNA methylation as a therapeutic
strategy in AML. We have discovered a novel synthetic small molecule, named XB05
that has potent activity against myeloid leukemia cell lines. Its precise molecular
mechanism is not yet fully understood; however, we believe that it induces leukemia cell
death through multiple mechanisms, including reactivation of tumor suppressor genes
via inhibition of DNA methylation. In this study, mechanistic studies were performed with
this novel agent in previously established leukemia cell lines.

ACKNOWLEDGEMENTS

I am using this opportunity to express my sincere gratitude to Dr. Tariq Malik who
supported me throughout the course of this Masters project. I am thankful for his
aspiring guidance, invaluably constructive criticism and friendly advice during the project
work.
I would also like to thank Dr. David Thorne and Dr. John Gerlach at the Michigan State
University for their guidance and direction in the completion of my thesis project.

iii

TABLE OF CONTENTS

LIST OF TABLES………………………………………………………………………..vi
LIST OF FIGURES................................................................................................ ix
KEY TO ABBREVIATIONS ..................................................................................xii
CHAPTER 1 ...........................................................................................................1
INTRODUCTION .........................................................................................1
Acute Myeloid Leukemia – Brief Review .................................................1
Epigenetic Mechanisms in AML – DNA Methylation ..............................7
CHAPTER 2 ..........................................................................................................11
XB05 – A Novel Synthetic Small Molecule .............................................11
CHAPTER 3 ..........................................................................................................17
SPECIFIC AIMS .........................................................................................17
Materials and Methods .............................................................................18
AML Cell lines .................................................................................... 18
Culturing Media ..............................................................................19
Culture Methods ............................................................................19
Drugs ..............................................................................................20
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide)
Assay ..............................................................................................20
Polymerase Chain Reaction (PCR) – Reverse Transcription PCR and
Real-time PCR ................................................................................22
EpiTect Methyl II PCR Array (Qiagen®)........................................24
Western Blot...................................................................................26
Protein Quantitation by Coomassie (Bradford) protein assay...27
Immunohistochemistry .................................................................28
RESULTS ...................................................................................................29
MTT-ASSAYS .................................................................................29
POLYMERASE CHAIN REACTION (PCR). ....................................47
EpiTect Methyl II PCR Array (Qiagen®)........................................51
Western Blots .................................................................................53
Immunohistochemistry (IHC) .......................................................57
Discussion ................................................................................................61
Conclusions ..............................................................................................68
APPENDIX ............................................................................................................69
iv

BIBLIOGRAPHY ..................................................................................................115

v

LIST OF TABLES

Table 1. Targeted agents in development in AML ..................................................6
Table 2. Oncogenes and tumor suppressor (TS) genes in 22-panel EpiTect Methyl II
PCR Panel ............................................................................................................25
Table 3. First MTT-assay performed on Kasumi-1 cells ........................................29
Table 4. Second MTT-assay performed on Kasumi-1 cells ...................................30
Table 5. Third MTT-assay performed on Kasumi-1 cells .......................................31
Table 6. Results of combined MTT-assays performed on Kasumi-1 cells .............32
Table 7. First MTT-assay performed on U937 cells ..............................................38
Table 8. Second MTT-assay performed on U937 cells .........................................39
Table 9. Third MTT-assay performed on U937 cells .............................................40
Table 10. Results of combined MTT-assays performed on U937 cells .................41
Table 11. Serial dilutions of XB05 for treatment of Kasumi-1 cell line ...................71
Table 12. Calculations for serial XB05 concentrations to be used for treatment of
Kasumi-1 cell line ..................................................................................................72
Table 13. Serial dilutions of XB05 for treatment of U937 cell line..........................72
Table 14. Calculations for serial XB05 concentrations to be used for treatment of U937
cell line ..................................................................................................................73
Table 15. Serial dilutions of azacytadine for treatment of Kasumi-1 and U937 .....74
Table 16. Calculations for serial XB05 concentrations to be used for treatment of U937
cell line ..................................................................................................................75
Table 17. Materials needed for RNA isolation .......................................................78
Table 18. Kit components for high capacity RNA-to-cDNA PCR ...........................81
Table 19. Volumes for each components of RT-PCR reaction ..............................82
vi

Table 20. Time settings for three steps of RT-PCR reaction .................................82
Table 21. Components of SYBR Green master mix ..............................................82
Table 22. Volumes for components of SYBR Green qRT-PCR reaction ...............84
Table 23. Time settings for the three steps of qRT-PCR reaction .........................85
Table 24. Volume for components of EpiTect Methyl II PCR reaction ...................88
Table 25. Volumes for four reactions in EpiTect Methyl II PCR assay ..................89
Table 26. Final reaction volume for four reactions in EpiTect Methyl II PCR assay
..............................................................................................................................90
Table 27. Time settings for EpiTect Methyl II PCR assay .....................................91
Table 28. Plate design for Coomassie assay and BSA serial dilutions .................92
Table 29. Components of Buffer B ........................................................................96
Table 30. Components of Buffer C ........................................................................96
Table 31. List of primary antibodies and dilutions used for Western Blot and
Immunohistochemistry ..........................................................................................97
Table 32. List of secondary antibodies used for Western Blot and Immunohistochemistry
..............................................................................................................................98
Table 33. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (0 and 100nM) and serial azacytadine ............101
Table 34. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine ........101
Table 35. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (800 and 1000nM) and serial azacytadine ......102
Table 36. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (0 and 100nM) and serial azacytadine
.............................................................................................................................102
Table 37. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (200 and 400nM) and serial azacytadine
.............................................................................................................................102
vii

Table 38. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (800nM) and serial azacytadine .......103
Table 39. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (0 and 50nM) and serial azacytadine ..............103
Table 40. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (100 and 200nM) and serial azacytadine ........103
Table 41. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (400 and 800nM) and serial azacytadine ........104
Table 42. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (0 and 100nM) and serial azacytadine ............104
Table 43. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine ........104
Table 44. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine ........105
Table 45. MTT-assays on HCT116 (colon cancer cell line) following treatment with
XB05 and DMSO ..................................................................................................105
Table 46. PCR data for Kasumi-1 cell line – 48 hour treatment with XB05 and 5’AZA
.............................................................................................................................107
Table 47. PCR data for U937 cell line – 48 hour treatment with XB05 and 5’AZA ..
.............................................................................................................................108
Table 48. PCR data for Kasumi-1 cell line – 96 hour treatment with XB05 and 5’AZA
.............................................................................................................................109
Table 49. PCR data for U937 cell line – 96 hour treatment with XB05 and 5’AZA ..
.............................................................................................................................110
Table 50. Average O.D.’s of serial dilutions of BSA for standard curve for cytoplasmic
protein ..................................................................................................................111
Table 51. Cytoplasmic protein calculations for western blot .................................112
Table 52. Average O.D.’s of serial dilutions of BSA for standard curve for nuclear
protein ..................................................................................................................112
Table 53. Cytoplasmic protein calculations for western blot .................................114
viii

LIST OF FIGURES

Figure 1. Survival curve of 428 consecutive patients with AML treated at Cardarelli
Hospital in Naples, Italy ..........................................................................................4
Figure 2. Gene silencing that occurs in AML as a result of DNMT, HDAC and HMT, and
specific inhibitors of these enzymes .......................................................................8
Figure 3. Molecular structure of XB05 ...................................................................12
Figure 4. Outline of the principle of PCR ...............................................................23
Figure 5. Percent cell proliferation in Kasumi-1 cell line ........................................30
Figure 6. Percent cell proliferation in Kasumi-1 cell line ........................................31
Figure 7. Percent cell proliferation in Kasumi-1 cell line ........................................32
Figure 8. Percent cell proliferation in Kasumi-1 cell line ........................................33
Figure 9. Using the ComuSyn software, IC50 for XB05 in Kasumi-1 cell line is calculated
..............................................................................................................................34
Figure 10. Using the ComuSyn software, IC50 for 5’AZA in Kasumi-1 cell line is
calculated ..............................................................................................................35
Figure 11. Results of the first MTT-assay performed in Kasumi-1 cell line following
combination treatment ...........................................................................................36
Figure 12. Results of the second MTT-assay performed in Kasumi-1 cell line following
combination treatment ...........................................................................................37
Figure 13. Percent cell proliferation in U937 cell line ............................................38
Figure 14. Percent cell proliferation in U937 cell line ............................................39
Figure 15. Percent cell proliferation in U937 cell line ............................................40
Figure 16. Percent cell proliferation in U937 cell line ............................................42
Figure 17. Using the ComuSyn software, IC50 for XB05 in U937 cell line is calculated
..............................................................................................................................43
ix

Figure 18. Using the ComuSyn software, IC50 for 5’AZA in U937 cell line is calculated
..............................................................................................................................44
Figure 19. Results of the first MTT-assay performed in U937 cell line following
combination treatment ...........................................................................................45
Figure 20. Results of the second MTT-assay performed in U937 cell line following
combination treatment ...........................................................................................46
Figure 21. Results of PCR performed on Kasumi-1 cell line without treatment and
following treatment with XB05 and azacytidine at 48-hour interval .......................47
Figure 22. Results of PCR performed on U937 cell line without treatment and following
treatment with XB05 and azacytidine at 48-hour interval ......................................48
Figure 23. Results of PCR performed on Kasumi-1 cell line without treatment and
following treatment with XB05 and azacytidine at 96-hour interval .......................49
Figure 24. Results of PCR performed on U937 cell line without treatment and following
treatment with XB05 and azacytidine at 96-hour interval ......................................50
Figure 25. EpiTect Methyl II PCR array results of percent methylation of CpG islands in
the promoter regions of selected genes in Kasumi cell line...................................51
Figure 26. EpiTect Methyl II PCR array results of percent methylation of CpG islands in
the promoter regions of selected genes in U937 cell line ......................................52
Figure 27. Western blot performed on nuclear proteins from Kasumi-1 cell line ...53
Figure 28. Western blot performed on nuclear proteins from U937 cell line ..........54
Figure 29. Western blot performed on cytoplasmic proteins from Kasumi-1 cell line
..............................................................................................................................55
Figure 30. Western blot performed on cytoplasmic protein fractions of U937 cell line
..............................................................................................................................56
Figure 31. Power 40x. DNMT1 staining for Kasumi – cell line...............................57
Figure 32. Power 40x. DNMT1 staining for Kasumi – cell line...............................57
Figure 33. Power 40x. HIC1 staining for Kasumi – cell line ...................................58
Figure 34. Power 40x. HIC1 staining for Kasumi – cell line ...................................58
x

Figure 35. Power 40x. DNMT1 staining for U937 – cell line ..................................59
Figure 36. Power 40x. DNMT1 staining for U937 – cell line ..................................59
Figure 37. Power 40x. HIC1 staining for U937 – cell line ......................................60
Figure 38. Power 40x. HIC1 staining for U937 – cell line ......................................60
Figure 39. Outline of the plan for MTT-assays ......................................................75
Figure 40. Plate Design for Kasumi-1 MTT experiment.........................................76
Figure 41. Plate Design for U937 MTT experiment ...............................................76
Figure 42. Plate design for drug combination experiments ...................................77
Figure 43. Outline of experiments for RNA extraction, cDNA and RT-PCR ..........80
Figure 44. General overview of RT-PCR experiment ............................................81
Figure 45. Plate design for qRT-PCR reaction for the study .................................83
Figure 46. Plate design for qRT-PCR reaction for the study .................................84
Figure 47. Overview of the qRT-PCR reaction for the study .................................85
Figure 48. EpiTect Methyll II PCR Procedure overview.........................................86
Figure 49. Plate design for EpiTect Methyl II PCR assay ......................................90
Figure 50. Plate design for Coomassie assay .......................................................93
Figure 51. Overview of western blot experiment for the study...............................95
Figure 52. Outline of IHC experiment for the study ..............................................100
Figure 53. Graphical representation of the MTT-assays on HCT116 (colon cancer cell
line) following treatment with XB05 and DMSO ....................................................106
Figure 54. Standard curve for BSA for quantitation of cytoplasmic protein ..........111
Figure 55. Standard curve for BSA for quantitation of nuclear protein .................113

xi

KEY TO ABBREVIATIONS

5'AZA

5'azacytadine

AFF1

AF4/FMR2 family, member 1

AML

Acute myeloid leukemia

ARE

Antioxidant response elements

ASXL1

Additional Sex Combs protein 1

ATCC

American Type Culture Collection

BHA

Butylated hydroxyanisole

CALGB

Cancer and Leukemia Group B

CBFB

Core-binding factor, beta subunit

CD

Cluster differentiation

CDKN2A

Cyclin-dependent kinase inhibitor 2A

CEBPA

CCAAT-enhancer binding protein – alpha

CEBPD

CCAAT/enhancer binding protein (C/EBP), delta

CR

Complete remission

CSF1R

Colony stimulating factor 1 receptor

CTNNA1

Catenin (cadherin-associated protein), alpha 1

DAPK

Death-associated protein kinase

DMSO

Dimethyl sulfoxide

DNMT

DNA methyltransferases

DRD2

Dopamine receptor D2

xii

ER-α

Estrogen receptor α

ESI-MS

Electrospray ionization mass spectrometry

ETO

Eight-Twenty-One

EXT1

Exostosin glycosyltransferase 1

FANCC

Fanconi anemia, complementation group C

FANCL

Fanconi anemia, complementation group L

FBS

Fetal bovine serum

FGFR

Fibroblast growth factor receptor

FLT-3

Fms-like tyrosine kinase

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

G-CSF

Granulocyte colony-stimulating factor

GM-CSF

Granulocyte macrophage colony-stimulating factor

GSH

Cysteine and glutathione

HDAC

Histone deacetylase

HIC-1

Hypermethylated in cancer – 1

HMT

Histone methyltransferase

HOXA7

Homeobox A7

HOXB5

Homeobox B5

HRP

Horseradish peroxidase

HSCT

Hematopoietic stem cell transplantation

IDH

Isocitrate dehydrogenase

IHC

Immunohistochemistry

xiii

IL

Interleukin

JUNB

Jun B proto-oncogene

Keap1

Kelch-like ECH-associated protein 1

KIT

v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog

LMNA

Lamin A/C

MAFB

v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B

MDS

Myelodysplastic syndrome

MEN1

Multiple endocrine neoplasia I

MGMT

O-6-methylguanine-DNA methyltransferase

MLL

Myeloid/lymphoid, or mixed-lineage, leukemia

MTG8

Myeloid translocation gene on 8

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MYH1

Myosin, heavy chain 1

NCI

National Cancer Institute

NFATC1

Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1

NMR

Nuclear magnetic resonance

NPM1

Nucleophosmin -1

Nrf2

Nuclear factor, erythroid 2-like 2

O.D.

Optical density

OS

Overall survival

PCR

Polymerase chain reaction

PDGFR

Platelet derived growth factor receptor

xiv

PER2

Period circadian clock 2

PML

Pro-myelocytic leukemia

PS

Performance status

qRT-PCR

Quantitative real-time PCR

RARα

Retinoic acid receptor – alpha

RAR-β

Retinoic acid receptor – beta

RAS

Rat sarcoma viral oncogene homolog

ROS

Reactive oxygen species

RPMI

Roswell Park Memorial Institute medium

RT-PCR

Reverse Transcription PCR

RUNX1

Runt-related transcription factor 1

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SLC4A8

Solute carrier family 4, sodium bicarbonate cotransporter, member 8

SOX9

SRY (sex determining region Y)-box 9

SPOCK2

Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2

TET2

Ten eleven translocation protein 2

TLE1

Transducin-like enhancer of split 1 (E(sp1) homolog, Drosophila)

TP53

Tumor protein p53

UPR

Unfolded protein responses

VEGF

Vascular endothelial growth factor

xv

CHAPTER 1
INTRODUCTION
Acute Myeloid Leukemia – Brief Review
Acute myeloid leukemia (AML) is the most common acute leukemia in adults
especially in patients older than 65 years. It accounts for approximately 50 percent of all
cases of leukemia in this age group [1], with five year survival rate <10% [2]. It is a
heterogeneous disease characterized by a clonal proliferation of myeloid precursors
with a reduced capacity to differentiate into more mature cellular elements resulting in
accumulation of leukemic blasts or immature cells in the bone marrow and peripheral
blood.[1] This leads to pancytopenia (anemia, neutropenia and thrombocytopenia), a
reduction in the number of mature blood cells, which accounts for the symptoms in
AML—anemia, infections, and bleeding [1].
Underlying etiology of AML cannot be determined in most cases; however, it has
been hypothesized that exposure to environmental carcinogens may play a role [1].
Association of AML to chemotherapy has clearly been demonstrated. Two distinct
subtypes of chemotherapy – related AML have been described. First subtype is the
result of exposure to topoisomerase II inhibitors (etoposide, mitoxantrone and
teniposide), anthracyclines (doxorubicin) and epipodophyllotoxins (VP-16 and VM-26)
which can present within months to two years following treatment with these agents and
is associated with MLL (myeloid/lymphoid, or mixed-lineage, leukemia) gene locus
(11q23) abnormalities [1]. Second subtype presents five to seven years post exposure

1

to alkylator agents (cyclophosphamide, procarbazine and melphalan) which is preceded
by myelodysplastic syndrome (MDS) and is associated with complex karyotypic
abnormalities [1]. It is thought that various inherited polymorphisms may be associated
with differential metabolism of these chemotherapeutic agents resulting in exposure to
toxic by-products and DNA damage [1]. Also, various mutations, translocations and
epigenetic changes have been described in AML. Common mutations involve Fms-like
tyrosine kinase (FLT-3), CCAAT-enhancer binding protein – alpha (CEBPA),
nucleophosmin -1 (NPM1), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene
homolog (KIT) and rat sarcoma viral oncogene homolog (RAS) pathways [1, 3, 4]. And,
common chromosomal abnormalities involve RUNX1/ETO [t(8;21)] (Runt-related
transcription factor 1/ Eight-Twenty-One), CBFB/MYH11 [inv(16)] (core-binding factor,
beta subunit/ myosin, heavy chain 1), and PML/RARα [t(15;17)] (Pro-myelocytic
leukemia/ Retinoic acid receptor – alpha) translocations which result in constitutively
active fusion gene products [1, 5]. Epigenetic silencing of genes related to DNAhypermethylation from up-regulation of DNA methyltransferases 1, 3A and 3B
(DNMT3A) and other epigenetic regulators such as ten eleven translocation protein 2
(TET2) and Additional Sex Combs protein 1 (ASXL1) has been described resulting in
silencing of numerous genes including, isocitrate dehydrogenase 1 and 2 (IDH 1 and 2),
cyclin-dependent kinase inhibitor 2A (CDKN2A), hypermethylated in cancer – 1 (HIC-1),
retinoic acid receptor – beta (RAR-β), death-associated protein kinase (DAPK), O-6methylguanine-DNA methyltransferase (MGMT), and estrogen receptor α (ER-α). [1, 69]

2

Response to treatment and overall survival (OS) of patients with AML is
heterogeneous; patients with poor performance status (PS), complex karyotypes, and
older age have worse prognosis . Current treatment options include induction
chemotherapy with combination of daunorubicin and cytarabine (7+3 regimen) followed
by consolidation and hematopoietic stem cell transplantation (HSCT) . Induction
chemotherapy is given with the aim to achieve complete remission (CR) while
consolidation chemotherapy which includes intermediate- or high-dose cytarabine alone
is given to eliminate residual leukemic cells [1]. Patients with unfavorable risk disease
do poorly with 5-year OS of 15 to 40 percent with current chemotherapy regimens and
HSCT; thus, improved treatments are urgently needed [10]. Outcomes are even worse
in patients over 65 years with five-year survival rate of less than 10 percent with
treatment with intensive regimens (Figure. 1) [1, 7]. Although reduced intensity
conditioning regimens have been used in older patients to help them undergo alloHSCT, the results have been similar to myeloablative regimens. Older patients in these
studies who underwent induction chemotherapy had good performance status and only
5 percent of all the patients recruited could undergo allo-HSCT [1, 11] Current modality
of induction regimen has been in place for over 30 years. Numerous trials evaluating the
substitution of daunorubicin with other anthracyclines or addition of a third agent have
failed to demonstrate improvements in outcomes [1, 11, 12].

3

Figure. 1. Survival curve of 428 consecutive patients with AML treated at
Cardarelli Hospital in Naples, Italy [1]. Patients older than 65 years had significantly
poor overall survival than younger patients.

Over the years, investigators have tried to optimize the dose of daunorubicin.
Trials conducted so far have demonstrated that the 90mg/m2 dose of daunorubicin is
more effective than 45 or 60mg/m2 dose with similar toxicities in patients with
intermediate risk cytogenetics and CBFB-AML [1]. In younger patients, a second
induction is given to help achieve durable disease control. However, without further
treatment or consolidation majority of the patients relapse. Trial done by the Cancer and
Leukemia Group B (CALGB) has demonstrated that consolidation with high-dose
cytarabine at the dose of 3gm/m2 resulted in long-term relapse-free survival of 45% in
patients up to 60 years with favorable cytogenetics [1]. Optimal number of cycles of
cytarabine have not been determined but can vary from three to four cycles depending

4

on patient tolerability and adverse effects. In older patients, studies using low-dose
cytarabine have found survival advantage in patients with favorable cytogenetics [1, 11].
With further understanding of molecular mechanisms involved in pathogenesis
of AML, new therapies are being developed. In order to achieve meaningful results with
new targeted therapies, patients will need to be stratified based on their underpinning
molecular abnormalities. A recent trial from the United Kingdom – UK Medical Research
Council AML group showed that the addition of gemtuzumab ozogamicin (anti-CD33
monoclonal antibody conjugated with cytotoxic antibiotic, calicheamicin) to
chemotherapy resulted in survival advantage in patients with core binding factor AML
[1]. This was further verified by two other studies that demonstrated survival advantage
in patients with intermediate risk karyotypes [1]. However, there was no survival benefit
seen in patients with unfavorable cytogenetics. Gemtuzumab ozogamicin has been
withdrawn from market because of increased mortality seen in treated patients. Trials
are ongoing to test the efficacy of FLT-3 inhibitors [1, 13]. In addition, Phase I and
Phase II studies are ongoing evaluating the efficacy and safety profile of numerous
other targeted agents shown in (Table.1) [13].

5

Drug

Mechanism of Action

Sorafenib

Small-molecule inhibitor of Raf, c-kit, RET, FLT3, VEGF, PDGFR and
FGFR

Midostaurin

Small-molecule inhibitor of protein kinase C, VEGFR-2, PDGFR-α,
PDGFR-β, c-kit and FLT3

Quizartinib

Bis-aryl urea inhibitor of FLT3, KIT, PDGFR-α, PDGFR-β, RET and
CSF1R

Vosaroxin

Quinolone derivative which inhibits topoisomerase II

Elacytarabine

Nucleoside analog which inhibits DNA polymerase

Sapacitabine

Cytosine nucleoside analogue which leads to DNA strand breaks and
apoptosis

Table 1 (cont’d)
Flavopiridol

Synthetic flavone derivative which inhibits multiple serine-threonine
cyclin dependent kinases

Volasertib

Polo-like kinase inhibitor which leads to cell cycle arrest and apoptosis

CPX-315

Liposomal cytarabine and daunorubicin

Lintuzumab

Humanized monoclonal antibody against CD33

SGN33a

Monoclonal CD33 antibody-drug conjugate with pyrrolobenzodiazepine
which causes DNA crosslinking and cell death

Table 1. Targeted agents in development in AML.

DNA hypomethylating agents such as decytabine have been used to treat AML,
especially in older patients who cannot tolerate the side effects of standard
chemotherapies [1, 10]. The intent is to reduce DNA methylation (a mechanism that
“silences” the expression of many tumor suppressor genes in leukemia) and thereby
reactivate genes that will inhibit leukemic cell survival and progression [1, 10, 14].
These drug has shown significant clinical benefit in a recent Phase III trial in older
6

patients with newly diagnosed AML, with 15.7 percent complete remissions (CR) and a
clinically meaningful improvement in OS, and was generally well tolerated [10, 15]. This
proves the value of inhibiting DNA methylation as a therapeutic strategy in AML, but
clearly there remains a need for developing new hypomethylating drugs with better
activity and fewer side effects.[10, 14]
Epigenetic Mechanisms in AML – DNA Methylation
Epigenetic mechanisms are gene regulatory mechanisms which result in altered
gene expression without changes in the gene sequence. These mechanisms include
DNA methylation and histone deacetylation. Epigenetic changes are a normal part of
physiologic development and regulation of gene expression, which at times can results
in an abnormal phenotypes such as cancer, leading to silenced important critical genes
due to hypermethylated promoters regions [6, 16-18]. DNA methylation results in altered
gene expression via covalent addition of a methyl group to 5’-position of cytosine in the
CpG dinucleotide (regions of DNA which are GC rich or are concentrated in large
clusters called the CpG islands) resulting in epigenetic silencing of genes [6, 19, 20].
Methylation in mammalian cells is the function of three DNA methyltransferase (DNMTs)
– DNMT1a, DNMT3a and DNMT3b [6-9, 16, 18, 20]. Mice studies have shown that
DNMT1 helps to maintain pre-existing methylation patterns during DNA replication,
while DNMT3a and DNMT3b initiate de novo methylation [7, 16, 20]. Expression of
DNMT3a is ubiquitous. DNMT3b is present in the cells at low levels except in testes,
thyroid, and bone marrow [16].

7

DNA methylation once establ
established
ished in CpG islands is maintained after cell division
through the activity of DNMTs, which localize to the replication foci to work on newly
synthesized hemi-methylated
methylated DNA [6]. Methylation
ethylation of cytosine residues in CpG islands
with resultant binding of methyl
methyl-binding proteins (MBP) to DNA [8, 9, 18].. This further
attracts histone deacetylase (HDAC) and histone methyltransferase (HMT) resulting in
condensation of chromatin which prevents specific transcript
transcription
ion factors or DNADNA
dependent RNA polymerase from accessing the promoter regions to cause gene
silencing [8, 9, 18] (Figure. 2).. DNA methylation can be reversed physiologically and
resets in early embryogenesis.

Figure 2. Gene silencing that occurs in AML as a result of DNMT, HDAC and HMT, and
specific inhibitors of these enzymes.
enzymes.[6]

8

Hypermethylation of the promoter regions of important tumor suppressor genes
by the DNA methyltransferase results in gene silencing and hence cancer development.
DNA methylation not only results in alterations in promoter and repetitive sequences of
DNA but can also alter the expression and regulation of noncoding RNAs such as
micro-RNAs (mi-RNA) [19]. The CpG islands hypermethylated in AML span the
promoters of house-keeping genes and tumor suppressor genes. Multiple genes are
concurrently methylated including genes involved in cell cycle regulation, DNA repair,
drug resistance, detoxification, differentiation, apoptosis, angiogenesis and
metastasis.[6, 16-18, 21]
Methylated promoter regions of tumor suppressor genes are important targets in
cancer treatment as these epigenetic changes are reversible, this has been
demonstrated in MDS and AML especially in the elderly [19]. Currently, two
hypomethylating agents are approved for the treatment of MDS; azacytidine and
decytabine. Both agents have been demonstrated to be equally effective in the
treatment of MDS; however, azacytidine has a better safety profile than decytabine [22].
Azacytidine or 5’Azacytidine (Vidaza®) was approved by the FDA for the treatment of
MDS in 2004 [7, 23]. It is a chemical analogue of cytosine nucleoside. This drug causes
DNA hypomethylation resulting in reactivation of tumor suppressor genes, and direct
cytotoxicity through incorporation into the tumor DNA and RNA. Decytabine or 5-Aza-2’deoxycytadine (Dacogen®) was approved by the FDA in 2006 for the treatment of MDS
and was recently approved for the treatment of AML in elderly patients [7]. It is an
analogue of the nucleoside 2’-deoxycytidine. It exerts its antineoplastic effects after
phosphorylation and direct incorporation into the tumor DNA, and inhibition of the DNA
9

methyltransferases, causing hypomethylation and cellular differentiation or apoptosis. In
clinical trials both of these agents were compared with best supportive care or low-dose
cytarabine in patients with low-, intermediate- or high-risk AML, and showed better OS
compared to both modalities [20]. Even though the epigenetic changes and gene
silencing is reversible with the use of these agents; however, aberrant methylation of
genes returns as soon as they are stopped [17].

10

CHAPTER 2
XB05 – A Novel Synthetic Small Molecule
XB05 (1-bromo-1,1-difluoro-non-2-yn-4-ol) is a novel synthetic small molecule
originally developed to introduce fluorine containing groups in to organic molecules [24].
This molecule was found to be selectively cytotoxic to certain malignant cells compared
to non-malignant cells, and had particularly potent activity against myeloid leukemia cell
lines [24]. XB05 was not originally designed as an anti-cancer agent—a serendipitous
observation led to the discovery of its effects on cancer cells—so its precise molecular
mechanism is not yet fully understood; nonetheless, our studies have identified several
activities that contribute to its selective anti-cancer effects . Based on the data collected
so far, we propose that XB05 induces leukemia cell death through multiple
mechanisms, including reactivation of tumor suppressor genes via inhibition of DNA
methylation, induction of lethal oxidative and proteotoxic stress through down regulation
of the transcription factor nuclear factor, erythroid 2-like 2 (Nrf2) [24-27]. This last
activity is particularly significant because Nrf2 is frequently over expressed in AML cells
and is known to contribute to the resistance of AML against a variety of
chemotherapies.[24, 26-28] In fact, based on our preliminary data, we predict that XB05
will sensitize AML cells to the effects of chemotherapy, while simultaneously protecting
healthy cells from side effects [29].

11

Figure 3. Molecular structure of XB05
XB05[29]

XB05 has a novel chemical structure which prompted initial anti
anti-cancer
cancer studies
showing cancer selectivity [24
24]. Following this, the agent was sent to the National
Cancer Institute (NCI) for further testing. NCI
NCI-60
60 screen was performed where the
activity of the agent is tested on 60 human cancer cell lines. XB05 was shown to be
cytotoxic in some cell lines while not in others. The specific activity of XB05 can be
compared to 40,000 other compounds screened by NCI in terms of mechanism of
action. On further analysis and comparative studies, we came across number of
interesting findings. XB05 was selectively cytotoxic to malignant cells and had no
cytotoxicity against non-malignant
malignant human cells such as fibroblast cell line “Hs27”,
“Hs27”
mammary cell line “MCF-10A
10A and lung fibroblast cell line “IMR
“IMR-90”.[29] Mechanism of
action of XB05 was unique and not related to any existing chemotherapeutic agents.
XB05 was closely related to a naturally occurring marine product called “halomon”
which inhibits the activity of DNMT
DNMTs.

12

One of the major concerns while studying XB05 was that a molecule like it will be
highly reactive and non-specific [24]. However, studies done using fluorine-19 nuclear
magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS)
showed that XB05 was not reactive with single stranded DNA oligonucleotides or with
basic amino acids. These studies did reveal that XB05 was slowly reactive with thiolcontaining amino acids such as cysteine and glutathione (GSH) [24]. Further studies
showed that addition of glutathione and N-acetyl cysteine inhibited the anti-proliferative
activity of XB05 but addition of non-thiol antioxidants such as ascorbic acid or butylated
hydroxyanisole (BHA) had no effect [24].
One of the important determinants that can help guide therapy and response to
XB05 is the transcription factor SRY (sex determining region Y)-box 9 (SOX9) [24].
SOX9 gene is located on chromosome 17q23 and encodes for a high mobility group
box transcription factor that plays a critical role in embryogenesis and is required for
development, differentiation, and lineage commitment in various tissues especially
skeletal and reproductive systems [30]. SOX9 is over expressed in many cancer cells
which correlates with malignant potential of the tumor and possibly its response to anticancer agents [30].
XB05 has also been shown to increase the sensitivity of cancer cells to oxidative
stress. Cancer cells have increased levels of reactive oxygen species (ROS) and are
constantly under increased oxidative stress secondary to elevated metabolism,
increased expression of oncogenic pathways, dysfunctional mitochondrial machinery
and loss of tumor suppressor genes [25-27]. In order to counteract the increased levels
13

of oxidative stress, cancer cells up regulate anti-oxidative mechanisms [26, 27]. One of
the major pathways that is involved in oxidative stress response is Kelch-like ECHassociated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2)antioxidant response elements (ARE) or Keap1-Nrf2-ARE pathway and protects cancer
cells from apoptosis [26-28]. When cells are exposed to oxidative and electrophilic
stress, Nrf2 induces transcriptional activation of cytoprotective and detoxification genes
preventing damage to DNA, proteins and lipids [26-28]. In AML, Nrf2 is present in the
nucleus where it is primed to up-regulate transcription of genes involved in detoxification
[26-28].
In non-malignant cells, the inhibitor of Nrf2 or Keap1 or iNrf2 mediates
ubiquitination and proteasomal degradation of Nrf2 [26-28]. In-vitro when cells are
exposed to increased oxidative stress such as exposure to chemotherapeutic agents,
Keap-1 mediated proteasomal degradation of Nrf2 is blocked [26-28]. The activated
Nrf2 then translocate to the nucleus and forms a complex with Maf proteins which
facilitates its binding to the ARE and stimulate transcription of cytoprotective and
detoxification genes. Nrf2 is regulated by another control element called the Bach1
which is a transcriptional repressor and binds to ARE enhancers blocking its activity
until cells are exposed oxidative stress [26-28]. In normal cells, this pathway helps to
protect cells from oxidative stress and damage to cellular machinery. On the contrary,
this pathway is also beneficial to the cancer cells protecting them from increased
oxidative stress in the presence of chemotherapeutic agents [26-28]. Up-regulation of
this pathway in the cancer cells helps them acquire growth advantage and chemoresistance [26-28]. Also, it has been recently shown that up-regulation of Nrf2 in AML
14

cells is directly related to higher nuclear levels of NF-κB indicating that it regulates the
transcription of Nrf2 m-RNA [26-28].
Cancer cells are prone to increased cellular and oxidative stress through
increase in metabolism and increased oncogene activation which results in the
production of ROS [25]. ROS can damage DNA, proteins and lipids inducing oxidative
and proteotoxic stress and causing cell death by both apoptotic and non-apoptotic
pathways. In order to survive this stress, cancer cells upregulate anti-stress
mechanisms such as GSH, heat shock proteins and unfolded protein responses (UPR)
[25]. Studies have demonstrated that inducing cellular stress and inhibiting stress
response pathways can selectively induce cancer cell death.
In addition to the above described mechanisms, we believe that XB05
suppresses cancer proliferation by suppressing epigenetic modulation by DNMTs. This
mechanism of action of XB05 is likely related to the naturally occurring marine
compound called “halomon”. Preliminary in vitro studies done in our laboratory have
confirmed that XB05 inhibits DNMT1 activity. Detailed epigenetic modulation via DNMTs
is described in the previous chapter (chapter 1). However, studies looking at the reexpression of tumor suppressor genes silenced by methylation are lacking. In this study,
we performed studies attempting to elucidate the mechanism of action of XB05 from the
perspective of inhibition of DNMT activity.
We believe that anti-cancer effects of XB05 are associated with its ability to
induce cellular stress and down-regulation of DNMTs. Based on preliminary studies, we
have determined that XB05 is highly selective and can induce cell death via both
15

apoptotic and non-apoptotic mechanisms in cancer cells but has no activity in benign
cells [24]. XB05 can generate high levels of ROS in cancer cells increasing cellular
stress and inducing DNA damage . XB05 can reduce the level of glutathione and Nrf2 in
malignant cells exposing them to increased oxidative stress and inhibiting cellular
cytoprotective mechanisms [24]. Also, it inhibits the activity of DNMTs resulting in reexpression of tumor suppressor genes silenced in various cancers including AML.

16

CHAPTER 3
SPECIFIC AIMS
Aim 1: Determination of the inhibitory concentration or IC50 for XB05 in two AML
cell lines.
Rationale: XB05 was studied in the NCI-60 screen where it was found to be cytotoxic in
leukemia cell lines. Expanding on the existing data and preliminary studies done in our
laboratory, MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-assays
were planned to determine the IC50 for XB05 in two AML cell lines (Kasumi-1 and
U937). Comparative MTT-assays with 5’azacytidine, a known hypomethylating agent,
and combination drug (XB05 and 5’azacytidine) MTT-assays were also planned to
evaluate the synergistic activity of XB05.
Aim 2: Assess the expression of DNA-methyltransferases and tumor suppressor
genes in AML following treatment with XB05 and 5’-azacytidine at different time
intervals.
Rationale: Preliminary in vitro studies done in our laboratory had suggested that XB05
is a DNA-hypomethylating agent. We plan to perform polymerase chain reaction (PCR)
to assess the expression of DNMTs and commonly methylated tumor suppressor genes
in AML following treatment with XB05 and 5’AZA at 48- and 96-hour intervals. Treated
and non-treated specimens will also be assessed for percent promoter methylated of
many tumor suppressor genes using Methyl PCR-assay. Furthermore, Western Blots

17

will be performed on nuclear and cytoplasmic extracts and immunohistochemistry (IHC)
will be performed on whole cells following treatment to assess for protein expression.
Materials and Methods
AML Cell lines
Following cell lines were chosen because of our past experience working with these cell
lines and they are easy to culture with rapid turnover. DNMT (especially DNMT1)
expression has been reported to be high in Kasumi-1 and U937 cell lines [31-34].
1. Kasumi-1 – Established cell line obtained from the American Type Culture Collection
(ATCC-CRL-2724) (http://atcc.org/Products/All/CRL-2724.aspx).
This cell line was established from the peripheral blood of a seven year old juvenile
Japanese patient with AML. The leukemic cells have t(8;21) chromosomal translocation
which juxtaposes the AML1 gene on chromosome 21 with ETO (eight twenty one) or
MTG8 (myeloid translocation gene on 8) gene on chromosome eight resulting in a
fusion gene AML1-ETO (also known as AML1-MTG or RUNX1-CBF2T1), and thus
producing a chimeric AML1-ETO protein. This protein down-regulates CEBPA mRNA,
protein and DNA binding activity, which is crucial for the differentiation of granulocytes.
The cells are positive for myeloperoxidase and in proliferation assay the cells in culture
show response to interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (GCSF), and granulocyte macrophage CSF (GM-CSF), but not to IL-1 or IL-5 [35, 36].
The base media for these cells is RPMI supplemented with 10% FBS and 1% penicillin
and streptomycin. Cells are seeded at the density of 1 to 300,000 cells/mL.
18

2. U937 – Established cell line obtained from the ATCC (ATCC-CRL-1593.2)
(http://atcc.org/Products/All/CRL-1593.2.aspx)
The U-937 cell line was established in 1974 from malignant cells obtained from the
pleural effusion of a 37 year old Caucasian male patient with histiocytic lymphoma.
These cells can be induced to terminal monocytic differentiation by supernatants from
human mixed lymphocyte cultures. These cells are negative for immunoglobulin
production and Epstein Barr virus expression. These cells express lysozyme, beta-2microglobulin (beta 2 microglobulin) and tumor necrosis factor alpha (TNF-alpha), after
stimulation with phorbol myristic acid (PMA). The cells also express the Fas antigen,
and are sensitive to TNF and anti-Fas antibodies [37].
The base media for these cells is RPMI supplemented with 10% FBS and 1% penicillin
and streptomycin. Cells are seeded at the density of 1 to 300,000 cells/mL.
Culturing Media
RPMI (Roswell Park Memorial Institute medium) supplemented with 10% fetal bovine
serum (FBS) and 1% PenStrep (Penicillin-Streptomycin) containing 5000 units of
penicillin and 5000 µg of streptomycin per ml.
Culture Methods
Each cell line was grown in RPMI (supplemented with 10% FBS and 1% Pen-Strep) for
three days in AutoFlow NU-4750 water jacket CO2 incubator before splitting. Cells were
seeded in the media at recommended concentration of 1 to 300,000 cells/mL.

19

Drugs
1. XB05: Stock concentration of 15.7mM in 100% DMSO.
2. 5’Azacytidine (5’AZA) (Sigma Aldrich: A1287) Stock concentration of 500µM in
RPMI.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) Assay
Principle: MTT is a colorimetric assay that measures the reduction of the oxidized
substrate MTT which is yellow in color by the mitochondrial succinate dehydrogenase.
The MTT enters the cells and passes into the mitochondria where it is reduced to an
insoluble formazan product which is dark purple in color. The cells are then solubilized
with cell lysis buffer and the released, solubilized formazan product is measured by
spectrophotometer. Since the reduction of MTT can only occur in metabolically active
cells, the level of activity is a measure of cell viability. This assay is broadly used to
measure in vitro cytotoxic effects of drugs on cell lines or primary patient cells. When
the amount of purple formazan produced by cells treated with an agent is compared
with the amount of formazan produced by untreated control cells, the effectiveness of
the agent in causing death of cells can be deduced, through the production of a doseresponse curve.
Assay Design: Refer to appendix for experimental details and design.
Initial studies involved optimizing conditions for the experiment. Cells from both cell
lines were plated at 1000, 1500, 2000, 2500 and 3000 cells per well per in 100µl of

20

culturing media. Results from these experiments suggested that Kasumi-1 cells should
be plated at 1500 cells per well and should be treated on the same day. However, U937
cells should be plated at 2000 cells per well and treated on the next day. Cells were
counted using an automatic cell counter (TC20 Automated Cell Counter System.
BioRad) according to manufacturer instruction. Viable cell count was more than 90% at
plating. Different XB05 and 5’azacytidine concentrations were added in quadruplicates
to determine inhibitory concentration (IC50) for each drug. Concentration of each drug
added to wells was three times. Volume of drug added was 50uL which resulted in the
total volume of 150uL and originally planned final concentration in each well. Also, drug
concentrations were determined based on pilot MTT-assays. Based on the pilot assays,
different serial concentrations for both XB05 and 5’AZA were determined. Control for
XB05 was dimethyl sulfoxide (DMSO) and for 5’AZA was RPMI. Cells were incubated
for 72 hours. At 96 hours, MTT reagent at 10% of the well volume or 15uL was added
and allowed to incubate for four hours. Then cell lysis buffer or sodium dodecyl sulfate
(SDS) at 50% of the well volume or 75uL was added and allowed to incubate overnight.
The following day, absorbance is read with spectrophotometer at wavelength of 570nm.
In addition, MTT-assays for combination treatment with XB05 and azacytidine were
performed. Cells were treated with constant concentration of XB05 along with serial
concentrations of 5’azacytidine.

21

Polymerase Chain Reaction (PCR) – Reverse Transcription PCR and Real-time
PCR
Principle: PCR is a method DNA amplification developed by Kary Mullis in the 1980s. It
is based on the ability of DNA polymerase to synthesize new strand of DNA
complementary to the offered template strand. Because DNA polymerase can add a
nucleotide only onto a preexisting 3'-OH group, a primer is needed which attaches to
the complimentary sequence on the DNA and provides the polymerase with a starting
point where the first nucleotide is added. This requirement helps the researcher to
identify and amplify a specific sequence of the template DNA segment. At the end of the
PCR reaction, the specific sequence will be accumulated in billions of copies or
amplicons. (Figure. 1)
Reverse Transcription PCR (RT-PCR): This method of PCR is used to amplify DNA
from mRNA. Reverse transcriptase transcribes RNA to cDNA and is then amplified by
PCR. It is used for expression profiling and determine the expression of a gene.
Quantitative real-time PCR (qRT-PCR): This method is used to determine the level of
expression of a gene in real-time. It can quantitate the starting amounts of DNA, cDNA
(obtained from RT-PCR), or RNA. qPCR has a very high degree of precision. It uses
fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA
probes, such as TaqMan, to measure the amount of amplified product in real time.

22

Figure 4. Outline of the principle of PCR. (Adapted from NCBI.
http://www.ncbi.nlm.nih.gov/genome/probe/doc/TechPCR.shtml)

Assay Design: Refer to appendix for experimental design and details
After determining the IC50 of XB05 and 5’AZA for both cell lines, they were treated in
flasks to collect post-treatment RNA. Cells were plated in flasks at the recommended
count of 1×105 cells/ml. Both the cell lines were treated with both XB05 and 5’AZA at
their respective IC50. RNA from cells was collected at different time intervals – 48- and
23

96-hours. Cells collected were homogenized with Trizol reagent. RNA was extracted as
per manufacturer’s protocol. (Outlined in Appendix) Extracted RNA was quantified using
NanoDrop. Using RT-PCR, 1ug/uL of RNA was converted to complimentary DNA
(cDNA). Protocol as per manufacturer – outlined in appendix. cDNA obtained with this
method was then quantified using qRT-PCR to determine the expression of various
genes of interest or the genes which are highly methylated in AML. Also, levels of the
three DNA methyltransferases was also determined to evaluate if the expression of
these genes decreases after treatment with XB05 and 5’AZA. We quantified the
expression of the following genes; Hypermethylated in Cancer – 1 (HIC-1), estrogen
receptor – alpha (ERα), p16, Methylguanine-DNA Methyltransferase (MGMT), DeathAssociated Protein Kinase 1 (DAPK1), DNMT1, DNMT3a, DNMT3b and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR experiments were
performed to elucidate that at what time interval, does XB05 result in down-regulation of
DNMTs and in turn result in re-expression of the hypermethylated genes. qRT-PCR
protocol as described by the manufacturer and outlined in appendix.
EpiTect Methyl II PCR Array (Qiagen®): Refer to appendix for experimental details
This method is based on detection of remaining input DNA after cleavage with a
methylation-sensitive and/or a methylation-dependent restriction enzyme. The
restriction enzymes digest unmethylated and methylated DNA. Following digestion, the
remaining DNA in each individual enzyme reaction is quantified by RT-PCR using
primers that flank a promoter for the gene region of interest. The relative fractions of
methylated and unmethylated DNA are subsequently determined by comparing the
24

amount in each digest with that of a mock (no enzymes added) digest using a ∆CT
method. We used a 22 gene panel array to evaluate the methylation pattern of promoter
sequences of commonly methylated genes in leukemia at 48 & 96 hour intervals in nontreated cells, & following treatment with XB05 & azacytidine.

AFF1 (Oncogene)

AF4/FMR2 family, member 1

CD9 (TS)

Cluster differentiation 9

CEBPD (TS)

CCAAT/enhancer binding protein (C/EBP), delta

CTNNA1 (TS)

Catenin (cadherin-associated protein), alpha 1

DRD2 (TS)

Dopamine receptor D2

EXT1 (TS)

Exostosin glycosyltransferase 1

FANCC (Oncogene)

Fanconi anemia, complementation group C

FANCL (Oncogene)

Fanconi anemia, complementation group L

HCK (Oncogene)

HCK proto-oncogene, Src family tyrosine kinase

HOXA7 (Oncogene)

Homeobox A7

HOXB5 (Oncogene)

Homeobox B5

JUNB (Oncogene)

Jun B proto-oncogene

LMNA (TS)

Lamin A/C

MAFB (Oncogene)

v-maf avian musculoaponeurotic fibrosarcoma oncogene
homolog B

MEN1 (TS)

Multiple endocrine neoplasia I

NFATC1 (Oncogene)

Nuclear factor of activated T-cells, cytoplasmic, calcineurindependent 1

Table 2. Oncogenes and tumor suppressor (TS) genes in 22-panel EpiTect Methyl II
PCR Panel.
Refer to http://www.ncbi.nlm.nih.gov/gene for detailed gene functions
25

Table 2 (cont’d)
NPM1 (Oncogene)

Nucleophosmin (nucleolar phosphoprotein B23, numatrin)

PER2 (TS)

Period circadian clock 2

SLC4A8 (Oncogene)

Solute carrier family 4, sodium bicarbonate cotransporter,
member 8

SPOCK2 (TS)

Sparc/osteonectin, cwcv and kazal-like domains
proteoglycan (testican) 2

TLE1 (Oncogene)

Transducin-like enhancer of split 1 (E(sp1) homolog,
Drosophila)

TP53 (TS)

Tumor protein p53

Western Blot
Principle: Primarily used to detect specific proteins expressed in a tissue or cell lysate.
Cytoplasmic or nuclear proteins extracted with different protocols are separated using
gel electrophoresis based on size and electrical conductivity. The separated proteins
are then transferred to a nitrocellulose or PVDF membrane where they are stained with
antibodies specific to that target protein. Then secondary antibody tagged with a HRPchemiluminesence agent is added which attaches to the primary antibody and intensity
of the chemiluminesence can be measured using photographic film.
Assay Design: Refer to appendix for experimental design and details

26

Western Blot was performed on the protein samples obtained after treatment with the
two drugs at the time interval which showed maximal DNMT inhibition and reexpression of methylated genes based on the results of PCR. Proteins selected for
Western Blot assay were based on the results of PCR and available antibodies.
Western blot was performed for the following proteins – DNMT1, DNMT3a, DNMT3b,
HIC1, ER-α, MGMT, p16, p21, alpha-tubulin, beta-actin and SOX9. Cytoplasmic and
nuclear proteins were extracted from both treated and untreated cells and quantified
using Coomassie Blue (BioRad) or Bradford protein assay (BioRad). Using
commercially available Bio-Rad SDS-PAGE gels, the nuclear and cytoplasmic protein
were separated. After separation, proteins were transferred to PVDF membranes using
wet transfer method. Membranes were incubated with primary antibody overnight at
4ºC. After appropriate washings, membranes were incubated with secondary antibody
tagged with horseradish peroxidase (HRP). Using enhanced chemiluminescence (ECL,
GE Healthcare Life Science) substrate, amount of protein expression was quantified on
X-ray films. GAPDH was used as a control for cytoplasmic proteins and histone H3 was
used as a control for nuclear proteins.
Protein Quantitation by Coomassie (Bradford) protein assay
Coomassie (Bradford) protein assay was used for colorimetric detection and
quantitation of total protein. Protocol as per manufacturer’s recommendations – outlined
in appendix.

27

Immunohistochemistry
Principle: Immunohistochemistry or IHC involves detecting an expression of a protein
in a cell or a tissue specimen by targeting the antigen with primary antibody followed by
incubation with fluorescence or peroxidase tagged secondary antibody to grade the
protein’s expression in the tissue.

Assay Design: Refer to appendix for experimental design and details
Using cytospin machine, treated and untreated cells from both cell lines were plated on
the slides and fixed with ice cold methanol for 15 minutes. Permeabilize cells with 0.5%
TritonX. Using the Pierce peroxidase detection kit (Thermo Scientific®), slides were
stained. DNMT1 and HIC1 expression was probed in non-treated and cells treated at
48- and 96-hour intervals. Mouse and goat serum was used as negative control.
Primary antibodies were used at 1:25 and secondary antibodies were used at 1:50
dilution. Protocol outlined in appendix.

28

RESULTS
MTT-ASSAYS
MTT-assay results for Kasumi-1 cell line reported in both tabulated and graphical
illustrations.
nM
XBO5
XBO5
XBO5
XBO5
AVG

0.00
0.57
0.63
0.59
0.62
0.60

50.00
0.56
0.60
0.60
0.65
0.60

100.00
0.57
0.61
0.63
0.57
0.59

200.00
0.49
0.56
0.56
0.58
0.55

400.00
0.45
0.50
0.50
0.57
0.50

800.00
0.30
0.38
0.43
0.47
0.40

99.79

98.46

90.73

83.79

65.79

0.00
0.58
0.65
0.61
0.70
0.64

50.00
0.56
0.60
0.58
0.55
0.57

100.00
0.47
0.52
0.53
0.47
0.50

200.00
0.43
0.42
0.43
0.39
0.42

400.00
0.36
0.38
0.40
0.35
0.37

800.00
0.34
0.35
0.32
0.33
0.33

100.00

89.78

77.98

65.28

58.83

52.38

% Cell Proliferation 100.00
nM
AZA
AZA
AZA
AZA
AVG
% Cell Proliferation

Table 3. First MTT-assay performed on Kasumi-1 cells with serial concentrations of
XB05 and 5’azacytidine. Percent proliferation is reported with serial concentration of
XB05 and 5’azacytidine.

29

Figure 5.. Percent cell proliferation in Kasumi-1 cell line is represented on graph
showing decreased proliferation with increasing concentration.

nM
XBO5
XBO5
XBO5
XBO5
Avg

0.00
0.48
0.50
0.50
0.48
0.49

100.00
0.47
0.48
0.48
0.51
0.48

200.00
0.45
0.47
0.45
0.45
0.46

400.00
0.38
0.41
0.43
0.41
0.41

600.00
0.35
0.42
0.36
0.37
0.38

800.00
0.37
0.35
0.34
0.33
0.35

1000.00
0.33
0.31
0.33
0.33
0.32

1200.00
0.29
0.29
0.28
0.29
0.29

1600.00
0.31
0.29
0.28
0.31
0.30

98.88

92.81

82.61

76.70

70.47

66.04

58.80

60.63

0.00
0.49
0.59
0.57
0.53
0.54

100.00
0.40
0.41
0.45
0.45
0.43

200.00
0.33
0.34
0.36
0.32
0.34

400.00
0.28
0.29
0.30
0.31
0.30

600.00
0.28
0.29
0.29
0.31
0.29

800.00
0.27
0.30
0.31
0.28
0.29

1000.00
0.25
0.28
0.27
0.28
0.27

1200.00
0.24
0.27
0.26
0.26
0.26

1600.00
0.25
0.25
0.24
0.25
0.25

100.00

78.78

62.29

54.34

53.70

52.92

49.61

47.36

45.38

% Cell Proliferation 100.00
nM
AZA
AZA
AZA
AZA
Avg
% Cell Proliferation

Table 4. Second MTT-assay
assay performed on Kasumi
Kasumi-1 cells with serial concentrations
of XB05 and 5’azacytidine.
dine. Percent proliferation is reported with serial concentration of
XB05 and 5’azacytidine.

30

Figure 6.. Percent cell proliferation in Kasumi-1 cell line is represented on graph
showing decreased proliferation with increasing concentration.

nM
XBO5
XBO5
XBO5
XBO5
Avg

0.00
0.51
0.49
0.50
0.50
0.50

100.00
0.45
0.46
0.48
0.41
0.45

200.00
0.47
0.47
0.42
0.45
0.45

400.00
0.41
0.43
0.41
0.39
0.41

600.00
0.36
0.34
0.38
0.38
0.37

800.00
0.35
0.31
0.35
0.38
0.35

1000.00
0.31
0.29
0.30
0.31
0.30

1200.00
0.28
0.25
0.28
0.27
0.27

1600.00
0.27
0.24
0.28
0.25
0.26

90.18

90.68

82.26

73.18

69.67

60.05

54.39

51.83

0.00
0.52
0.52
0.52
0.53
0.52

100.00
0.39
0.38
0.39
0.40
0.39

200.00
0.32
0.33
0.34
0.33
0.33

400.00
0.27
0.31
0.30
0.31
0.30

600.00
0.26
0.29
0.29
0.32
0.29

800.00
0.28
0.29
0.31
0.31
0.30

1000.00
0.26
0.27
0.28
0.32
0.28

1200.00
0.25
0.26
0.27
0.27
0.26

1600.00
0.24
0.24
0.25
0.23
0.24

100.00

75.12

63.25

56.60

54.98

56.70

53.16

50.19

45.89

% Cell Proliferation 100.00
nM
AZA
AZA
AZA
AZA
Avg
% Cell Proliferation

Table 5. Third MTT-assay
assay performed on Kasumi
Kasumi-1 cells with serial concentrations of
XB05 and 5’azacytidine.
dine. Percent proliferation is reported with serial concentration of
XB05 and 5’azacytidine.

31

Figure 7.. Percent cell proliferation in Kasumi-1 cell line is represented on graph
showing decreased proliferation with increasing concentration.

XBO5 (nM)
% Cell Proliferation
% Cell Proliferation
% Cell Proliferation
Avg % Cell Proliferation
Effect

0.00
100.00
100.00
100.00
100.00
0.00

50.00
99.79

AZA (nM)
% Cell Proliferation
% Cell Proliferation
% Cell Proliferation
Avg % Cell Proliferation
Effect

0.00
100.00
100.00
100.00
100.00
0.00

50.00
89.78

99.79
0.00

89.78
0.10

100.00
98.46
98.88
90.18
95.84
0.04

200.00
90.73
92.81
90.68
91.41
0.09

400.00
83.79
82.61
82.26
82.89
0.17

600.00

100.00
77.98
78.78
75.12
77.29
0.23

200.00
65.28
62.29
63.25
63.61
0.36

400.00
58.83
54.34
56.60
56.59
0.43

600.00

76.70
73.18
74.94
0.25

53.70
54.98
54.34
0.46

800.00
65.79
70.47
69.67
68.65
0.31

1000.00

1200.00

1600.00

66.04
60.05
63.04
0.37

58.80
54.39
56.59
0.43

60.63
51.83
56.23
0.44

800.00
52.38
52.92
56.70
54.00
0.46

1000.00

1200.00

1600.00

49.61
53.16
51.38
0.49

47.36
50.19
48.78
0.51

45.38
45.89
45.63
0.54

Table 6. Results of combined MTT-assays performed on Kasumi-1
1 cells with serial
concentrations of XB05 and 5’
5’azacytidine.
dine. Percent proliferation is reported with serial
concentration of XB05 and 5’azac
azacytidine.

32

Figure 8.. Percent cell proliferation in Kasumi-1 cell line is represented on graph
showing decreased proliferation with increasing concentration. Graphical representation
repre
of the combined results of three MTT
MTT-assays.

These results demonstrate that the cytotoxicity of both drugs increases with increasing
concentration; however, the
he rate of decline in percent viability of cells with azacytidine is
greater than seen with XB05.
These results were then exported to CompuSyn® software to calculate the IC50 for both
drugs in Kasumi-1 cell line.

33

Figure 9. Using the ComuSyn software, IC50 for XB05 in Kasumi-1 cell line is
calculated which was 1345.88nM. Dose-effect curve is shown which based on the
existing data points shows predicted decrease in cell proliferation with increasing dose.
Also, based on increasing dose, log decrease in cell proliferation is also shown.

34

Figure 10. Using the ComuSyn software, IC50 for 5’AZA in Kasumi-1 cell line is
calculated which was 932.198nM. Dose-effect curve is shown which based on the
existing data points shows predicted decrease in cell proliferation with increasing dose.
Also, based on increasing dose, log decrease in cell proliferation is also shown.

35

Figure 11. Results of the first MTT
MTT-assay performed in Kasumi-1
1 cell line
following combination treatment with XB05 and 5’azacytidine.
dine. XB05 concentration
was kept constant and 5’azac
azacytidine was added in serial concentrations. Refer to
supplementary data.

36

Figure 12. Results of the second MTT-assay performed in Kasumi-1
1 cell line
following combination treatment with XB05 and 5’azacytidine.
dine. XB05 concentration
was kept constant and 5’azac
azacytidine was added in serial concentrations. Refer to
supplementary data.

37

nM
XBO5
XBO5
XBO5
XBO5
AVG
% Cell
Proliferation
nM
AZA
AZA
AZA
AZA
AVG
% Cell
Proliferation

0.00
1.01
0.90
0.89
0.90
0.92

50.00
0.91
0.91
0.86
0.84
0.88

100.00
0.81
0.75
0.83
0.77
0.79

200.00
0.74
0.84
0.78
0.72
0.77

400.00
0.64
0.68
0.61
0.55
0.62

800.00
0.38
0.24
0.19
0.35
0.29

1000.00
0.24
0.23
0.16
0.45
0.27

100.00

95.53

85.71

83.46

67.36

31.39

29.30

0.00
0.91
0.89
0.88
0.91
0.90

50.00
0.79
0.82
0.80
0.87
0.82

100.00
0.78
0.69
0.77
0.78
0.75

200.00
0.63
0.61
0.74
0.70
0.67

400.00
0.50
0.52
0.61
0.64
0.57

800.00
0.35
0.40
0.44
0.44
0.41

1000.00
0.37
0.41
0.40
0.41
0.40

100.00

90.98

84.04

74.49

63.19

45.50

44.19

Table 7. First MTT-assay
assay performed on U937 cells with serial concentrations of XB05
and 5’azacytidine.
dine. Percent proliferation is reported with serial concentration of XB05
and 5’azacytidine.

Figure 13.. Percent cell proliferation in U937 cell line is represented on graph
showing decreased proliferation with increasing concentration.

38

nM
XBO5
XBO5
XBO5
XBO5
Avg
% Cell
Proliferation
nM
AZA
AZA
AZA
AZA
Avg
% Cell
Proliferation

0.00
0.65
0.77
0.61
0.75
0.70

50.00
0.71
0.93
0.75
0.79
0.80

100.00
0.64
0.81
0.70
0.77
0.73

200.00
0.58
0.64
0.58
0.61
0.60

400.00
0.39
0.29
0.33
0.38
0.35

600.00
0.17
0.15
0.22
0.39
0.23

800.00
0.29
0.15
0.08
0.01
0.13

1000.00
0.22
0.08
0.07
0.15
0.13

100.00

114.36

104.92

86.22

49.71

33.56

19.20

18.38

0.00
0.77
0.73
0.75
0.75
0.75

50.00
0.66
0.64
0.64
0.59
0.63

100.00
0.64
0.58
0.58
0.55
0.59

200.00
0.58
0.55
0.51
0.53
0.54

400.00
0.47
0.45
0.38
0.44
0.43

600.00
0.36
0.34
0.36
0.35
0.35

800.00
0.30
0.29
0.32
0.35
0.31

1000.00
0.22
0.26
0.29
0.29
0.27

100.00

83.84

78.19

72.17

57.68

46.84

41.72

35.24

Table 8. Second MTT-assay
assay performed on U937 cells with serial concentrations of
XB05 and 5’azacytidine.
dine. Percent proliferation is reported with serial concentration of
XB05 and 5’azacytidine.

Figure 14.. Percent cell proliferation in U937 cell line is represented on graph
showing decreased proliferation with increasing concentration.

39

nM
XBO5
XBO5
XBO5
XBO5
Avg
% Cell
Proliferation
nM
AZA
AZA
AZA
AZA
Avg
% Cell
Proliferation

0.00
0.72
0.75
0.63
0.74
0.71

50.00
0.75
0.85
0.69
0.66
0.74

100.00
0.66
0.81
0.75
0.64
0.72

200.00
0.53
0.59
0.67
0.61
0.60

400.00
0.34
0.31
0.33
0.40
0.34

600.00
0.13
0.33
0.13
0.05
0.16

800.00
0.03
0.06
0.04
0.41
0.14

1000.00
0.01
0.02
0.12
0.35
0.12

100.00

103.95

101.16

84.40

48.45

22.41

19.05

17.50

0.00
0.72
0.69
0.68
0.71
0.70

50.00
0.64
0.66
0.63
0.69
0.66

100.00
0.63
0.59
0.65
0.68
0.64

200.00
0.55
0.53
0.54
0.50
0.53

400.00
0.42
0.44
0.41
0.45
0.43

600.00
0.35
0.34
0.40
0.40
0.37

800.00
0.33
0.31
0.36
0.35
0.34

1000.00
0.25
0.25
0.28
0.29
0.27

100.00

94.15

91.42

75.64

61.57

53.53

48.15

38.46

Table 9. Third MTT-assay
assay performed on U937 cells with serial concentrations of
XB05 and 5’azacytidine.
dine. Percent proliferation is reported with serial concentration of
XB05 and 5’azacytidine.

Figure 15.. Percent cell proliferation in U937 cell line is represented on graph
showing decreased proliferation with increasing concentration.

40

XBO5 (nM)
% Cell
Proliferation
% Cell
Proliferation
% Cell
Proliferation
Avg % Cell
Proliferation
Effect
AZA (nM)
% Cell
Proliferation
% Cell
Proliferation
% Cell
Proliferation
Avg % Cell
Proliferation
Effect

0.00

50.00

100.00

200.00

400.00

100.00

95.53

85.71

83.46

67.36

100.00

114.36

104.92

86.22

49.71

100.00

103.95

101.16

84.40

100.00
0.00

104.61
-0.05

84.69
0.15

97.27
0.03

800.00

1000.00

31.39

29.30

33.56

19.20

18.38

48.45

22.41

19.05

17.50

55.17
0.45

27.98
0.72

23.22
0.77

21.73
0.78

0.00

50.00

100.00

200.00

400.00

100.00

90.98

84.04

74.49

63.19

100.00

83.84

78.19

72.17

57.68

100.00

94.15

91.42

75.64

100.00
0.00

89.66
0.10

84.55
0.15

74.10
0.26

600.00

600.00

800.00

1000.00

45.50

44.19

46.84

41.72

35.24

61.57

53.53

48.15

38.46

60.81
0.39

50.19
0.50

45.13
0.55

39.30
0.61

Table 10. Results of combined MTT-assays performed on U937 cells with serial
concentrations of XB05 and 5’azacytidine. Percent proliferation is reported with serial
concentration of XB05 and 5’azacytidine.

41

Figure 16.. Percent cell proliferation in U937 cell line is represented on graph
showing decreased proliferation with increasing concentration. Graphical representation
of the combined results of three MTT
MTT-assays.
Results of the assay show that with the increasing concentrations of both XB05 and
5’azacytidine,
dine, percent cell proliferation decreases; however, the rate of decline in
percent cell proliferation off cells with XB05 is greater than seen with 5’azac
azacytidine.
These results were then exported to CompuSyn® software to calculate the IC50 for both
drugs in U937 cell line. The results obtained from the CompuSyn® software are
reported below. IC50 for XB05 is 463.297nM and for 5’azacytidine
dine is 633.538nM.

42

Figure 17. Using the ComuSyn software, IC50 for XB05 in U937 cell line is
calculated which was 463.297nM. Dose-effect curve is shown which based on the
existing data points shows predicted decrease in cell proliferation with increasing dose.
Also, based on increasing dose, log decrease in cell proliferation is also shown.

43

Figure 18. Using the ComuSyn software, IC50 for 5’AZA in U937 cell line is
calculated which was 633.538nM. Dose-effect curve is shown which based on the
existing data points shows predicted decrease in cell proliferation with increasing dose.
Also, based on increasing dose, log decrease in cell proliferation is also shown.

44

Figure 19.. Results of the first MTT
MTT-assay
assay performed in U937 cell line following
combination treatment with XB05 and 5’azacytidine.
dine. XB05 concentration was kept
constant and 5’azacytidine
dine was added in serial concentrations. Refer to supplementary
data.

45

Figure 20.. Results of the second MTT
MTT-assay
assay performed in U937 cell line following
combination treatment with XB05 and 5’azacytidine.
dine. XB05 concentration was kept
constant and 5’azacytidine
dine was added in serial concentrations. Refer to supplementary
data.

46

POLYMERASE CHAIN REACTION
EACTION (PCR):

Figure 21.
1. Results of PCR performed on Kasumi
Kasumi-1
1 cell line without treatment and
following treatment with XB05 and 5’azacytdine at 48-hour interval are shown.
shown At
48 hours, following treatment with XB05, expression of three DNMTs (DNMT1, 3a and
3b) decreased in Kasumi-1
1 cell line as compared to non
non-treated
treated cells. Increased
expression of methylated genes was not seen following XB05 treatment at 48 hours.
Following treatment with 5’azacytidine
azacytidine at 48 hours, expression of DNMT1 increased;
there was no change in the expression of DNMT3a and expression of DNMT3b
increased. However, expression of genes thought to methylated in leukemia increased
following treatment with 5’azacytidine
azacytidine at 48 hours. Refer to supplementary data.

.

47

Figure 22.. Results of PCR performed on U937 cell line without treatment and
following treatment with XB05 and 5’azacytidine at 48-hour interval are shown.
shown At
48 hours, following treatment with XB05, expression of all three DNMTs (DNMT1, 3a
and 3b) decreased. Again, there was no increased expression of methylated genes
noted at 48 hours following treatment with XB05. Following treatment with 5’azacytidine,
expression
pression all three DNMTs increased. Also, expression of genes except MGMT
increased at 48 hours after 5’azacytidine
azacytidine treatment. Refer to supplementary data.

48

Figure 23. Results of PCR performed on Kasumi
Kasumi-1
1 cell line without treatment and
following treatment with XB05 and 5’azacytidine at 96-hour interval are shown. At
96 hours following treatment with XB05, expression of DNMT1 increased. However,
expression of DNMT3a and 3b decreased. Also, expression of genes methylated in
leukemia increased following treatment with XB05 at 96 hours. Following treatment with
5’azacytidine at 96 hours, expression of DNMT1 increased but the expression of
DNMT3a and 3b decreased. Expression of only ER-alpha
alpha and p16 increased, but
expression of other genes decreased following treatment with 5’azacytidine.
dine. Refer to
supplementary data.

49

Figure 24. Results of PCR performed on U937 cell line without treatment and
following treatment with XB05 and 5’azacytidine at 96-hour interval are shown. At
96 hours, following treatment with XB05 expression of DNMT3a decreased while
expression of DNMT1 and 3b inc
increased.
reased. Expression of tumor suppressor genes
methylated in leukemia increased following XB05 treatment at 96 hours. Following
treatment with 5’azacytidine,, expression of DNMT3a decreased again, while expression
of DNMT1 and 3b increased. Expression of gene
genes
s methylated in leukemia also
increased following treatment with 5’azacytidine. Refer to supplementary data.

50

EpiTect Methyl II PCR Array (Qiagen®)

Figure 25.. EpiTect Methyl II PCR array results of percent methylation of CpG
islands in the promoter regions of selected genes in Kasumi cell line.
line In Kasumi-1
cell line, following treatment with XB05, percent methylation of promoter regions of
oncogenes such as AFF1, FANCL and SLC5A8 increased, percent methylation of TLE1
did not change and percent methylation of FANCC and HCK decreased. However, there
was no significant change in percent methylation of oncogenes such as HOXA7,
HOXB5, JUNB, MAFB, NFATC1 and NPM1. Percent methylation of promoter regions of
tumor suppressor genes such
ch as CD9, CEBPD, CTNNA1, DRD2, EXT1, LMNA, MEN1,
PER2 and SPOCK2 decreased following treatment with XB05 in Kasumi
Kasumi--1 cell line
while it remained unchanged for TP53. Following treatment of Kasumi-1
1 cell line with
5’AZA, there was increased percent methylation of promoter regions of oncogenes such
as AFF1 and FANCL while percent methylation decreased in FANCC, HCK, HOXA7,
HOXB5, JUNB, MAFB, NFATC1, NPM1, SLC4A8 and TLE1
TLE1.. 5’AZA treatment also
resulted in decreased promoter methylation in tumor suppressor genes such as CD9,
CEBPD, CTNNA1, DRD2, EXT1, LMNA, MEN1, PER2, SPOCK2 and TP53.

51

Figure 26.. EpiTect Methyl II PCR array results of percent methylation of CpG
islands in the promoter regions
ions of selected genes in U937 cell line. In U937 cell
line, following treatment with XB05, percent methylation of promoter regions of
oncogenes such as HOXB5, MAFB and SLC5A8 increased and percent methylation of
AFF1, FANCC, FANCL, HCK, HOXA7, JUNB, NFATC1, NPM1 and TLE1 decreased.
FANCC and HOXA7 promoter methylation decreased at a relatively small level. Percent
methylation of promoter regions of tumor suppressor genes such as CD9, CEBPD,
MEN1, PER2, SPOCK2 and TP53 decreased following treatment with XB05 in KasumiKasumi
1 cell line while it remained relatively unchanged for CTNNA1 and EXT1 and increased
for DRD2 and LMNA. Following treatment of Kasumi
Kasumi-1
1 cell line with 5’AZA, there was
increased percent methylation of promoter regions of oncog
oncogenes
enes such as MAFB while
percent methylation decreased in AFF1, FANCC, FANCL, HCK, HOXA7, HOXB5,
JUNB, NFATC1, NPM1, SLC5A8 and TLE1. 5’AZA treatment also resulted in
decreased promoter methylation in tumor suppressor genes such as CD9, DRD2,
EXT1, LMNA, MEN1,
N1, PER2, SPOCK2 and TP53 while percent promoter methylation
increased in CEBPD and CTNNA1.

52

Western Blots:

Figure 27. Western blot performed on nuclear proteins from Kasumi-1 cell line.
After treatment with XB05, the expression of DNMT3a and 3b and ER-alpha increased
at 48 and 96hours; however, there was no change noted in the expression of MGMT or
HIC1. The expression of DNMT1 decreased at 48 hours. Treatment with 5’azacytidine
did not result in decrease in DNMT1, 3a or 3b. ER-alpha expression was slightly
increased at 48hours. Histone protein H3 was used as the internal control for this
experiment.

53

Figure 28. Western blot performed on nuclear proteins from U937 cell line. After
treatment with XB05, there was increased expression of DNMT3a and 3b at 48hours.
However, the expression of DNMT3b decreased at 96hours. There was no change
noticed in the expression of DNMT1 after treatment with XB05. HIC1 and ER-alpha
expression slightly increased at 48hours. There was no change in the expression of
MGMT after both XB05 and 5’azacytidine treatments. H3 histone expression was used
as an internal control. Treatment with 5’azacytidine did decrease the expression of
DNMT1 at 48hours and DNMT3b at 96hours. There was no increase in the expression
of HIC1 or ER-alpha noticed. Histone protein H3 was used as the internal control for this
experiment.

54

Figure 29. Western blot performed on cytoplasmic proteins from Kasumi-1 cell
line. Based on the western blot results, no correlation was found between the
expression of SOX-9 and susceptibility to XB05. However, expression of SOX-9
decreased at 48 hours both after XB05 and 5’azacytidine treatments. We did notice that
the expression of alpha tubulin decreased following treatment with XB05 at 48 and
96hours, and with 5’azacytidine at 48hours. Changes in the expression of beta-actin
were also noted after treatment with both XB05 and 5’AZA. Expression of p16 slightly
increased at 96 hour after treatment with XB05 and with 5’azacytidine at 48hours. There
was no change noticed in the expression of p21 both with XB05 and 5’azacytidine
treatments. GAPDH was used as internal control for cytoplasmic protein expression.

55

Figure 30. Western blot performed on cytoplasmic protein fractions of U937 cell
line showed that baseline level of SOX-9 was lower which may explain its susceptibility
to XB05. SOX9 expression increased at 96 hour following treatment with 5’azacytidine.
There was no expression of p16 or p21 noticed in non-treated and treated cells.
Expression of alpha tubulin decreased at 48hours following treatment with XB05 and
both at 48 and 96 hours following treatment with 5’azacytidine. Expression of beta-actin
did not change following treatment with XB05; however, it first increased at 48 hours
and then decreased after 96 hour treatment with 5’azacytidine. GAPDH was used as
internal control for cytoplasmic protein expression.

*Previous studies performed in our laboratory have demonstrated that when
DMSO (vehicle for XB05) is used at high concentration alone, it does not affect
the expression of various cellular proteins.[29]

56

Immunohistochemistry (IHC):
*Despite extreme care, the slides accumulated bubbles. This is likely related the
mounting media we used for these slides.
Kasumi cell line:

Figure 31. Power 40x. DNMT1 staining for Kasumi – cell line; NT (A), AZA 48hrs (B)
and AZA 96hrs (C). IHC staining of non-treated and 5’AZA treated cells at 48hrs and
96hrs did not show change in nuclear expression of DNMT1.

Figure 32. Power 40x. DNMT1 staining for Kasumi – cell line; NT (A), XB05 48hrs
(B) and XB05 96hrs (C). IHC staining of non-treated and 5’AZA treated cells at 48hrs
and 96hrs did not show change in nuclear expression of DNMT1.

57

Figure 33. Power 40x. HIC1 staining for Kasumi – cell line; NT (A), AZA 48hrs (B)
and AZA 96hrs (C). Non-treated Kasumi-1 cells did not show any positive staining for
HIC1. Treatment with 5’azacytidine at 48 hours did not result in re-expression of HIC1;
however, at 96 hours, slightly increased expression is noted mostly in the cytoplasm of
the cells.

Figure 34. Power 40x. HIC1 staining for Kasumi – cell line; NT (A), XB05 48hrs (B)
and XB05 96hrs (C). Non-treated Kasumi-1 cells did not show any positive staining for
HIC1. Treatment with XB05 at 48- and 96-hours did not result in re-expression of HIC1.

58

U937 cell line:

Figure 35. Power 40x. DNMT1 staining for U937 – cell line; NT (A), AZA 48hrs (B)
and AZA 96hrs (C). Non-treated U937 cells showed nuclear expression of DNMT1 on
IHC staining. The expression of DNMT1 was lost following treatment with 5’azacytidine
both at 48 and 96 hours.

Figure 36. Power 40x. DNMT1 staining for U937 – cell line; NT (A), XB05 48hrs (B)
and XB05 96hrs (C). Non-treated U937 cells showed nuclear expression of DNMT1 on
IHC staining. The expression of DNMT1 was lost following treatment with XB05 both at
48 and 96 hours.

59

Figure 37. Power 40x. HIC1 staining for U937 – cell line; NT (A), AZA 48hrs (B) and
AZA 96hrs (C). Non-treated U937 cells showed no nuclear expression of HIC1 on IHC
staining. At 48 hours, treatment with 5’azacytidine did not result in re-expression of
HIC1; however, at 96 hours, strong expression of HIC1 is seen.

Figure 38. Power 40x. HIC1 staining for U937 – cell line; NT (A), XB05 48hrs (B) and
XB05 96hrs (C). Non-treated U937 cells showed no nuclear expression of HIC1 on IHC
staining. At 48 hours, treatment with XB05 did not result in re-expression of HIC1;
however, at 96 hours, strong expression of HIC1 is seen.

60

Discussion
AML is a clonal stem cell malignancy resulting from proliferation and
accumulation of immature hematopoietic cells in the bone marrow, peripheral blood and
other tissues. Current therapeutic options are highly toxic and very poorly tolerated in
elderly patients. Recently, FDA approved decytabine (a DNA hypomethylating agent) for
AML treatment in elderly patients. It was shown to provide complete response in 47
percent patients with excellent tolerability in this patient population.
We recently discovered a novel small molecule XB05 which on NCI-60 screen
was shown to be cytotoxic in certain cell lines while not in others. We have performed
preliminary studies in out laboratory demonstrating the in-vitro efficacy of XB05 in
certain cell lines. Expanding further on our current knowledge of the mechanisms of
action of XB05, we performed in-vitro studies to demonstrate its efficacy in AML cell
lines.
MTT-assays were performed on two AML cell lines (Kasumi-1 and U937) as
described in the methods and material section. Preliminary data form MTT-assays in
comparison with a known DNA-hypomethylating agent, 5’azacytidine showed promising
results. Results from three MTT-assays performed on Kasumi-1 cell lines following
treatment with XB05 and 5’azacytidine in serial concentrations were combined. XB05
demonstrated decrease in percent cell proliferation with increasing concentration
reaching IC50 of 1345.8nM based on calculations done with CompuSyn software.
5’Azacytidine which is a known nucleoside analogue and DNMT-hypomethylating agent
similarly showed declining percent cell proliferation with increasing concentration
61

reaching IC50 of 932.2nM. This clearly demonstrates the cytotoxicity of XB05 relative to
5’azacytidine and shows that IC50 for XB05 is very comparable to 5’azacytidine. Further
MTT-assays were performed where the concentration of XB05 was kept constant and
serial 5’azacytidine was added to the wells. Results of two MTT-assays performed
keeping the Xb05 drug concentration constant were combined. The results from these
MTT-assays demonstrated that XB05 further enhances the cytotoxicity of 5’azacytidine,
thereby lowering the IC50 for the drug. This finding may have significant implications for
the further development of the drug as it may be combined with 5’azacytidine in the
treatment of AML and help reduce treatment associated with side-effects.
Similarly designed MTT-assays were performed on U937 cell line. The results of
the comparative MTT-assays (assays where XB05 and 5’azacytidine cytotoxicity is
compared) showed that XB05 was more cytotoxic than 5’azacytidine. The IC50 for XB05
was 463.3nM and for 5’azacytidine was 633.5nM. MTT-assays performed while keeping
the XB05 concentration constant and using serial concentrations of 5’azacytidine (refer
to methods and materials for assay design) showed that XB05 sensitized the cells to the
cytotoxic effects of 5’azacytidine and thereby further enhancing its cytotoxicity. As
mentioned above for Kasumi-1 cell line, these results again demonstrate that further
development of XB05 is needed especially for combinatorial regimens where adverse
effects related to established cytotoxic agents can be decreased without sacrificing the
efficacy.
Analysis of gene expression following treatment with XB05 and 5’azacytidine at
48 and 96 hours was done with PCR. In the Kasumi-1 cell line, following treatment with
62

XB05 at 48 hours, expression of DNMT1, 3a and 3b decreased relative to non-treated
samples. We had expected XB05 not to affect the transcription of DNMTs as it is
thought to be inhibitory to the DNMT enzymes, thus reducing the methylation of the
promoter sequences of commonly suppressed genes in AML. Decreased expression of
DNMTs following treatment with XB05 may be the result of off-target effect of XB05
where it is likely inhibiting the transcription of different genes through a yet unknown
mechanism. Expression of genes methylated in AML also slightly decreased in
comparison to the untreated samples. Again this may be related to across the board
inhibition of gene transcription machinery. However, the results from PCR following 96
hour treatment with XB05 were completely opposite. Expression of DNMT3a and 3b
decreased, but the expression of all the other genes increased significantly. Again, we
don’t expect XB05 to inhibit or increase the transcription of DNMTs; however, increased
expression of commonly suppressed genes in AML does demonstrate that XB05 is
likely inhibiting the DNMT enzymes at the protein level and thus causing the reexpression of genes.
Following treatment with 5’azacytidine in the Kasumi-1 cell line at 48 hours, we
saw increased expression of DNMT1 and decreased expression of DNMT3a and 3b
relative to the non-treated samples. The expression of HIC1 also decreased; however,
the expression of rest of the genes increased following the treatment. 5’Azacytidine
definitely can cause decreased expression of certain genes directly as it also a
nucleoside analogue. Re-expression of commonly methylated genes in AML
demonstrates that 5’azacytidine can also inhibit the function of DNMTs. At 96 hours,
expression of DNMT3a and 3b is decreased significantly relative to the non-treated
63

samples. Expression of HIC1, DAPK1, MGMT and RAR-beta also decreased.
Expression of DNMT1, ER-alpha and p16 increased. This likely demonstrates that at 48
hours, 5’azacytidine is mostly a DNA-hypomethylating agent; however, at 96 hours, it
suppresses the transcription of various genes secondary to its anti-nucleoside
properties.
PCR analysis done on U937 cell line following treatment with XB05 for 48
hours showed that the expression of all the genes decreased relative to non-treated
samples. This effect as in the Kasumi-1 cell line may the result of off-target effect of
XB05 where it is inhibiting the expression of genes. However, at the 96 hours, it was
seen that the expression of DNMT3a continued to be suppressed, but expression of all
the other genes increased relative to the non-treated samples. This likely reflects the
DNA-hypomethylating effect of XB05 where there is increased expression of the
previously suppressed genes. Why the expression of DNMT3a continued to be
suppressed cannot be explained based on the experiments done so far but can be
hypothesized to be again the result of off-target effect of XB05.
Expression of all the genes except p16 increased in the U937 cell line following
treatment with 5’azacytidine at 48 hours. This could be the result of differential
hypomethylation of DNA following treatment with 5’azacytidine. However, at 96 hours,
expression of DNMT3a was suppressed but the expression of the other genes
increased relative to the non-treated samples. This effect can be explained partially
related to the hypomethylation and partially to anti-nucleoside effects of 5’azacytidine.

64

Results from EpiTect Methyl II PCR assay showed that XB05 affects the
promoter region methylation of both oncogenes and tumor suppressor genes. However,
demethylation of promoter regions in oncogenes was not as robust as noticed in tumor
suppressor genes. Increasing the methylation of promoters of oncogenes will result in
decreased expression of oncogenes and decreased methylation of tumor suppressor
genes will result in their increased expression and in essence inhibiting tumor growth.
Following treatment with 5’AZA, promoter region methylation decreased in most
oncogenes and tumor suppressors at the same level. This shows that 5’AZA is not
selective in targeting promoter region methylation in tumor suppressor genes and can
result in increased expression of oncogenes through demethylation of their promoter
regions. XB05 showed better targeting of promoter region methylation in tumor
suppressor genes as opposed to similar regions in oncogenes. This observation is quite
striking and provides additional evidence to further support the development of XB05 for
clinical use.
Western blot experiments were performed to further understand the mechanism
of action of XB05 at protein level. Nuclear and cytoplasmic fractions were separated as
per protocol described in the appendix. 30ug of protein was loaded for both the nuclear
and cytoplasmic fractions. Both fractions were pure with R2 of over 95% in reference to
BSA standards.
Western blot was performed for nuclear fractions extracted from Kasumi-1 cell
line following treatment with XB05 and 5’azacytidine at 48- and 96-hours. Following
treatment with XB05, both at 48- and 96-hours, the expression of DNMT1 decreased
65

although more pronounced at 48-hours. There was no change in DNMT1 expression
noted following treatment with 5’azacytidine. Expression of DNMT3a and 3b was low to
absent in the non-treated cell line. No change in the level of HIC1 or MGMT was noted
following treatment with both drugs at both time intervals. Level of ER-alpha expression
increased at 48 hours following treatment with both XB05 and 5’azacytidine. Western
blot performed on the cytoplasmic fraction showed that levels of beta-actin and p16
increased slightly following treatment with XB05. Increase in their level was more
following treatment with 5’azacytidine. No increased expression of other protein was
noted following treatment with XB05. These results to some extent demonstrate that
treatment with XB05 can suppress the activity of DNMTs and cause re-expression of
genes methylated in AML. The results of western blot were comparable for both the
drugs which substantiates that XB05 can be developed as an effective adjunct to
currently existing treatment regimens and thus decrease the treatment associated sideeffects.
In the U937 cell line, contrary to our expectation, we did not see the decrease in
the level of DNMTs following treatment with XB05. Instead, levels increased following
treatment. At the same time, increase in the level of HIC1 and ER-alpha was noted.
Treatment with 5’azacytidine did not result in increased level of proteins such as HIC.
ER-alpha or MGMT. DNMT1 level had decreased at 48 hours. Treatment with XB05 did
not result in significant change in the levels of cytoplasmic proteins evaluated.
Level of SOX9 protein was also interrogated with western blot in both cell lines.
U937 cell line had slightly lower level of SOX9 expression in the non-treated samples
66

which may be associated with its increased susceptibility to XB05 cytotoxicity. We did
not see a similar correlation with baseline SOX9 expression in Kasumi-1 cell line and its
susceptibility to XB05.
On IHC, slight re-expression of HIC1 was noted in the Kasumi-1 cell line
following treatment with 5’azacytidine at 48 hours but not at 96 hours. No baseline
increased expression of DNMT1 was noted and no change in expression was noted
following treatment with XB05 or 5’azacytidine. U937 cells did show increased nuclear
expression of DNMT1 at baseline which was lost following treatment with both XB05
and 5’azacytidine. Also, as we had expected the HIC1 was re-expressed following
treatment with XB05 and 5’azacytidine at 96 hours.
Validation in vitro experiments performed evaluating the efficacy a novel small
molecule XB05 were described in this thesis paper. Cytotoxic effects of XB05 were
clearly demonstrated in both cell lines and the cytotoxicity was comparable to the known
DNA-hypomethylating agent 5’azacytidine. Also, when used in combination, XB05
increased the cytotoxicity of 5’azacytidine. The ability to lower the dose of a cytotoxic
agent without losing the efficacy is particularly important from clinical perspective. This
observation substantiates our belief that further development of XB05 would help in
combinatorial therapy approaches thus reducing treatment related side-effects. Also, in
terms of inducing the expression of previously suppressed genes in AML, XB05 fared
well and its effects at mRNA and protein level were again comparable to 5’azacytidine.
Some of the results of the experiments performed did not turn out to be as
expected. This may entirely be secondary to technical issues or related to poor quality
67

of some of the antibodies used especially for western blots and IHC. Further repetition
of these experiments may be needed to optimize the conditions and hopefully, results
will be more consistent and reflective of hypothesized mechanism of action of XB05.
Also, other gene targets and proteins may need to be evaluated. Regardless, these
experiments demonstrate that XB05 has cytotoxic effects in AML and further
development of this agent may be needed especially from the perspective of
combinatorial treatment approaches and reduction of adverse effects.
Conclusions
Management of AML in elderly is challenging with limited treatment options which are
poorly tolerated. DNA-hypomethylating agents have been successfully employed in the
treatment of this patient subset. XB05 is a novel synthetic molecule which is selectively
toxic to malignant cells. In-vitro studies have demonstrated that it is active in leukemia &
suppresses the expression of DNMTs resulting in re-expression of tumor suppressor
genes. Further studies to elucidate the mechanisms of action of this novel agent may be
needed for eventual development for clinical use.

68

APPENDIX

69

MTT-Assay
XBO Drug Dilutions:
Weigh 1.5ml Eppendorf tube: x gm
Weight of the tube with 2uL of XBO5: y gm
Weight of the drug: y – x = z gm
Then add 250uL of DMSO (Dimethyl sulfoxide) to each mg of XBO5, which will
give 15.7mM of XBO5.
Further make a 10,000ul stock of 1mM of XBO5 from 15.7mM in 100% DMSO
M1 * V1 = M2 * V2
1mM * 10,000µl = 15.7mM * V2
Vol = 636.9ul
=> 636.9ul of 15.7mM XBO5 + 9363.1ul of 100% DMSO = 10,000ul of 1mM
of XBO5
In order to make XBO5 more soluble in RPMI, make further dilutions each time
prior to using the drug for MTT assay or treatment in the flask.
Make 100uM XBO5:
M1 * V1 = M2 * V2
100uM * 2000uL = 1000uM * V2
V2 = 200uL
=> 200uL of 1mM of XBO5 + 1800uL of RPMI = 2000uL of 100uM of XBO5
Now make another dilution of 10uM
M1 * V1 = M2 * V2
70

10uM * 5000uL = 100uM * V2
V2 = 500uL
=> 500uL of 100uM of XBO5 + 4500uL of RPMI = 5000uL of 10uM of XBO5
Now make further dilutions for treatment in wells. Make three times concentrated
drug dilutions for desired final concentration of the drug in each well. Add 50uL of
drug to 100uL of media in wells to get desired concentration:

For Kasumi 1 cell line ->
Make 150nM for 50nM in well
300nM for 100nM in well
600nM for 200nM in well
1200nM for 400nM in well
1800nM for 600nM in well
2400nM for 800nM in well
3000nM for 1000nM in well
3600nM for 1200nM in well
4800nM for 1600nM in well
Table 11. Serial dilutions of XB05 for treatment of Kasumi-1 cell line.

71

Drug
Stock
Concentration
(nM)

Stock Volume
(uL)

RPMI volume
(uL)

Total Volume
(uL)

4800

10uM
(10,000nM)

720

780

1500

3600

10uM
(10,000nM)

540

960

1500

3000

10uM
(10,000nM)

450

1050

1500

2400

4800nM

750

750

1500

1800

3600nM

750

750

1500

1200

2400nM

750

750

1500

600

1200nM

750

750

1500

300

600nM

750

750

1500

150

300nM

750

750

1500

Table 12. Calculations for serial XB05 concentrations to be used for treatment of
Kasumi-1 cell line.

For U937 cell line ->
Make 150nM for 50nM in well
300nM for 100nM in well
600nM for 200nM in well
1200nM for 400nM in well
1800nM for 600nM in well
2400nM for 800nM in well
3000nM for 1000nM in well
Table 13. Serial dilutions of XB05 for treatment of U937 cell line.

72

Drug
Concentration
(nM)

Stock

Stock Volume
(uL)

RPMI volume
(uL)

Total Volume
(uL)

3000

10uM

450

1050

1500

2400

10uM
(10,000nM)

360

1140

1500

1800

10uM
(10,000nM)

270

1230

1500

1200

2400nM

750

750

1500

600

1200nM

750

750

1500

300

600nM

750

750

1500

150

300nM

750

750

1500

Table 14. Calculations for serial XB05 concentrations to be used for treatment of
U937 cell line.
DMSO will be used as the control. Concentration of DMSO in the control wells
will correspond to the highest drug concentration used in the experiment to
ensure that the cells are not being directly killed by the vehicle (DMSO).
First, 10% DMSO was made from 100% DMSO following the dilutions used for
XBO5.
= 200uL 100% DMSO + 1800uL RPMI
Then, 1% DMSO was made from 10% DMSO again following the XBO5 dilutions.
= 500uL 10% DMSO + 4500uL of RPMI
Then for Kasumi-1 MTT assay;
Dilute 720uL of 1% DMSO in 780uL of RPMI
For U937 MTT-assay;
Dilute 450uL of 1% DMSO in 1050uL of RPMI
73

5’Azacytadine Drug Dilutions:
Stock concentration 5’AZA = 500uM in RPMI
Make working concentration of 10uM from 500uM stock to use for further drug
dilutions to be used in MTT assays.
M1 * V1 = M2 * V2
10uM * 5000uL = 500uM * V2
V2 = 100uL
100uL of 500uM of 5’AZA + 4900uL RPMI = 10uM of 5’AZA
Make further dilutions as described above for XBO5.
Control for 5’azacytadine will be RPMI.
For combination drug treatments, XB05 concentration was kept constant and
serial concentrations of azacytadine were used. Six time concentrated drug was
added to each well in the volume of 25uL for a total volume of 150uL after
addition of both drugs.
XB05 and azacytadine drug concentrations for Kasumi 1 and U937 cell
lines using working drug concentration of 10uM->
Make 300nM for 50nM in well
600nM for 100nM in well
1200nM for 200nM in well
2400nM for 400nM in well
4800nM for 800nM in well
6000nM for 1000nM in well
Table 15. Serial dilutions of azacytadine for treatment of Kasumi-1 and U937.

74

Drug
Concentration
(nM)

Stock

Stock Volume
(uL)

RPMI volume
(uL)

Total Volume
(uL)

6000

10uM
(10,000nM)

6000

4000

10,000

4800

10uM
(10,000nM)

7200

7800

15,000

2400

4800nM

7500

7500

15,000

1200

2400nM

7500

7500

15,000

600

1200nM

7500

7500

15,000

300

600nM

7500

7500

15,000

Table 16. Calculations for serial XB05 concentrations to be used for treatment of
U937 cell line.

Figure 39. Outline of the plan for MTT-assays.

75

1 (0nM)

2 (50nM)

8
3 (100nM) 4 (200nM) 5 (400nM) 6 (600nM) 7 (800nM) (1000nM)

9
(1200nM)

10
(1600nM)

11

12

A (XBO5)

B (XBO5)

C (XBO5)

D (XBO5)

E (5'AZA)

F (5'AZA)

G (5'AZA)

Blank

H (5'AZA)

Figure 40. Plate Design for Kasumi-1 MTT experiment.

1 (0nM)

2 (50nM)

8
3 (100nM) 4 (200nM) 5 (400nM) 6 (600nM) 7 (800nM) (1000nM)

9

10

11

12

A (XBO5)

B (XBO5)

C (XBO5)

D (XBO5)

E (5'AZA)

F (5'AZA)

G (5'AZA)

Blank

H (5'AZA)

Figure 41. Plate Design for U937 MTT experiment.

76

Figure 42. Plate design for drug combination experiments.
XB05 concentration was increased accordingly in the other plates to maximum
concentration of 800nM.

RNA Extraction Protocol – TRIzol® Reagent:
TRIzol® Reagent is a ready-to-use reagent, designed to isolate high quality total
RNA (as well as DNA and proteins) from cell and tissue samples within one hour.
It is a monophasic solution of phenol, guanidine isothiocyanate, and other
proprietary components which facilitate the isolation of a variety of RNA species
of large or small molecular size. It maintains the integrity of the RNA due to
highly effective inhibition of RNase activity while disrupting cells and dissolving
cell components during sample homogenization.

77

• Chloroform
• Isopropyl alcohol
• 75% ethanol (in DEPC-treated water)
• RNase-free water or 0.5% SDS
• Centrifuge and rotor capable of reaching up to 12,000 × g
• Polypropylene microcentrifuge tubes
• Water bath or heat block (55–60°C)
Table 17. Materials needed for RNA isolation.

Sample preparation – suspension cells:
1. Harvest cells by centrifugation and remove media.
2. Add 0.75 mL of TRIzol® Reagent per 0.25 mL of sample. Do not wash cells
before addition of TRIzol® Reagent to avoid increased chance of mRNA
degradation.
3. Lyse cells in sample by pipetting up and down several times.
Phase separation
1. Incubate the homogenized sample for 5 minutes at room temperature to permit
complete dissociation of the nucleoprotein complex.
2. Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent used for
homogenization. Cap the tube securely.
3. Shake tube vigorously by hand for 15 seconds.
4. Incubate for 2–3 minutes at room temperature.
5. Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.

78

Note: The mixture separates into a lower red phenol-chloroform phase, an
interphase, and a colorless upper aqueous phase. RNA remains exclusively in
the aqueous phase. The upper aqueous phase is ~50% of the total volume.
6. Remove the aqueous phase of the sample by angling the tube at 45° and
pipetting the solution out. Avoid drawing any of the interphase or organic layer
into the pipette when removing the aqueous phase.
7. Place the aqueous phase into a new tube and proceed to the RNA Isolation
Procedure.
RNA Isolation Procedure
RNA precipitation
1. Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol®
Reagent used for homogenization.
2. Incubate at room temperature for 10 minutes.
3. Centrifuge at 12,000 × g for 10 minutes at 4°C.
4. Proceed to RNA wash.
RNA wash
1. Remove the supernatant from the tube, leaving only the RNA pellet.
2. Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol® Reagent used
in the initial homogenization.
3. Vortex the sample briefly, then centrifuge the tube at 7500 × g for 5 minutes at
4°C. Discard the wash.
4. Vacuum or air dry the RNA pellet for 5–10 minutes.
5. Proceed to RNA resuspension.
79

RNA resuspension
1. Resuspend the RNA pellet in RNase-free water by passing the solution up and
down several times through a pipette tip.
2. Incubate in a water bath

Figure 43. Outline of experiments for RNA extraction, cDNA and RT-PCR.

80

High Capacity RNA-to-cDNA Kit

PCR INSTRUMENT: Applied Biosystems 9800 Fast Thermal Cycler with 96-Well
Aluminum Sample Block Module

Table 18. Kit components for high capacity RNA-to-cDNA PCR.

Figure 44. General overview of RT-PCR experiment.
81

Table 19. Volumes for each components of RT-PCR reaction.

Table 20. Time settings for three steps of RT-PCR reaction.

Fast SYBR® Green Master Mix Protocol
Fast SYBR® Green Master Mix, supplied in a 2✕ concentration, is a convenient
premix to perform real-time PCR using SYBR® Green I dye.
• SYBR® Green I Dye
• AmpliTaq® Fast DNA Polymerase, UP (Ultra Pure)
• Uracil-DNA Glycosylase (UDG)
• ROX™ dye Passive Reference
• dNTPs
• Optimized buffer components
Table 21. Components of SYBR Green master mix.

82

Primers, template, and water are the only components needed by the
investigator.
INSTRUMENT NEEDED
• Applied Biosystems 7500 Fast Real-Time PCR System
Principle:
The SYBR Green I dye chemistry uses the SYBR Green I dye to detect PCR
products by binding to double-stranded DNA formed during PCR.
The process works as follows:
1. When Fast SYBR® Green Master Mix is added to a sample, SYBR Green I
dye immediately binds to all double-stranded DNA.
2. During the PCR, AmpliTaq® Fast DNA Polymerase, UP amplifies the target
sequence, which creates the PCR product, or “amplicon.”
3. The SYBR Green I dye then binds to each new copy of double stranded DNA.
4. As the PCR progresses, more amplicon is created. Because the SYBR Green
I dye binds to all double-stranded DNA, the result is an increase in fluorescence
intensity proportional to the amount of double-stranded PCR product produced.

Figure 45. Plate design for qRT-PCR reaction for the study.
83

Figure 46. Plate design for qRT-PCR reaction for the study.

Component

Volume for One 20-µL Reaction (µL)

Fast SYBR® Green
Master Mix (2✕
✕)

10.0

Forward and Reverse
Primers

Variable

cDNA template + RNasefree water

Variable

Total Volume

20.0

Table 22. Volumes for components of SYBR Green qRT-PCR reaction.

84

Figure 47. Overview of the qRT-PCR reaction for the study.

Table 23. Time settings for the three steps of qRT-PCR reaction.

85

EpiTect Methyl II PCR (adapted from Qiagen EpiTect Methyl II PCR Handbook)

Figure 48. EpiTect Methyll II PCR Procedure overview.

86

Procedure overview:
The input genomic DNA is aliquoted into four equal portions and subjected to
mock (no enzyme), methylation-sensitive (MSRE), methylation-dependent
(MDRE), and double (MSRE and MDRE) restriction endonuclease digestion.
After digestion, the enzyme reactions are mixed directly with qPCR master mix
and are dispensed into a PCR Array plate containing pre-aliquoted primer mixes.
RT-PCR is carried out using cycling conditions provided by the manufacturer.
The data obtained (∆CT values) are exported into the data analysis spreadsheet
provided by the manufacturer which automatically calculates the relative amount
of methylated and unmethylated DNA fractions.
The product of the mock (no enzyme) digestion represents the total amount of
input DNA. In the methylation-sensitive digestion (Ms) reaction, the MSRE will
digest unmethylated and partially methylated DNA. The remaining
hypermethylated DNA — DNA in which all CpG sites are methylated — will be
detected by real-time PCR. In the methylation-dependent digestion (Md) reaction,
the MDRE will preferentially digest methylated DNA. The remaining
unmethylated DNA will be detected by real-time PCR. In the double digestion
(Msd) reaction, both enzymes are present, and all DNA molecules (both
methylated and unmethylated) will be digested. This reaction measures the
background and the fraction of input DNA refractory to enzyme digestion.

87

The assay provides gene methylation status as percentage unmethylated (UM)
and percentage methylated (M) fraction of input DNA. Unmethylated represents
the fraction of input genomic DNA containing no methylated CpG sites in the
amplified region of a gene. Methylated represents fraction of input genomic DNA
containing two or more methylated CpG sites in the targeted region of a gene.
Each well of EpiTect Methyl II PCR Array plate contains a different primer assay
mixed with an inert dye. This dye does not affect assay performance or
fluorescence detection.
EXPERIMENT:
Restriction digestion
-

Restriction enzyme digestions are performed using the EpiTect Methyl II
DNA Restriction Kit (cat. no. 335452).

-

Reaction mix without enzymes as below:

Table 24. Volume for components of EpiTect Methyl II PCR reaction.

88

-

RNase-/DNase-free water is added to make the final volume 120 µl.
Vortex and centrifuge the components.

-

Four enzyme digestion reactions are set up as below and should contain
equal amounts of genomic DNA – MMo, Ms, Md, and Msd.

Table 25. Volumes for four reactions in EpiTect Methyl II PCR assay.
-

Mix the reaction components thoroughly by pipetting up and down to
gently but do not vortex.

-

All four tubes are incubated at 37°C for 6 h in a heating block or thermal
cycler.

-

Stop the reaction after incubation by heat-inactivating the enzymes at
65°C for 20 mins.

PCR
-

PCR reaction mix for for each of the 4 digestions (Mo, Ms, Md, and Msd)
is prepared in a 1.5 ml tube as below:
89

Table 26. Final reaction volume for four reactions in EpiTect Methyl II PCR assay.
-

Vortex and centrifuge the contents to the bottom of the tube.

-

Add 25 µl of the Mo reaction to each well in rows A and B of the 96-well
EpiTect Methyl II Signature PCR Array. Add 25 µl of the Ms reaction to
each well in rows C and D. Add 25 µl of the Md reaction to each well in
rows E and F. Finally, add 25 µl of the Msd reaction to each well in rows G
and H.

Figure 49. Plate design for EpiTect Methyl II PCR assay.
90

-

Seal the wells of the plate and centrifuge the plate for 1 min at 2000 rpm
to remove any air bubbles.

-

Program the thermal cycler as per manufacturer’s instructions described
below:

Table 27. Time settings for EpiTect Methyl II PCR assay.
Coomassie (Bradford) Protein Assay Kit
Bradford protein assay is a quick and ready-to-use modification of the
Bradford coomassie-binding, colorimetric method for total protein
quantitation. When coomassie dye binds protein in an acidic medium, an
immediate shift in absorption maximum occurs from 465nm to 595nm
with a concomitant color change from brown to blue.
Standard Microplate Protocol
1. Pipette 5µL of each standard or unknown sample into 96-well
microplate.

91

2. Add 250µL of the Coomassie Reagent to each well and mix with plate
shaker for 30 seconds.
3. Remove plate from shaker and incubate plate for 10 minutes at room
temperature (RT).
4. Measure the absorbance at 595nm with a plate reader.
5. Prepare a standard curve by plotting the average Blank-corrected
595nm measurement for each BSA standard vs. its concentration in
µg/mL. Use the standard curve to determine the protein concentration of
each unknown sample.

Table 28. Plate design for Coomassie assay and BSA serial dilutions.

92

Figure 50. Plate design for Coomassie assay.
Western Blot
Protocol for treatment of cells in flasks, protein extraction and western blot:
Step 1: Plate ~100,000 cells per ml or 1,500,000 cells per flask in a T-150 flask
(total volume of media = 15ml) on day one.
Step 2: On day two, add XBO5 and 5’AZA at IC50 concentrations to the individual
flasks to get desired molar concentration.
Step 3: Incubate for three days and extract the cell pellet by centrifuging at 1000
rpm for five minutes. Repeat the same steps for untreated cells.
Step 4: Extract nuclear and cellular proteins using protocol separately.
Step 5: Add different concentrations of the extracted proteins to commercially
available 10-well Bio-Rad (4-20%) SDS-PAGE gel and run at 100volts for 1 hour.

93

Step 6: Transfer proteins from gel to the PVDF membrane using XCell II™ Blot
Module (Invitrogen®) and transfer media at 100mA for 2 hours. Activate the
membrane with methanol for 30 secs.
Step 7: After transfer, block the membrane with 5% milk at 4ºC overnight.
Step 8: Wash the membrane with PBS-T for five minutes times three.
Step 9: Incubate the membrane with commercially available primary antibody at
1:500 at 4*C overnight.
Step 10: Wash with PBS-T for five minutes times three.
Step 11: Incubate the membrane with commercially available secondary antibody
at 1:1000 for 1 hour at room temperature.
Step 12: Wash with PBS-T every five minutes times three.
Step 13: Using ECL Western Blot detection kit and darkroom development
techniques, develop film to detect and quantify the expression of these proteins
in untreated and treated cells.
Protein Extraction Protocol:
Wash pellet twice in cold 5ml of 1x PBS
Add 100-500µl of ice-cold extraction buffer B to the pellet.
Transfer contents to Eppendorf tubes.
Perform three cycles of freeze-thaw. Freeze the tubes at -80ºC for five
minutes and thaw at room temperature (37ºC).
Recover cytoplasmic extracts (supernatant) by centrifuging at 12,000rpm
for one minute.
Resuspend pellets in 40-60µl of buffer C.
94

Incubate at 40ºC for 30 minutes.
Recover nuclear extracts (supernatant) by centrifuging at 12,000rpm for
five minutes.

Figure 51. Overview of western blot experiment for the study.

95

Content

Volume

Molar
concentration

1M Hepes (pH 7.9)

100µl

10nM

1M MgCl2

15µl

1.5nM

2M KCl

50µl

10mM

Leupeptin (1mg/ml)

10µl

1µg/ml

Aprotinin (5mg/ml)

2µl

1µg/ml

100mM PMSF
(phenylmethanesulfonylfluoride or
phenylmethylsulfonyl fluoride)

100µl

1mM

H2O

9723µl

Total

10,000µL

Table 29. Components of Buffer B.
Content

Volume

Molar concentration

1M Hepes (pH 7.9)

200µl

20nM

1M MgCl2

15µl

1.5nM

2M KCl

2100µl

420mM

Leupeptin (1mg/ml)

10µl

1µg/ml

Aprotinin (5mg/ml)

2µl

1µg/ml

EDTA

4µl

0.2mM

Glyceral

2500µl

25%

100mM PMSF
100µl
(phenylmethanesulfonylfluoride
or phenylmethylsulfonyl
fluoride)
H2O

5069µl

Total

10,000µl

Table 30. Components of Buffer C.

96

1mM

Primary Product
Antibody Number

Idiotype

Dilution

Company

Experime
nt

Histone
H3

(FL-136): sc10809

Rabbit
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

GAPDH

(G-9): sc365062

Mouse
monoclonal
IgG1

1:500

Santa Cruz
Biotech

WB

MGMT

(C-20): sc8825

Goat
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

DNMT1

(C-17): sc10222

Goat
polyclonal IgG

1:500 for
WB &
1:25 for
IHC

Santa Cruz
Biotech

WB & IHC

DNMT3a

(H-295): sc20703

Rabbit
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

DNMT3b

(T-16): sc10236

Goat
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

HIC1

(H-6): sc271499

Mouse
monoclonal
IgG2b

1:500 for
WB &
1:25 for
IHC

Santa Cruz
Biotech

WB & IHC

SOX9

(H-90): sc20095

Rabbit
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

Betaactin

A3853

Moue
monoclonal
IgG

1:500

Sigma Aldrich WB

ERAlpha

ab2746

Mouse
monoclonal
IgG

1:100

Abcam

WB

P16INK4A

P16D25

Rabbit
polyclonal

1:500

Cell Signaling

WB

Table 31. List of primary antibodies and dilutions used for Western Blot and
Immunohistochemistry.

97

Table 31. (cont’d)
P21

(0.N.488): sc71811

Mouse
monoclonal
IgG2a

1:500

Santa Cruz
Biotech

WB

α
Tubulin

(H-300): sc5546

Rabbit
polyclonal IgG

1:500

Santa Cruz
Biotech

WB

Primary
Antibody

Product
Number

Idiotype

Dilution

Company

Experiment

Goat anti-rabbit
IgG-HRP

sc-2030

IgG

1:1000

Santa Cruz
Biotech

WB

Goat anti-mouse
IgG-HRP

sc-2031

IgG

1:1000 for
WB & 1:50
for IHC

Santa Cruz
Biotech

WB & IHC

Donkey anti-goat
IgG-HRP

sc-2020

IgG

1:1000 for
WB & 1:50
for IHC

Santa Cruz
Biotech

WB & IHC

Table 32. List of secondary antibodies used for Western Blot and
Immunohistochemistry.

Immunohistochemistry (IHC)
Materials Required
• Wash Buffer (Tris-buffered saline with 0.05% Tween-20 Detergent): Dissolve
contents of one BupH TBS Pack in 500mL of ultrapure water. Add 2.5mL of 10%
Tween-20 Detergent and mix well. Store reconstituted buffer at 4°C.
• Primary Antibody: Dilute antibody with Universal Blocker Blocking Buffer at
1:25.

98

• HRP-conjugated secondary antibody: Dilute antibody with Universal Blocker
Blocking Buffer at 1:50.
• Coverslips
Protocol:
1. Permeabilize cells on slide with 0.05% TritonX.
2. Wash slide two times for 3 minutes with Wash Buffer.
3. Add blocking buffer to the slide and incubate for 30 minutes.
4. Blot excess blocker from the tissue sections. Apply the Primary Antibody and
incubate tissue at 4*C overnight.
5. Wash slide two times for 3 minutes with Wash Buffer.
6. Apply the HRP Secondary Antibody and incubate tissue for 30 minutes.
7. Wash slides three times for 3 minutes each with Wash Buffer.
8. Remove the DAB/Metal Concentrate (10X) from -20°C storage and mix well by
inverting the bottle. Remove quantity required for use and immediately return
bottle to -20°C.
9. Prepare a 1X working solution of the DAB/Metal Concentrate (10X) by adding
the Stable Peroxide Buffer and mixing well.
10. Add the Metal Enhanced DAB Substrate Working Solution to the slide and
incubate for 15mins.
11. Wash slide two times for 3 minutes each with Wash Buffer and counterstain
with hematoxylin.
12. Mount slide with Mounting Media and apply coverslip after washing with
distilled water.
99

Figure 52. Outline of IHC experiment for the study.

100

SUPPLEMENTARY DATA

Results of the first MTT-assays performed in Kasumi-1 cell line following combination
treatment with XB05 and azacytadine are outlined below. XB05 concentration was kept
constant and azacytadine was added in serial concentrations.
XB05 in nM XB05 0
0.00

XB05 100

XB05 100

XB05 100

XB05 100

XB05 100

XB05 100

100.00

200.00

400.00

800.00

1000.00

0.00

100.00

200.00

400.00

800.00

1000.00

0.51

0.55

0.35

0.43

0.36

0.36

0.68

0.51

0.45

0.36

0.35

0.27

0.72

0.66

0.56

0.50

0.40

0.41

0.78

0.57

0.50

0.33

0.39

0.36

0.72

0.58

0.54

0.48

0.41

0.41

0.78

0.57

0.52

0.42

0.42

0.73

0.58

0.55

0.51

0.46

0.40

0.85

0.59

0.54

0.48

0.48

0.68

0.53

0.56

0.51

0.47

0.41

0.84

0.57

0.55

0.46

0.43

0.38

0.68

0.52

0.50

0.42

0.43

0.36

0.73

0.51

0.45

0.41

0.42

0.33

0.67

0.57

0.51

0.47

0.42

0.39

0.78

0.55

0.50

0.41

0.41

0.33

AZA 9nM)->

XB05 0 XB05 0

XB05 0

XB05 0

XB05 0

Table 33. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (0 and 100nM) and serial azacytadine.

XB05 in nM XB05200
0.00

AZA 9nM)->

XB05200 XB05200

XB05 400

XB05 400

XB05 400

XB05 400

XB05 400

XB05 400

100.00

200.00

XB05200
400.00

XB05200
800.00

XB05200
1000.00

0.00

100.00

200.00

400.00

800.00

1000.00

A

0.54

0.50

0.38

0.41

0.36

0.32

0.61

0.51

0.40

0.37

0.36

0.31

B

0.65

0.55

0.44

0.39

0.34

0.34

0.62

0.49

0.39

0.36

0.36

0.39

C

0.63

0.55

0.44

0.41

0.34

0.35

0.58

0.50

0.40

0.40

0.35

D

0.68

0.53

0.46

0.39

0.36

0.34

0.61

0.49

0.40

0.40

0.39

0.44

E

0.62

0.53

0.45

0.39

0.32

0.33

0.58

0.52

0.40

0.39

0.36

0.43

F

0.63

0.55

0.45

0.37

0.40

0.35

0.60

0.48

0.46

0.39

0.40

G

0.62

0.53

0.44

0.40

0.35

0.34

0.60

0.50

0.41

0.38

0.37

Table 34. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine.

101

0.39

XB05 in nM XB05 800
0.00

XB05 800 XB05 800

AZA 9nM)->

XB05 800

XB05 800

100.00

200.00

400.00

800.00

XB05 800
1000.00

XB05 1000
0.00

XB05 1000
100.00

XB05 1000 XB05 1000
200.00

400.00

XB05 1000

XB05 1000

800.00

A

0.50

0.48

0.36

0.33

0.29

0.28

0.56

0.44

0.35

0.33

B

0.57

0.48

0.37

0.29

0.26

0.26

0.53

0.40

0.33

0.28

C

0.52

0.44

0.36

0.32

0.26

0.26

0.50

0.39

0.33

0.28

0.27

D

0.60

0.44

0.36

0.31

0.25

0.26

0.46

0.41

0.33

0.29

0.30

E

0.57

0.47

0.37

0.32

0.27

0.25

0.49

0.43

0.35

0.32

0.31

F

0.57

0.46

0.34

0.32

0.27

0.21

0.51

0.43

0.38

0.34

0.31

G

0.55

0.46

0.36

0.32

0.27

0.25

0.51

0.42

0.35

0.30

0.30

1000.00
0.28
0.30
0.31

0.30

Table 35. Results of the first MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 (800 and 1000nM) and serial azacytadine.

Combined results of two MTT-assays performed in Kasumi-1 cell line following
combination treatment with XB05 and azacytadine are outlined below. XB05
concentration was kept constant and azacytadine was added in serial concentrations.

XB05 in nM XB05 0
AZA in
0.00
nM->

AVG

XB05 0

XB05 0

100.00

0.67
0.29
0.48

0.57
0.23
0.40

XB05 0

200.00

0.51
0.23
0.37

XB05 0

400.00

0.47
0.21
0.34

800.00

0.42
0.20
0.31

XB05 100

XB05 100

XB05 100

XB05 100

XB05 100

0.00

100.00

200.00

400.00

800.00

0.78
0.26
0.52

0.55
0.25
0.40

0.50
0.23
0.36

0.41
0.20
0.31

0.41
0.18
0.30

Table 36. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (0 and 100nM) and serial azacytadine.

XB05 in nM XB05 200 XB05 200 XB05 200 XB05 200 XB05 200 XB05 400 XB05 400 XB05 400 XB05 400 XB05 400
AZA in
0.00
100.00
200.00
400.00
800.00
0.00
100.00
200.00
400.00
800.00
nM->
0.62
0.53
0.44
0.40
0.35
0.60
0.50
0.41
0.38
0.37
0.26
0.24
0.23
0.21
0.18
0.26
0.24
0.24
0.21
0.19
AVG
0.44
0.39
0.33
0.30
0.27
0.43
0.37
0.32
0.30
0.28
Table 37. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (200 and 400nM) and serial azacytadine.

102

XB05 in nM XB05 800 XB05 800 XB05 800 XB05 800 XB05 800
AZA in
0.00
100.00
200.00
400.00
800.00
nM->
0.55
0.46
0.36
0.32
0.27
0.26
0.24
0.20
0.20
0.17
AVG
0.41
0.35
0.28
0.26
0.22
Table 38. Combined results of two MTT-assays performed in Kasumi-1 cell line
following combination treatment with XB05 (800nM) and serial azacytadine.

Results of the first MTT-assays performed in U937 cell line following combination
treatment with XB05 and azacytadine are outlined below. XB05 concentration was kept
constant and azacytadine was added in serial concentrations.
XB05 in nM

AZA nM ->

AVG

XBO5 0

XBO5 0

XBO5 0

XBO5 0

XBO5 0

XBO5 0

XBO5 50 XBO5 50 XBO5 50 XBO5 50 XBO5 50 XBO5 50

0.00

50.00

100.00

200.00

400.00

800.00

0.00

50.00

100.00

200.00

400.00

800.00

0.41

0.33

0.29

0.29

0.24

0.23

0.35

0.31

0.29

0.26

0.26

0.26

0.39

0.31

0.33

0.28

0.27

0.20

0.37

0.32

0.30

0.27

0.25

0.22

0.38

0.36

0.31

0.28

0.26

0.23

0.39

0.36

0.31

0.32

0.24

0.23

0.37

0.32

0.32

0.25

0.27

0.23

0.39

0.35

0.35

0.32

0.26

0.26

0.38

0.34

0.32

0.30

0.26

0.22

0.35

0.32

0.32

0.30

0.28

0.26

0.37

0.31

0.28

0.25

0.25

0.21

0.33

0.29

0.29

0.28

0.24

0.23

0.38

0.33

0.31

0.28

0.26

0.22

0.36

0.33

0.31

0.29

0.25

0.24

Table 39. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (0 and 50nM) and serial azacytadine.

XB05 in nM XBO5 100 XBO5 100 XBO5 100 XBO5 100 XBO5 100 XBO5 100 XBO5 200

AZA nM ->

XBO5 200 XBO5 200 XBO5 200 XBO5 200 XBO5 200

0.00

50.00

100.00

200.00

400.00

800.00

0.00

50.00

100.00

200.00

400.00

800.00

0.37

0.32

0.31

0.24

0.21

0.21

0.34

0.30

0.27

0.24

0.20

0.23

0.36

0.30

0.35

0.25

0.24

0.21

0.33

0.32

0.27

0.25

0.23

0.23

0.36

0.33

0.32

0.26

0.23

0.21

0.33

0.27

0.28

0.24

0.23

0.22

0.36

0.32

0.30

0.24

0.23

0.23

0.31

0.27

0.27

0.25

0.24

0.23

0.35

0.31

0.29

0.25

0.21

0.21

0.33

0.29

0.26

0.24

0.23

0.22

0.32

0.30

0.29

0.23

0.20

0.22

0.32

0.28

0.26

0.24

0.21

0.19

0.35

0.31

0.31

0.24

0.22

0.21

0.33

0.29

0.27

0.25

0.22

0.22

Table 40. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (100 and 200nM) and serial azacytadine.

103

XB05 in nM

AZA nM ->

AVG

XBO5 400 XBO5 400 XBO5 400 XBO5 400 XBO5 400 XBO5 400 XBO5 800 XBO5 800 XBO5 800 XBO5 800 XBO5 800 XBO5 800
0.00

50.00

100.00

200.00

400.00

800.00

0.00

50.00

100.00

200.00

400.00

800.00

0.35

0.30

0.29

0.25

0.23

0.15

0.20

0.28

0.23

0.22

0.21

0.19

0.34

0.30

0.27

0.27

0.22

0.21

0.28

0.28

0.27

0.23

0.21

0.19

0.35

0.32

0.27

0.26

0.23

0.21

0.29

0.26

0.25

0.23

0.21

0.19

0.33

0.32

0.27

0.25

0.23

0.19

0.29

0.27

0.26

0.24

0.20

0.22

0.35

0.32

0.28

0.25

0.22

0.20

0.30

0.27

0.27

0.22

0.20

0.20

0.35

0.28

0.27

0.24

0.21

0.19

0.26

0.24

0.24

0.21

0.18

0.34

0.31

0.28

0.25

0.22

0.19

0.27

0.25

0.23

0.21

0.19

0.27

Table 41. Results of the first MTT-assays performed in U937 cell line following
combination treatment with XB05 (400 and 800nM) and serial azacytadine.

Combined results of two MTT-assays performed in U937 cell line following combination
treatment with XB05 and azacytadine are outlined below. XB05 concentration was kept
constant and azacytadine was added in serial concentrations.
XB05 in
nM
XB05 0

AZA nM
->

0.00

XB05 0
100.00

XB05 0
200.00

XB05 0
400.00

XB05 0
800.00

XB05 100 XB05 100 XB05 100 XB05 100 XB05 100
0.00

100.00

200.00

400.00

800.00

0.68
0.63
0.55
0.51
0.42
0.66
0.52
0.48
0.38
0.39
0.38
0.31
0.28
0.26
0.22
0.35
0.31
0.24
0.22
0.21
0.53
0.47
0.41
0.38
0.32
0.51
0.41
0.36
0.30
0.30
Table 42. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (0 and 100nM) and serial azacytadine.

XB05 in
nM
XB05 200 XB05 200 XB05 200 XB05 200 XB05 200 XB05 400 XB05 400 XB05 400 XB05 400 XB05 400

AZA nM
->

0.00

100.00

200.00

400.00

800.00

0.00

100.00

200.00

400.00

800.00

0.69
0.66
0.52
0.46
0.40
0.75
0.60
0.54
0.48
0.38
0.33
0.27
0.25
0.22
0.22 0.34
0.28
0.25
0.22
0.19
0.51
0.47
0.38
0.34
0.31
0.55
0.44
0.39
0.35
0.29
Table 43. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine.

104

XB05 in
nM
XB05 800 XB05 800 XB05 800 XB05 800 XB05 800

AZA nM
->

0.00

100.00

200.00

400.00

800.00

0.65
0.56
0.46
0.39
0.35
0.27
0.25
0.23
0.21
0.19
0.46
0.41
0.35
0.30
0.27
Table 44. Combined results of two MTT-assays performed in U937 cell line following
combination treatment with XB05 (200 and 400nM) and serial azacytadine.

Previously, we had performed MTT-assays on HCT116 (colon cancer cell line) following
treatment with XB05 and DMSO to evaluate if the concentration of DMSO that was used
for reconstitution of XB05 was toxic to cells. The results below demonstrate that DMSO
even at high concentrations was not cytotoxic to cells.
Concentration ->
HCT116 vehicle control (DMSO)

0.00
100.00
200.00
400.00
800.00 1600.00
1.00
0.93
0.92
0.92
0.87
0.88
1.00
0.90
0.91
0.87
1.07
0.83
avg
1.00
0.92
0.92
0.89
0.97
0.86
stdev
0.00
0.02
0.01
0.03
0.15
0.04
Hct116 treated with XBO5
1.00
0.89
0.80
0.53
0.24
0.10
1.00
1.03
0.92
0.57
0.26
0.08
avg
1.00
0.96
0.86
0.55
0.25
0.09
stdev
0.00
0.10
0.08
0.03
0.01
0.02
Table 45. MTT-assays on HCT116 (colon cancer cell line) following treatment with
XB05 and DMSO.

105

Figure 53. Graphical representation of the MTT-assays
assays on HCT116 (colon cancer cell
line)) following treatment with XB05 and DMSO.

106

Target Name Cт
Cт Mean Cт SD ∆Cт ∆Cт Mean ∆Cт SE ∆∆Cт Gene Expression
GAPDH
24.62
24.46
0.22
GAPDH
24.31
24.46
0.22
GAPDH
24.00
23.99
0.02
GAPDH
23.97
23.99
0.02
GAPDH
27.56
27.44
0.17
GAPDH
27.33
27.44
0.17
DNMT1
26.56
26.63
0.10
2.17
0.00
1.00
DNMT1
26.70
26.63
0.10
DNMT1
27.35
27.28
0.09
3.30
-1.13
0.46
DNMT1
27.22
27.28
0.09
DNMT1
26.81
27.02
0.30
-0.42
2.59
6.02
DNMT1
27.23
27.02
0.30
DNMT3a
24.59
24.65
0.09
0.19
0.00
1.00
DNMT3a
24.71
24.65
0.09
DNMT3a
24.80
24.86
0.08
0.87
-0.68
0.62
DNMT3a
24.91
24.86
0.08
DNMT3a
27.69
27.70
0.02
0.25
-0.07
0.96
DNMT3a
27.71
27.70
0.02
DNMT3b
21.71
21.92
0.31
-2.54
0.00
1.00
DNMT3b
22.14
21.92
0.31
DNMT3b
22.35
22.59
0.34
-1.39
-1.15
0.45
DNMT3b
22.83
22.59
0.34
DNMT3b
25.70
25.70
0.01
-1.75
-0.79
0.58
DNMT3b
25.69
25.70
0.01
HIC1
25.13
25.04
0.13
-1.08
0.00
1.00
HIC1
24.94
25.04
0.13
HIC1
26.62
26.57
0.07
0.37
-2.01
0.25
HIC1
26.52
26.57
0.07
HIC1
28.80
28.76
0.06
-0.25
-0.74
0.60
HIC1
28.72
28.76
0.06
ER alpha
25.72
25.69
0.05
-0.43
0.00
1.00
ER alpha
25.66
25.69
0.05
ER alpha
25.70
25.99
0.41
-0.20
-0.78
0.58
ER alpha
26.28
25.99
0.41
ER alpha
27.41
27.23
0.26
-1.79
1.45
2.72
ER alpha
27.04
27.23
0.26
p16
27.06
27.24
0.25
2.77
0.00
1.00
p16
27.42
27.24
0.25
p16
27.41
27.54
0.18
3.56
-0.78
0.58
p16
27.67
27.54
0.18
p16
27.65
27.76
0.16
0.32
2.46
5.48
p16
27.88
27.76
0.16
DAPK
25.15
25.26
0.15
0.79
0.00
1.00
DAPK
25.37
25.26
0.15
DAPK
24.96
25.05
0.12
1.06
-0.27
0.83
DAPK
25.13
25.05
0.12
DAPK
27.81
27.73
0.12
0.28
0.51
1.42
DAPK
27.65
27.73
0.12
MGMT
33.11
33.11
0.00
8.64
0.00
1.00
MGMT
33.11
33.11
0.00
MGMT
33.09
32.85
0.35
8.86
-0.22
0.86
MGMT
32.60
32.85
0.35
MGMT
33.08
32.81
0.37
5.37
3.27
9.68
MGMT
32.55
32.81
0.37
RARb
26.80
26.81
0.02
2.34
0.00
1.00
RARb
26.82
26.81
0.02
RARb
26.68
26.72
0.06
2.74
-0.40
0.76
RARb
26.76
26.72
0.06
RARb
27.42
27.38
0.06
-0.07
2.41
5.31
RARb
27.34
27.38
0.06

Table 46. PCR data for Kasumi-1 cell line – 48 hour treatment with XB05 and 5’AZA.

107

Sample Name
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza
U937-NT
U937-NT
U937-XB
U937-XB
U937-Aza
U937-Aza

Target Name
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
HIC1
HIC1
HIC1
HIC1
HIC1
HIC1
ER alpha
ER alpha
ER alpha
ER alpha
ER alpha
ER alpha
p16
p16
p16
p16
p16
p16
DAPK
DAPK
DAPK
DAPK
DAPK
DAPK
MGMT
MGMT
MGMT
MGMT
MGMT
MGMT
RARb
RARb
RARb
RARb
RARb
RARb

Cт
26.56
28.03
24.74
24.59
28.94
29.17
27.13
27.01
27.43
27.45
27.80
27.94
27.50
27.67
25.02
25.28
28.18
28.01
28.23
28.33
25.85
25.97
27.66
28.15
29.47
29.34
27.82
27.61
28.56
28.83
27.57
27.18
26.70
26.59
27.25
27.01
27.70
27.57
26.74
26.83
24.71
25.40
28.51
28.93
26.06
26.01
29.08
28.94
Undetermined
35.53
38.33
34.51
37.34
37.72
27.74
27.63
27.44
27.70
27.50
27.55

Cт Mean Cт SD ∆Cт
27.29
1.04
27.29
1.04
24.67
0.11
24.67
0.11
29.05
0.16
29.05
0.16
27.07
0.08
27.07
0.08
27.44
0.01
27.44
0.01
27.87
0.10
27.87
0.10
27.58
0.12
27.58
0.12
25.15
0.18
25.15
0.18
28.09
0.12
28.09
0.12
28.28
0.07
28.28
0.07
25.91
0.09
25.91
0.09
27.90
0.34
27.90
0.34
29.41
0.09
29.41
0.09
27.72
0.15
27.72
0.15
28.69
0.19
28.69
0.19
27.38
0.28
27.38
0.28
26.65
0.08
26.65
0.08
27.13
0.17
27.13
0.17
27.64
0.10
27.64
0.10
26.78
0.07
26.78
0.07
25.05
0.48
25.05
0.48
28.72
0.30
28.72
0.30
26.04
0.04
26.04
0.04
29.01
0.10
29.01
0.10
35.53
35.53
36.42
2.70
36.42
2.70
37.53
0.27
37.53
0.27
27.68
0.08
27.68
0.08
27.57
0.19
27.57
0.19
27.52
0.03
27.52
0.03

∆Cт Mean

∆Cт SE ∆∆Cт

Gene Expression

-0.23

0.00

1.00

2.77

-3.00

0.12

-1.19

0.96

1.94

0.29

0.00

1.00

0.49

-24.38

0.00

-0.96

1.25

2.38

0.98

0.00

1.00

1.24

-0.26

0.84

-1.15

2.14

4.39

-0.33

0.00

1.00

0.98

-0.94

0.52

-2.60

2.47

5.55

-2.35

0.00

1.00

-0.09

-1.90

0.27

-4.16

2.01

4.02

0.34

0.00

1.00

2.12

-1.78

0.29

-4.00

4.34

20.27

1.43

0.00

1.00

1.37

0.06

1.04

-0.05

1.48

2.78

8.24

0.00

1.00

11.75

-3.51

0.09

8.47

-0.24

0.85

0.39

0.00

1.00

2.90

-2.52

0.17

-1.53

1.92

3.79

Table 47. PCR data for U937 cell line – 48 hour treatment with XB05 and 5’AZA.

108

Sample Name
Kasumi-NT
Kasumi-NT
Kasumi-XBO
Kasumi-XBO
Kasumi-AZA
Kasumi-AZA
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XBO
Kasumi-XBO
Kasumi-AZA
Kasumi-AZA
Kasumi-NT
Kasumi-NT
Kasumi-XBO
Kasumi-XBO
Kasumi-AZA
Kasumi-AZA
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza
Kasumi-NT
Kasumi-NT
Kasumi-XB
Kasumi-XB
Kasumi-Aza
Kasumi-Aza

Target Name
Cт
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
HIC-1
HIC-1
HIC-1
HIC-1
HIC-1
HIC-1
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
p16
p16
p16
p16
p16
p16
DAPK
DAPK
DAPK1
DAPK1
DAPK1
DAPK1
MGMT
MGMT
MGMT
MGMT
MGMT
MGMT
RARb
RARb
RARb
RARb
RARb
RARb

26.06
26.57
27.98
27.95
26.95
26.54
26.56
26.70
27.45
26.97
26.76
26.88
24.59
24.71
32.45
31.82
31.88
32.58
21.71
22.14
28.02
27.87
26.82
26.95
25.76
25.63
26.86
26.95
27.41
27.73
25.71
25.55
25.81
25.83
25.82
25.95
27.06
27.42
26.72
26.88
26.55
26.76
25.15
25.37
26.15
26.39
26.17
25.97
33.11
33.11
34.44
34.49
34.40
38.54
26.80
26.82
27.45
27.59
27.61
27.73

Cт Mean
Cт SD
∆∆Cт
Gene Expression
26.32
0.36
26.32
0.36
27.97
0.03
27.97
0.03
26.75
0.29
26.75
0.29
26.63
0.10
0.00
1.00
26.63
0.10
27.21
0.34
1.07
2.10
27.21
0.34
26.82
0.09
0.25
1.19
26.82
0.09
24.65
0.09
0.00
1.00
24.65
0.09
32.13
0.44
-5.83
0.02
32.13
0.44
32.23
0.49
-7.15
0.01
32.23
0.49
21.92
0.31
0.00
1.00
21.92
0.31
27.94
0.11
-4.37
0.05
27.94
0.11
26.88
0.10
-4.53
0.04
26.88
0.10
25.69
0.09
0.00
1.00
25.69
0.09
26.91
0.06
0.44
1.35
26.91
0.06
27.57
0.22
-1.45
0.37
27.57
0.22
25.63
0.11
0.00
1.00
25.63
0.11
25.82
0.01
1.46
2.74
25.82
0.01
25.88
0.09
0.18
1.13
25.88
0.09
27.24
0.25
0.00
1.00
27.24
0.25
26.80
0.11
2.08
4.24
26.80
0.11
26.66
0.15
1.01
2.02
26.66
0.15
25.26
0.15
0.00
1.00
25.26
0.15
26.27
0.17
0.64
1.56
26.27
0.17
26.07
0.14
-0.38
0.77
26.07
0.14
33.11
0.00
0.00
1.00
33.11
0.00
34.47
0.04
0.29
1.22
34.47
0.04
36.47
2.93
-2.93
0.13
36.47
2.93
26.81
0.02
0.00
1.00
26.81
0.02
27.52
0.10
0.93
1.91
27.52
0.10
27.67
0.09
-0.43
0.74
27.67
0.09

Table 48. PCR data for Kasumi-1 cell line – 96 hour treatment with XB05 and 5’AZA.

109

Target Name
Cт
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
GAPDH
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT1
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3a
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
DNMT3b
HIC-1
HIC-1
HIC-1
HIC-1
HIC-1
HIC-1
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
ER-Alpha
p16
p16
p16
p16
p16
p16
DAPK
DAPK
DAPK1
DAPK1
DAPK1
DAPK1
MGMT
MGMT
MGMT
MGMT
MGMT
MGMT
RARb
RARb
RARb
RARb
RARb
RARb

25.87
25.61
27.95
28.21
28.45
27.81
27.13
27.01
27.33
27.32
27.16
26.99
27.50
27.67
32.40
32.36
32.32
32.27
28.23
28.33
27.19
27.40
27.48
27.19
26.97
26.82
26.71
26.66
26.56
26.47
25.74
25.77
25.82
25.98
25.91
26.51
27.70
27.57
27.67
27.94
27.94
27.66
28.51
28.93
25.89
25.77
25.89
25.87
35.53
35.53
31.22
30.89
31.40
31.88
27.74
27.63
26.44
26.45
26.45
26.53

Cт Mean
Cт SD
∆∆Cт
Gene Expression
25.74
0.18
25.74
0.18
28.08
0.19
28.08
0.19
28.13
0.46
28.13
0.46
27.07
0.08
0.00
1.00
27.07
0.08
27.32
0.01
2.08
4.24
27.32
0.01
27.07
0.12
2.39
5.24
27.07
0.12
27.58
0.12
0.00
1.00
27.58
0.12
32.38
0.04
-2.46
0.18
32.38
0.04
32.29
0.04
-2.32
0.20
32.29
0.04
28.28
0.07
0.00
1.00
28.28
0.07
27.29
0.15
3.33
10.04
27.29
0.15
27.34
0.20
3.34
10.11
27.34
0.20
26.89
0.11
0.00
1.00
26.89
0.11
26.69
0.04
2.55
5.85
26.69
0.04
26.51
0.07
2.77
6.84
26.51
0.07
25.76
0.02
0.00
1.00
25.76
0.02
25.90
0.11
2.20
4.59
25.90
0.11
26.21
0.43
1.94
3.84
26.21
0.43
27.64
0.10
0.00
1.00
27.64
0.10
27.80
0.19
2.17
4.51
27.80
0.19
27.80
0.20
2.23
4.70
27.80
0.20
28.72
0.30
0.00
1.00
28.72
0.30
25.83
0.08
5.23
37.64
25.83
0.08
25.88
0.01
5.24
37.76
25.88
0.01
35.53
0.00
1.00
35.53
31.05
0.24
6.82
113.05
31.05
0.24
31.64
0.34
6.29
78.31
31.64
0.34
27.68
0.08
0.00
1.00
27.68
0.08
26.44
0.01
3.58
11.96
26.44
0.01
26.49
0.05
3.59
12.03
26.49
0.05

Table 49. PCR data for U937 cell line – 96 hour treatment with XB05 and 5’AZA

110

Standard curve obtained from serial concentrations of BSA (mg/mL) using the Buffer B
for dilutions, the same buffer that was used to extra
extract
ct cytoplasmic fractions.

0.000

0.025

0.125

0.250

0.500

0.750

1.000

1.500

2.000

BSA

0.001

0.015

0.080

0.171

0.323

0.429

0.566

0.743

0.829

BSA

-0.001

0.018

0.084

0.182

0.327

0.417

0.580

0.744

0.827

AVG

0.000

0.017

0.082

0.177

0.325

0.423

0.573

0.744

0.828

Table 50. Average O.D.’s of serial dilutions of BSA for standard curve for cytoplasmic
protein.

Figure 54. Standard curve for BSA for quantitation of cytoplasmic protein.

111

Calculation performed to calculate the volume to be loaded in each well for 30ug of
protein for cytoplasmic fractions.

KNT
0.075

K XB 48
0.055

K XB 96
0.095

K A 48
0.059

K A 96
0.123

UNT
0.091

U XB 48
0.096

U XB 96
0.122

U A 48
0.075

U A 96
0.103

0.069

0.059

0.096

0.053

0.123

0.079

0.094

0.113

0.067

0.109

AVG

0.072

0.057

0.096

0.056

0.123

0.085

0.095

0.118

0.071

0.106

Protein Quantity (1:20)

0.076

0.069

0.087

0.069

0.099

0.082

0.086

0.097

0.075

0.091

Protein quantity (mg/mL)

1.515

1.379

1.732

1.370

1.976

1.632

1.722

1.930

1.506

1.822

Protein quantity (ug/uL)

1.515

1.379

1.732

1.370

1.976

1.632

1.722

1.930

1.506

1.822

30uq protein

19.8

21.8

17.3

21.9

15.2

18.4

17.4

15.5

19.9

16.5

Laemelli Reagent

19.8

21.8

17.3

21.9

15.2

18.4

17.4

15.5

19.9

16.5

H2O

10.4

6.5

15.3

6.2

19.6

13.2

15.2

18.9

10.1

17.1

50.0

50.0

50.0

50.0

Total Volume
50.0
50.0
50.0
50.0
50.0
50.0
Table 51. Cytoplasmic protein calculations for western blot.

Standard curve obtained from serial concentrations of BSA (mg/mL) using the Buffer C
for dilutions, the same buffer that was used to extract nuclear fractions.

0

0.025

0.125

0.25

0.5

0.75

1

1.5

2

BSA

-0.002

0.016

0.077

0.156

0.303

0.422

0.55

0.749

0.831

BSA

0.002

0.012

0.079

0.157

0.314

0.408

0.541

0.739

0.819

AVG

0

0.014

0.078

0.1565

0.3085

0.415

0.5455

0.744

0.825

Table 52. Average O.D.’s of serial dilutions of BSA for standard curve for nuclear
protein.

112

Figure 55.. Standard curve for BSA for quantitation of nuclear protein.

113

Calculation performed to calculate the volume to be loaded in each well for 30ug of
protein for nuclear fractions.

KNT

K XB 48

K XB 96

K A 48

K A 96

UNT

U XB 48

U XB 96

U A 48

U A 96

0.203

0.131

0.34

0.128

0.159

0.187

0.142

0.181

0.128

0.156

0.184

0.122

0.347

0.133

0.152

0.179

0.141

0.155

0.136

0.127

0.194

0.127

0.344

0.131

0.156

0.183

0.142

0.168

0.132

0.142

Protein quantity (1:20)

0.123

0.093

0.19

0.095

0.106

0.118

0.1

0.111

0.095

0.1

Protein quantity (mg/mL)

2.462

1.86

3.81

1.896

2.12

2.363

1.995

2.228

1.905

1.995

Protein quantity (ug/uL)

2.46

1.86

3.81

1.9

2.12

2.36

1.99

2.23

1.9

1.99

uL for 30ug

12.2

16.1

7.9

15.8

14.1

12.7

15

13.5

15.8

15

Laemelli Reagent

12.2

16.1

7.9

15.8

14.1

12.7

15

13.5

15.8

15

H2O

25.6

17.7

34.3

18.4

21.7

24.6

19.9

23.1

18.5

19.9

Total Volume

50

50

50

50

50

50

50

50

50

50

AVG

Table 53. Cytoplasmic protein calculations for western blot.

114

BIBLIOGRAPHY

115

BIBLIOGRAPHY

1.

Ferrara, F. and C.A. Schiffer, Acute myeloid leukaemia in adults. Lancet, 2013.
381(9865): p. 484-95.

2.

Kern, W. and E.H. Estey, High-dose cytosine arabinoside in the treatment of
acute myeloid leukemia: Review of three randomized trials. Cancer, 2006.
107(1): p. 116-24.

3.

Shih, A.H., et al., The role of mutations in epigenetic regulators in myeloid
malignancies. Nat Rev Cancer, 2012. 12(9): p. 599-612.

4.

Kelly, L.M. and D.G. Gilliland, Genetics of myeloid leukemias. Annu Rev
Genomics Hum Genet, 2002. 3: p. 179-98.

5.

Figueroa, M.E., et al., DNA methylation signatures identify biologically distinct
subtypes in acute myeloid leukemia. Cancer Cell, 2010. 17(1): p. 13-27.

6.

Oki, Y. and J.P. Issa, Epigenetic mechanisms in AML - a target for therapy.
Cancer Treat Res, 2010. 145: p. 19-40.

7.

Gutierrez, S.E. and F.A. Romero-Oliva, Epigenetic changes: a common theme in
acute myelogenous leukemogenesis. J Hematol Oncol, 2013. 6: p. 57.

8.

Esteller, M., Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum
Mol Genet, 2007. 16 Spec No 1: p. R50-9.

9.

Ordway, J.M., et al., Comprehensive DNA methylation profiling in a human
cancer genome identifies novel epigenetic targets. Carcinogenesis, 2006. 27(12):
p. 2409-23.

10.

MP, C., Decitabine: A Review of its Use in Older Patients with Acute Myeloid
Leukaemia. Drugs Aging, 2013. 30(6): p. 447-58.

11.

Estey, E.H., Acute myeloid leukemia: 2013 update on risk-stratification and
management. Am J Hematol, 2013. 88(4): p. 318-27.

12.

O'Donnell, M.R., et al., Acute myeloid leukemia. J Natl Compr Canc Netw, 2012.
10(8): p. 984-1021.

116

13.

Sweet, K. and J.E. Lancet, Novel therapeutics in acute myeloid leukemia. Curr
Hematol Malig Rep, 2014. 9(2): p. 109-17.

14.

Yan P, F.D., Murphy M, Tam HH, Rodriguez B, Curfman J, Trimarchi M, Geyer
S, Wu YZ, Whitman SP, Metzeler K, Walker A, Klisovic R, Jacob S, Grever MR,
Byrd JC, Bloomfield CD, Garzon R, Blum W, Caligiuri MA, Bundschuh R,
Marcucci G, Genome-wide methylation profiling in decitabine-treated patients
with acute myeloid leukemia. Blood, 2012. 120(12): p. 2466-74.

15.

Conway O'Brien, E., S. Prideaux, and T. Chevassut, The epigenetic landscape of
acute myeloid leukemia. Adv Hematol, 2014. 2014: p. 103175.

16.

Jiang, S., et al., Reactivating aberrantly hypermethylated p15 gene in leukemic T
cells by a phenylhexyl isothiocyanate mediated inter-active mechanism on DNA
and chromatin. J Hematol Oncol, 2010. 3: p. 48.

17.

Lakshmikuttyamma, A., et al., Reexpression of epigenetically silenced AML
tumor suppressor genes by SUV39H1 inhibition. Oncogene, 2010. 29(4): p. 57688.

18.

Clark, S.J. and J. Melki, DNA methylation and gene silencing in cancer: which is
the guilty party? Oncogene, 2002. 21(35): p. 5380-7.

19.

Kulis, M. and M. Esteller, DNA methylation and cancer. Adv Genet. 70: p. 27-56.

20.

Estey, E.H., Epigenetics in clinical practice: the examples of azacitidine and
decitabine in myelodysplasia and acute myeloid leukemia. Leukemia, 2013.
27(9): p. 1803-12.

21.

Geyer, C.R., Strategies to re-express epigenetically silenced p15(INK4b) and
p21(WAF1) genes in acute myeloid leukemia. Epigenetics, 2010. 5(8): p. 696703.

22.

Yun-Gyoo Lee, I.K., Sung-Soo Yoon, Seonyang Park, June Won Cheong, Yoo
Hong Min, Jeong-Ok Lee, Soo-Mee Bang, Hyeon Gyu Yi, Chul Soo Kim, Yong
Park, Byung-Soo Kim, Yeung-Chul Mun, Chu-Myoung Seong, Jinny Park, Jae
Hoon Lee, Sung-Yong Kim, Hong Ghi Lee, Yeo-Kyeoung Kim and Hyeoung-Joon
Kim, Comparative analysis between azacitidine and decitabine for the treatment
of myelodysplastic syndromes. British Journal of Haematology, 2013. 161: p.
339-347.

23.

Hagemann, S., et al., Azacytidine and decitabine induce gene-specific and nonrandom DNA demethylation in human cancer cell lines. PLoS One, 2011. 6(3): p.
e17388.
117

24.

Francesca R. Šalipur, E.M.R.-R., Bo Xu, Gerald B. Hammond and Paula J.
Bates, A Novel Small Molecule that Selectively Kills Malignant Cells via an
Oxidative Stress-Dependent Mechanism Free Radical Biology and Medicine,
2013. 68: p. 110-121.

25.

Irwin ME, R.-D.V.N., Chandra J, Redox control of leukemia: from molecular
mechanisms to therapeutic opportunities. Antioxid Redox Signal, 2013. 18(11): p.
1349-83.

26.

Rushworth, S.A., K.M. Bowles, and D.J. MacEwan, High basal nuclear levels of
Nrf2 in acute myeloid leukemia reduces sensitivity to proteasome inhibitors.
Cancer Res. 71(5): p. 1999-2009.

27.

Rushworth, S.A., et al., The high Nrf2 expression in human acute myeloid
leukemia is driven by NF-kappaB and underlies its chemo-resistance. Blood.
120(26): p. 5188-98.

28.

Wu, R.P., et al., Nrf2 responses and the therapeutic selectivity of electrophilic
compounds in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A. 107(16):
p. 7479-84.

29.

Salipur, F.R., et al., A novel small molecule that induces oxidative stress and
selectively kills malignant cells. Free Radic Biol Med, 2014. 68: p. 110-21.

30.

Sun L, M.L., Cabarcas SM, Zhang X, Yang A, Zhang Y, Young MR, Klarmann
KD, Keller JR, Farrar WL, Epigenetic Regulation of SOX9 by the NF-κB Signaling
Pathway in Pancreatic Cancer Stem Cells. Stem Cells, 2013: p. 1-25.

31.

Flotho, C., et al., The DNA methyltransferase inhibitors azacitidine, decitabine
and zebularine exert differential effects on cancer gene expression in acute
myeloid leukemia cells. Leukemia, 2009. 23(6): p. 1019-28.

32.

Shen, N., et al., A nucleolin-DNMT1 regulatory axis in acute myeloid
leukemogenesis. Oncotarget, 2014. 5(14): p. 5494-509.

33.

Garcia-Dominguez, P., et al., Synthetic approaches to DNMT inhibitor SGI-1027
and effects on the U937 leukemia cell line. Bioorg Med Chem Lett, 2013. 23(6):
p. 1631-5.

34.

Soultanas, P., et al., Modulation of human DNA methyltransferase activity and
mRNA levels in the monoblast cell line U937 induced to differentiate with
dibutyryl cyclic AMP and phorbol ester. J Mol Endocrinol, 1993. 11(2): p. 191200.

118

35.

Larizza, L., I. Magnani, and A. Beghini, The Kasumi-1 cell line: a t(8;21)-kit
mutant model for acute myeloid leukemia. Leuk Lymphoma, 2005. 46(2): p. 24755.

36.

Asou, H., et al., Establishment of a human acute myeloid leukemia cell line
(Kasumi-1) with 8;21 chromosome translocation. Blood, 1991. 77(9): p. 2031-6.

37.

Sundstrom, C. and K. Nilsson, Establishment and characterization of a human
histiocytic lymphoma cell line (U-937). Int J Cancer, 1976. 17(5): p. 565-77.

119