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Use of DNA methylation inhibitors for chemotherapy and chemoprevention of cancer
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Use of DNA methylation inhibitors for chemotherapy and chemoprevention of cancer
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Content
USE OF DNA METHYLATION INHIBITORS FOR CHEMOTHERAPY AND
CHEMOPREVENTION OF CANCER
by
Christine Bora Yoo
____________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2007
Copyright 2007 Christine Bora Yoo
ii
DEDICATION
Not to us, O Lord, not to us
But to your name be the glory,
Because of your love and faithfulness.
Psalm 115:1
iii
ACKNOWLEDGEMENTS
I would like to acknowledge the following people who have helped me over the years:
Dr. Peter A. Jones, my thesis advisor, for his wonderful guidance, dynamic leadership,
and never-ending support. Drs. Woojin An, Louis Debeau, Ite Laird-Offringa, and
Darryl Shibata for their helpful comments and constructive criticisms as my graduate
committee members. Dr. Victor E. Marquez for the unlimited supply of zebularine and
prodrugs. Drs. Allen S. Yang, Peter W. Laird, Sheldon Greer, Thomas Thykjaer, Torben
Orntoft, Chris McGuigan, Pasit Phiasivongsa, and Sanjeev Redkar for research
collaborations. Past and present members of the Jones’ laboratory with whom I have had
the pleasure of working and sharing great memories: Ana Aparicio, Lindsey Barske,
Jonathan Cheng, Jody Chuang, Connie Cortez, Gerda Egger, Sonia Escobar, Mehrnaz
Fatemi, Martin Frederick, Jeffrey Friedman, Einav Gal-Yam, Shinwu Jeong, Toni Li,
Gangning Liang, Joy Lin, Shiro Matsuro, Tina Miranda, Yoshimasa Saito, Daiya Takai,
Yvonne Tsai, Daniel Weisenberger, and Erika Wolff.
Lastly, I give my deepest thanks to my loving parents, my husband, Leo, and my brother,
Michael, whose love and support without which nothing would have been possible.
iv
TABLE OF CONTENTS
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures viii
Abstract xii
Chapter 1: Epigenetic Therapy of Cancer 1
Introduction 1
Enzyme families involved in epigenetic regulation 4
Cancer epigenetics 7
DNA methylation inhibitors 15
Histone deacetylase inhibitors 24
Perspectives 35
Overview of Thesis Research 38
Chapter 2: Mechanism of Action of Zebularine 43
Introduction 43
Materials and Methods 45
Results 54
Discussion 90
Chapter 3: Zebularine Prodrugs 95
Introduction 95
Materials and Methods 98
Results 102
Discussion 143
Chapter 4: Short Oligonucleotide DNA Methylation Inhibitors 147
Introduction 147
Materials and Methods 149
Results 156
Discussion 180
v
Chapter 5: Cancer Chemoprevention by Zebularine 185
Introduction 185
Materials and Methods 187
Results 190
Discussion 203
Chapter 6: Summary and Conclusions 208
Reference 214
vi
LIST OF TABLES
Table 1.1 DNA methylation inhibitors: nucleoside analogs. 17
Table 1.2 DNA methylation inhibitors: non-nucleoside analogs. 24
Table 1.3 Histone deacetylase inhibitors: short-chain fatty acids and 28
hydroxamic acids.
Table 1.4 Histone deacetylase inhibitors: cyclic peptides. 32
Table 1.5 Histone deacetylase inhibitors: benzamides. 34
Table 2.1 Effects of folic acid on the growth rate of HCT15 colon 63
cancer cells.
Table 2.2 Effects of zebularine on growth inhibition in normal 70
fibroblasts and cancer cells.
Table 2.3 Genes upregulated2 fold in all cancer cell lines, but not in 74
fibroblast cell lines eight days after continuous zebularine
treatment.
Table 2.4 Genes downregulated2 fold in all cancer cell lines, but 76
not in fibroblast cell lines eight days after continuous
zebularine treatment.
Table 2.5 Genes upregulated2 fold in all fibroblast cell lines, but 77
not in cancer cell lines eight days after continuous
zebularine treatment.
Table 2.6 Genes downregulated2 fold in all fibroblast cell lines, but 78
not in cancer cell lines eight days after continuous
zebularine treatment.
Table 3.1 Cytidine analogs; candidate DNA methylation inhibitors. 103
Table 3.2 Effects of zebularine prodrugs on the growth rate of cancer 104
cells.
Table 3.3 Cytidine analogs; candidate DNA methylation inhibitors. 110
vii
Table 3.4 Effects of zebularine prodrugs on the growth rate of cancer 111
cells.
Table 3.5 Zebularine phosphoramidite prodrugs. 114
Table 3.6 Effects of zebularine phosphoramidate prodrugs on the 121
growth rate of T24 bladder cancer cells.
Table 3.7 Effects of zebularine phosphoramidate prodrugs on the 122
growth rate of HCT15 colon cancer cells.
Table 3.8 Effects of zebularine phosphoramidate prodrugs on the 128
growth rate of Cf-Pac-1 pancreatic cancer cells.
Table 3.9 DNA methylation analysis of Cf-Pac-1 cells treated with 130
zebularine phosphoramidites.
Table 3.10 Cyclic deoxyzebularine monophosphate dimers. 134
Table 3.11 Effects of cyclic d(ZpZ) monophosphate dimer on the 137
growth rate of T24 bladder and Cf-Pac-1 pancreatic cancer
cells.
Table 3.12 Effects of cyclic d(ZpT) monophosphate dimer on the 138
growth rate of Cf-Pac-1 pancreatic cancer cells.
Table 4.1 5-Aza-2’-deoxycytidine analogs and quinoline derivatives 156
from SuperGen.
Table 4.2 Short oligonucleotide DNA methylation inhibitors. 161
Table 5.1 Genes upregulated in colonic epithelial cells after chronic 197
zebularine treatment of Apc
min/+
mice.
Table 5.2 Genes downregulated in colonic epithelial cells after 198
chronic zebularine treatment of Apc
min/+
mice.
viii
LIST OF FIGURES
Figure 1.1 Epigenetic gene silencing in cancer. 10
Figure 1.2 Reactivation of aberrantly silenced genes by DNA 12
methylation inhibitors.
Figure 1.3 Mechanism-based inhibition of DNA methylation by 20
cytosine analogues.
Figure 1.4 Effects of HDAC inhibitors in human cancer. 26
Figure 2.1 Rate of DNA incorporation of 2-[
14
C]-zebularine in normal 56
fibroblasts cultured in 10 or 20% fetal bovine serum.
Figure 2.2 The rate of DNA incorporation of 2-[
14
C]-zebularine in T24 58
and LD419 cells grown in media containing 20% fetal calf
serum.
Figure 2.3 Effects of folate on the level of gene induction by 60
zebularine in HCT15 colon cancer cells.
Figure 2.4 Effects of folate on the level of gene expression by 62
zebularine in HCT15 colon cancer cells.
Figure 2.5 Effects of zebularine on DNMT protein levels in normal 65
fibroblasts and cancer cells.
Figure 2.6 Effects of zebularine on DNA methylation in normal 67
fibroblasts and cancer cells.
Figure 2.7 Effects of zebularine on gene expression in normal 69
fibroblasts and cancer cells.
Figure 2.8 Level of incorporation of 2-[
14
C]-zebularine into DNA and 72
level of uridine/cytidine kinase activity in normal and
cancer cells.
Figure 2.9 Position of cancer-testis antigens on X chromosome. 80
Figure 2.10 CpG content and map of cancer-testis antigens. 81
ix
Figure 2.11 Effects of zebularine on the expression cancer-testis 82
antigens in normal fibroblasts and cancer cell lines.
Figure 2.12 Effects of zebularine on the expression cancer-testis 84
antigens in normal fibroblasts and cancer cell lines.
Figure 2.13 Effects of chronic zebularine treatment on the expression of 86
cancer-testis antigens.
Figure 2.14 Effects of chronic zebularine treatment on DNA 87
methylation.
Figure 3.1 Effects of 4-methylzebularine on gene expression in T24 105
cells.
Figure 3.2 Effects of 3-deaza-5-aza-2’-deoxycytidine on p16 106
expression in T24 cells.
Figure 3.3 Effects of NSC740467 and NSC 737452 on p16 expression 107
in T24 cells.
Figure 3.4 Effects of NSC737451 and NSC737453 on p16 expression 108
in Cf-Pac-1 cells.
Figure 3.5 Effects of CME-1 through CME-5 on p16 expression in 112
T24 cells.
Figure 3.6 Effects of 2’-zebularine deoxycytidine phosphoramidate 117
and 2’-F-zebularine deoxycytidine phosphoramidate on p16
expression in T24 cells.
Figure 3.7 Effects of 2’-zebularine deoxycytidine phosphoramidate 118
p16 expression in Cf-Pac-1 cells.
Figure 3.8 Effects of CPF190-192 on p16 expression in cancer cells. 120
Figure 3.9 Effects of CPF208 and CPF209 on p16 expression in 124
cancer cells.
Figure 3.10 Effects of CPF213, CPF214 and CPF242 on p16 expression 126
in cancer cells.
Figure 3.11 Effects of CPF213, CPF214 and CPF242 on p16 expression 127
in Cf-Pac-1 pancreatic cancer cells.
x
Figure 3.12 Effects of thymidine and uridine on CPF213 induction of 132
p16 expression in Cf-Pac-1 pancreatic cancer cells.
Figure 3.13 Mechanism of action of cyclic deoxyzebularine 133
monophosphate dimer.
Figure 3.14 Effects of cyclic deoxy-(ZpZ) monophosphate on p16 136
expression in T24 bladder and Cf-Pac-1 pancreatic cancer
cells.
Figure 3.15 Effects of cyclic deoxy-(ZpT) monophosphate dimer on 139
p16 expression in Cf-Pac-1 pancreatic cancer cells.
Figure 3.16 Effects of TpZ dinucleotide on p16 expression in T24 140
bladder cancer and Cf-Pac-1 pancreatic cancer cells.
Figure 4.1 Re-expression of p16 and inhibition of DNA methylation 162
by S54, S55, and S56 in T24 bladder cancer cells.
Figure 4.2 Inhibition of DNA methylation and re-expression of p16 by 164
S110 in cancer cells.
Figure 4.3 Effects of S110 treatment on DNMT1 and p16 protein 166
levels in cancer cells.
Figure 4.4 Inhibition of DNMT1 in vitro by S110. 168
Figure 4.5 Fractionation of DNMT1 in nuclear extracts using sucrose 170
density gradient ultracentrifugation.
Figure 4.6 Re-expression of p16 and inhibition of DNA methylation 172
by S53 but not by S52 in T24 cancer cells.
Figure 4.7 Comparison of stability of 5-aza-2’-deoxycytidine and 173
S110.
Figure 4.8 Comparison of cytotoxicity of 5-aza-2’-deoxycytidine and 175
S110 in T24 bladder cancer cells.
Figure 4.9 Comparison of rate of enzymatic degradation of 5-aza-2’- 176
deoxycytidine and S110 by cytidine deaminase (CDA).
xi
Figure 5.1 Gender-specific effects of chronic zebularine 190
administration on the formation of intestinal polyps in
Apc
min/+
mice.
Figure 5.2 H&E staining of intestinal polyps after chronic zebularine 191
treatment of Apc
min/+
mice.
Figure 5.3 Gender- and tissue-specific demethylation by zebularine in 193
Apc
min/+
mice.
Figure 5.4 Effects of chronic administration of zebularine on the 195
weights of male and female Apc
min/+
mice.
Figure 5.5 H&E staining of liver after chronic zebularine treatment of 200
Apc
min/+
mice.
Figure 5.6 H&E staining of small intestine after chronic zebularine 201
treatment of Apc
min/+
mice.
xii
ABSTRACT
Aberrant DNA methylation is a common feature of cancer, which has been targeted for
pharmacologic intervention because of its reversible nature. Nucleoside analogs such as
5-azacytidine, 5-aza-2’-deoxycytidine, and zebularine are mechanism-based inhibitors
of DNA methylation that are incorporated into the DNA during replication and deplete
the DNA methyltransferase enzymes. 5-Azacytidine and 5-aza-2’-deoxycytidine are
extremely potent inhibitors and may be ideal for use in chemotherapy of cancer.
Delivery of these inhibitors has been facilitated with the use of short oligonucleotides
that prevent enzymatic degradation. On the other hand, zebularine, a more stable and
less toxic surrogate of the other two compounds may be best utilized as a
chemopreventive agent. Chronic oral administration of zebularine in a murine colon
cancer model highlighted its low toxicity and gender- and tissue-specific action against
DNA methylation.
1
CHAPTER 1
EPIGENETIC THERAPY OF CANCER
INTRODUCTION
‘Epigenetic’ is a term used to describe mitotically and meiotically heritable
states of gene expression that are not due to changes in DNA sequence (Bird, 2002).
Epigenetic events are important in all aspects of biology, and research during the
past decade has shown that they have a key role in carcinogenesis and tumor
progression.
Two of the most studied epigenetic phenomena are DNA methylation and
histone tail modifications. DNA is methylated by DNA methyltransferases (DNMTs)
at the 5-position (C5) of the cytosine ring, almost exclusively in the context of a CpG
dinucleotides, which are poorly represented in the genome overall due to
spontaneous deamination of 5-methyl-cytosine into thymine (Takai and Jones, 2002).
Low or lack of DNA methylation in the promoter region is correlated with gene
expression. Approximately 50% of genes are associated with CpG islands in their
promoter regions and these CpGs are usually low in methylation and capable of
transcriptional activation (CpG islands found elsewhere such as the body of genes or
other non-coding regions of the genome are sometimes methylated in somatic tissues
yet do not block transcription elongation). By contrast, methylation near the
transcription start site inhibits gene expression. This is mediated by the recruitment
2
of transcription repressors such as methyl-binding proteins (MBDs), which are a part
of a large complex that includes histone deacetylases (HDACs) (Fujita et al., 1999;
Hendrich et al., 1999). DNA methylation can also inhibit transcription directly by
blocking binding of transcriptional factors such as myc (Perini et al., 2005).
The mammalian genome is compacted in a hierarchical structure that
involves highly conserved, basic proteins known as histones. A nucleosome, the
fundamental unit of chromatin, is composed of the core histones, an octamer
consisting of H3/H4 tetramer and two H2A/H2B dimers, and 147 base pairs of DNA
wrapped around it. Histones function as an important structural component in the
nucleus and as a regulator of the gene expression profile in various tissue types. The
histone tails protrude out of the nucleosomes and are subject to a number of
posttranslational modifications including acetylation, methylation, phosphorylation,
ubiquitination, sumoylation, ADP-ribosylation, glycosylation, biotinylation, and
carbonylation (Strahl and Allis, 2000). These modifications influence how tightly or
loosely the chromatin is compacted, and thereby play a regulatory role in gene
expression (Jenuwein and Allis, 2001). For example, acetyl groups neutralize the
positive charges on the basic histone tails, thereby weakening electrostatic
interactions between the histones and the negatively-charged phosphate backbone of
DNA (Margueron et al., 2005).
The ‘histone code’ hypothesis states that a given modification of a specific
histone residue is a prerequisite for a modification on the same histone or a
neighboring histone (Jenuwein and Allis, 2001). It is this combination of
3
modifications that allows the gene expression status to switch interchangeably from
activation to repression and vice versa. In one of the first examples demonstrating
the cooperativity of histone modifications in regulation of gene expression,
phosphorylation of H3-S10 together with H3-K14 acetylation was shown to prevent
the methylation of H3-K9 (Lo et al., 2000).
Histone modifications promote or prevent binding of proteins and protein
complexes that drive that particular region of the genome into active transcription or
repression. Proteins containing bromo- and chromodomains have been shown to
have affinity for acetylated and methylated lysine residues, respectively. It is
hypothesized that the modifications would recruit proteins or protein complexes,
which in turn would cause the chromatin to take on a certain conformation and
spread the pattern to its neighboring region (Margueron et al., 2005). For example,
heterochromatin protein1 (HP1) is a protein responsible for the formation of
heterochromatin whose chromodomain interact with a trimethylated H3-K9 and
recruits SUV39H1. SUV39H1 is a histone H3-K9 methylase that subsequently
methylates the H3 tail of the adjacent nucleosome at lysine 9 to mediate HP1 binding
and further spreading of heterochromatic regions (Lachner et al., 2001).
Most notably, the acetylation of lysine residues on histones H3 and H4 is
correlated with active or open chromatin, which allow various transcription factors
access to the promoters of target genes. In contrast, deacetylation of lysine residues
by HDACs results in chromatin compaction and inactivation of genes.
4
Unlike histone lysine acetylation, histone lysine methylation may result in
either activation or repression, depending on the residue on which it resides. In this
way, specific modifications of histone tail residues can be used as “markers” of
transcriptionally active or inactive chromatin. For example, histone H3 lysine 4 (H3-
K4) methylation is a well known “active” marker. “Inactive” markers include
methylation of H3-K9 (Nakayama et al., 2001)and trimethylation of H4-K20
(Schotta et al., 2004). While only one acetyl group is added to the lysine residue at a
time, up to 3 methyl groups per lysine residue can be present. Interestingly, mono-
and dimethylated H3-K9 are found in silent regions located within euchromatic
genes, whereas trimethylated H3-K9 is enriched in pericentromeric heterochromatin,
suggesting that the different methylated states mark distinct domains of
heterochromatin (Rice et al., 2003). However, methylated H3-K9, along with
heterochromatin protein1 (HP1), has recently been found in the region of active
transcription in mammalian chromatin (Vakoc et al., 2005). The effect of
methylation on H3-K9 on transcription activation is not clear and further studies are
necessary to define its precise role.
It is widely accepted that histone modification and DNA methylation are
intricately interrelated, working together to determine the status of gene expression
and to decide cell fate (Hashimshony et al., 2003). A number of DNMT inhibitors
and HDAC inhibitors have been shown to have antitumor effects and are being tested
in clinical trials (Tables 1.1-1.3).
5
ENZYME FAMILIES INVOLVED IN EPIGENETIC REGULATION
Several families of proteins are responsible for establishing epigenetic
patterns: DNA methyltransferases (DNMTs), histone acetylases (HATs), histone
deacetylases (HDACs), histone lysine methyltransferases (HMTs), and possibly
histone demethylases. Although most of them function as a part of larger protein
complexes, these proteins interact directly with DNA or histone tails adding
modification at appropriate sites.
DNA methylation. DNA methyltransferases (DNMT) are responsible for de novo as
well as maintenance of methylation. Currently, there are five known human DNMTs,
DNMT1, 2, 3a, 3b and 3L, all of which have enzymatic function except for DNMT2
and DNMT3L that lack the N-terminal regulatory domain and the catalytic domain,
respectively (Goll and Bestor, 2004). DNMT1 is known as the maintenance
methyltransferase and binds preferentially to hemimethylated DNA whereas
DNMT3a and 3b are known as de novo methylases and bind to both hemimethylated
and unmethylated CpG sites. DNMT2 has been shown to contain the conserved
methyltransferase motif as the other methyltransferases but without significant
activity (Goll and Bestor, 2004). The DNMT3-like (DNMT3L) enzyme, although
lacking a catalytic domain, participates in de novo methylation of retrotransposons in
pre-meiotic spermatogonial stem cells (Bourc'his and Bestor, 2004)and establishment
of maternal imprints (Bourc'his et al., 2001).
6
Histone acetylation. Acetyl groups are added to the histone tails by the histone
acetyltransferases (HATs) which are comprised of three superfamilies, GNAT
(Gcn5-related N-acetyltransferase), MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60), and
p300/CBP (Gibbons, 2005), and removed by HDACs which are organized into three
separate classes. Class I HDACs include HDAC1, HDAC2, HDAC3, and HDAC8
and are localized in the nucleus. The class II HDACs includes HDAC4, HDAC5,
HDAC6, HDAC7, HDAC9, and HDAC10 and move between the nucleus and the
cytoplasm. The third class of HDACs are the human sirtuin (SIRT) enzymes that
have sequence similarity with a yeast transcriptional repressor called Sir2 (Gibbons,
2005). There are 7 human SIRT proteins (SIRT1 through 7) whose cellular
localization include various organelles depending on the function of each protein
(Michishita et al., 2005).
Histone methylation. Histones are methylated at lysine residues by a group of
enzymes called histone lysine methyltransferases (HMT) which are characterized by
a conserved SET domain (Gibbons, 2005). The SET domain proteins are divided into
4 subgroups, depending on the homology within the SET domain, and they are
SUV3, SET1, SET2, and RIZ (Schneider et al., 2002). Each subgroup contains
several proteins with the ability to methylate histones on lysine residues (Kouzarides,
2002). Currently, there are 17 lysine residues on histone tails at which methylation is
observed, however, there are several specific HMTs for methylation of residues such
7
as histone H3-K4 and H3-K9 while none have so far been identified for others.
Examples of well-known HMTs are: SET7/9, SUV39H1, G9a, Eu-HMTase1,
SETDB1, EZH2, DOT1L, Pr-SET7, and SUV4-20 (Bannister and Kouzarides, 2005).
At present, lysine specific demethylase 1 (LSD1) is the only known histone
demethylase enzyme (Shi et al., 2004).
8
CANCER EPIGENETICS
DNA methylation and cancer. DNA methylation is an epigenetic mechanism used
for long-term silencing of gene expression. It can maintain differential gene
expression patterns in a tissue-specific and developmental stage-specific manner,
although the relationship between tissue- and developmental stage-specific DNA
methylation and expression still remains controversial. The methylation pattern is
established during development and is normally maintained throughout the life of an
individual. However, in DNA of older individuals, it is common to see deviations
from the expected methylation pattern which may lead to less stringent control of
gene expression and increases the likelihood of genome instability. It has been
shown that such epigenetic mechanisms can be important in initiating tumorigenesis
and sustaining the malignant state of cancer cells (Chen et al., 2004). Both promoter
hypermethylation and genome-wide hypomethylation have been observed in cancer
and are responsible for transcriptional inactivation of genes and genome instability,
respectively. Global DNA hypomethylation is closely linked to chromatin
restructuring and nuclear disorganization in cancer cells, leading to chromosomal
instability (Hoffmann and Schulz, 2005). In addition, histologically normal tissues
obtained from healthy individuals have also been shown to contain promoter
hypermethylation (Holst et al., 2003) and inhibition of DNMT1 by zebularine has
been shown to be effective in a mouse xenograft model and against murine T-cell
9
Figure 1.1. Epigenetic gene silencing in cancer. In mammalian cells, both DNA
methylation and histone modifications contribute to gene silencing. A
transcriptionally active gene is marked by DNA hypomethylation of the promoter
region of the gene and acetylation of the lysine residues on histone tails, both of
which promote the formation of euchromatin. During tumorigenesis, DNA
methylation accumulates in the promoter region, which attracts methyl-binding
proteins such as MBD which is coupled to other repressor proteins and complexes
such as H3-K9 methylase SETDB1. In addition, increased activity of histone
deacetylases and histone methylases leads to loss of active markers such as H3-K4
acetylation and H4-K16 acetylation and accumulation of inactive markers such as
H3-K9 methylation. Changes in DNA methylation and histone tail modification lead
to silencing or reduced activity of a gene, which in the case of a tumor suppressor
gene may play a pivotal role in carcinogenesis. With the use of epigenetic drugs, the
process of heterochromatinization can be reversed and gene expression restored,
although the level of gene expression may not fully recover. HAT, histone
acetyltransferase; HDAC, histone deacetylase; DNMT, DNA methyltransferase;
HMT, histone methyltransferase.
10
TUMORIGENESIS
EPIGENETIC THERAPY
Methylated CpG
Unmethylated CpG
Phosphorylation
Acetylation
Methylation
HDAC
HDAC
HDAC
HMT
HMT HMT
MBD
MBD
MBD
HAT
HAT
HAT
HAT
HAT
DNMT
11
lymphoma (Cheng et al., 2003; Herranz et al., 2005), suggesting that DNA
methylation most likely plays a crucial role in setting the stage for carcinogenesis.
The potential reversibility of DNA methylation patterns suggests that these
are a viable target for the treatment of cancer. One specific goal of epigenetic therapy
is to restore normal DNA methylation patterns and to prevent the cells from
acquiring further methylation in DNA which may lead to silencing of genes critical
for normal cell function (Fig 1). Treatment of cancer cells with demethylating agents
can reactivate a group of genes such as p16, MLH1, and RB that are often crucial in
controlling cell proliferation, differentiation, apoptosis and other key homeostatic
mechanisms (Fig 2).
Inactivation of DNMTs is the most effective way of inhibiting DNA
methylation and re-establishing more normal patterns. However, targeting the
methyltransferase enzyme leads to the problem of loss of specificity and
hypomethylation of the genome. Therefore, the inhibition of DNMTs is not expected
to direct the reactivation of a specific set of genes, but rather, results in a general
decrease in overall methylation level which may reactivate several genes at random.
Nevertheless, decreased activity of DNMT resulting from the administration of
DNMT inhibitor, 5-aza-2’-deoxycytidine, has been shown to result in the abrogation
of tumorigenicity in a mouse model of neoplasia (Laird et al., 1995).
While promoter hypermethylation is observed in cancer, the rest of the
genome suffers from loss of methylation which was the first aberrant epigenetic
12
Figure 1.2. Reactivation of aberrantly silenced genes by DNA methylation
inhibitors. Promoter hypermethylation and aberrant gene silencing are characteristic
features of cancer. With the use of demethylating agents such as 5-Aza-CR or
zebularine, genome-wide demethylation is initiated, which then leads to reactivation
of methylation-silenced genes. Upregulation of cell-cycle regulators such as p16
leads to inhibition of growth. Cells become more chemosenstive with the activation
of repair genes such as MLH1 and cell adhesion can be increased due to E-cadherin
reactivation. Potential activation of transposons may be beneficial as it would
increase interferon response. Activation of tumor antigens may lead to increased
immunogenicity, which may be helpful when considering combining immunotherapy
with epigenetic drugs.
13
change observed in transformed cells (Feinberg and Vogelstein, 1983; Riggs and
Jones, 1983). The use of demethylating agents may aggravate the situation by further
decreasing the level of methylation and activation of genes that are potentially
deleterious i.e. oncogenes (Eden et al., 2003; Gaudet et al., 2003). There are those
who believe that the use of these drugs may lead to inadvertent consequences and
therefore remain cautious (Gius et al., 2004), and others who are in favor of using
demethylating agents since genes are preferentially upregulated in malignant cells
after drug treatment as shown by microarray studies (Cheng et al., 2004b; Liang et
al., 2002b). Others have employed novel, genome-wide scanning techniques that
have proven to be valuable in assessing the validity of targeting DNA methylation as
a treatment for cancer. For example, restriction landmark genomic scanning (RLGS)
has been used to show that a significant proportion of the genes are subject to
promoter hypermethylation with a specific pattern in cancer (Costello et al., 2000).
Much effort has been put into identifying CpG island methylator phenotype (CIMP)
with the final goal of constructing global methylation profiles for different cancer
types (Toyota et al., 2000). Recent work by Weber et al. on the genomic methylation
profile of normal and cancer cells has suggested that there is a relatively small subset
of differentially methylated promoters in prostate cancer (Weber et al., 2005).
Additional studies utilizing epigenomics to evaluate the implications of
demethylation on cancer may be useful in determining the effectiveness of the
therapy and in further implementing it in the clinic for both diagnosis and treatment.
14
Histone modification in cancer. Similar to DNA methylation, loss and gain of both
histone lysine acetylation and methylation are observed in cancerous cells, implying
that the general pattern across the genome is disturbed (Fraga et al., 2005). In cancer
cells, losses of both the acetylation of H4-K16 and trimethylation of H4-K20 and
disruption of the constitutive heterochromatin structure have been shown (Fraga et
al., 2005). Certainly, histone modification patterns are not identical among different
types of cancer or even at different stages of progression (Seligson et al., 2005) and
classifying tumor types into different epigenetic patterns may help to differentiate
them. Diagnosis of cancer by analyzing histone modification patterns may be
possible in the future, although the cooperative nature of histone modifications
makes this approach difficult.
Loss of lysine acetylation, rather than the increase in histone methylation, has
been identified as the first step in gene silencing (Mutskov and Felsenfeld, 2004) and
HDAC, which are responsible for removing acetyl groups from histones, have
become the major target for therapy. This again is a reiteration of the theme on DNA
methylation shown in Fig 1: prevention of gene silencing and restoration of a more
normal state of gene expression. Loss of acetylation does not only result in gene
silencing, but can also lead to decreased DNA repair (Masumoto et al., 2005),
accelerating molecular events leading to the development of cancer. Furthermore,
HDA-1, a class II HDAC homolog in C. elegans, is involved in the regulation of
tissue-specific and extracellular matrix-related genes that can play a role in cancer
progression (Whetstine et al., 2005). HDAC inhibitors have a specific antitumor
15
effect and as their mechanism of action remains elusive, the possibility remains that
these drugs work on currently unidentified targets rather than on histone acetylation.
This indicates the need to evaluate the effects of HDAC inhibitors thoroughly in
order to understand the full potential and mechanisms of action of these drugs.
Methylation on lysine residue of histones was considered a quite static
modification until LSD1, a lysine-specific demethylase, was reported in 2004 (Shi et
al., 2004). The original report on LSD1 reported its ability to demethylate an active
marker, H3-K4, which leads to the downregulation of its target genes. However, an
independent study showed that LSD1 specifically colocalizes with the androgen
receptor (AR) to remove H3-K9 methylation and to activate AR target genes
(Metzger et al., 2005). The search for other histone demethylases is an area of
intense investigation as it is most likely that each enzyme interacts with a specific
lysine or arginine residue containing different degrees of methylation, and it may
even be possible that these demethylases interact with specific transcription factors,
similar to LSD1/AR interaction. Therefore, the use of histone demethylase inhibitors
in epigenetic therapy is not ideal for a number of reasons. As discussed, the methyl
marks exist as both active and inactive markers, which make it difficult to target for
inhibition of histone demethylases, as in the case of LSD1. For example, it may be
difficult to determine whether the inhibition of LSD1 would be beneficial for
patients since the enzyme demethylates both H3-K4 and H3-K9. There has been a
report of an abnormal telomere elongation which resulted after the loss of H3-K9
methylation in mice (Garcia-Cao et al., 2004) so the removal of an inactive marker
16
may prove beneficial or detrimental depending on the target region. Furthermore, it
appears that the loss of histone acetylation is the primary event leading to gene
silencing, whereas the accumulation H3-K9 methylation plays a secondary role
(Mutskov and Felsenfeld, 2004). It may be a while before the entire family of histone
demethylases is identified, the significance each enzyme plays in making up the
entire epigenetic patterns is fully understood, and the therapeutic potential of
targeting each enzyme, if any, is established.
DNA METHYLATION INHIBITORS
Nucleoside analogs. There are two classes of DNA methylation inhibitors:
nucleoside analogs and non-nucleoside analogs (Table 1). Nucleoside analogs have a
modified cytosine ring that is attached either to a ribose or a deoxyribose. They are
metabolized by kinases that convert the nucleosides into nucleotides for
incorporation into DNA and/or RNA. DNA methylation is thought to be inhibited
when the compounds are incorporated into DNA.
Ribonucleoside analogs such as 5-azacytidine (5-aza-CR) and zebularine are
phosphorylated by uridine/cytidine kinase and other kinases into a corresponding
mono-, di-, and triphosphate, which ultimately end up in RNA. The effects of RNA
incorporation of these compounds have not been well-studied. However,
ribonucleotide diphosphates can also be reduced by ribonucleotide reductase into a
deoxy-diphosphate which can then subsequently be incorporated into DNA. The
17
Table 1.1. DNA methylation inhibitors: nucleoside analogs.
Inhibitor Structure [Range] Clinical Trials
Nucleoside Analogs
5-Azacytidine
O
OH OH
H H
H H
HO
N
N
N
NH 2
O
µM
I, II, III:
Hematologic
malignancies
5-Aza-2’-
deoxycytidine
O
H OH
H H
H H
HO
N
N
N
NH 2
O
µM
I, II, III:
Hematologic
malignancies,
cervical,
NSCLC
5-F-2’-
deoxycytidine
O
H OH
H H
H H
HO
N
N
NH 2
O
F
µM I
5,6-Dihydro-5-
azacytidine
O
OH OH
H H
H H
HO
HN
N
N
NH 2
O
µM
I, II: Ovarian
and
lymphomas
Zebularine
O
OH OH
H H
H H
HO
N
N
O
µM~mM Preclinical
18
deoxyribonucleoside analogs, such as 5-aza-2’-deoxycytidine (5-aza-CdR) and 5-
fluoro-2’-deoxycytidine (5-F-CdR) are phosphorylated by deoxycytidine kinase and
other kinases and are incorporated into DNA. The deoxy- form of zebularine is not a
good substrate for deoxycytidine kinase and zebularine is only incorporated into
DNA via the ribonucleotide reductase pathway, which serves as a rate liming step for
the entire process. Furthermore, unlike 5-aza-CR and 5-aza-CdR which are subject to
deamination by cytidine deaminase and deoxycytidine deaminase, respectively, and
are thus rendered inactive, zebularine also acts as an inhibitor of the enzyme cytidine
deaminase therefore not subject to deactivation.
During DNA replication, the DNMTs flip out cytosine rings from the double
helix to form an intermediate complex in which S-adenosyl-
L
-methionine (Ado-Met)
is incorporated (Erlanson, 1993; Klimasauskas et al., 1994; Wu and Santi, 1987). In
a normal cytosine ring, a methyl group is transferred from Ado-Met to the C5 of the
base and the enzyme is released in a-elimination reaction (Erlanson, 1993) (Fig 3).
In the case of cytosine analogs, the modification at C5 of the ring prevents the
release of the enzyme and results in the formation of a covalent complex (Santi et al.,
1983; Santi et al., 1984; Zhou et al., 2002). This prevents further methylation of the
genome, and as a result, the DNA of the progeny cells is not methylated.
Interestingly, demethylating agents do not target cells for immediate death as do
most other chemotherapeutic drugs and the cells must be allowed to proliferate and
reactivate genes that have been methylation-silenced for these drugs to take effect.
Reactivation of genes including apoptotic genes and cell-cycle regulators takes place
19
Figure 1.3. Mechanism-based inhibition of DNA methylation by cytosine
analogs. The methylation of the 5-position (C5) of the cytosine ring in DNA is a
highly ordered process. A. During this process, a DNMT transfers a methyl group
from S-adenosyl-
L
-methionine to C5 of the cytosine residue in DNA. The cysteine
thiolate in the catalytic pocket of a DNMT is conjugated to the 6-carbon of the
cytosine ring, which is followed by transfer of a methyl group to C5. The free
enzyme is released from the substrate by a-elimination and moves to the next CpG
site. B. 5-Azacytidine (5-Aza-CR) and 5-Aza-2’-deoxycytidine (5-Aza-CdR).
Mechanism-based inhibitors of DNMT undergo one or more of the catalytic
reactions and form covalent complexes with the enzyme. In the case of 5-Aza-CR,
the catalytic thiolate reacts with the 6-carbon to form the covalent complex which is
followed by a methyl transfer by S-adenosyl-
L
-methionine (AdoMet), however, the
lack of H at C5 prevents the restoration of the trapped enzyme. It has been
demonstrated that the covalent complex may form in the presence or absence of
AdoMet, suggesting the nonessential role it plays in the inhibition of these enzymes.
The inhibition mechanism of 5-Aza-CdR is identical to that of 5-Aza-CR. C. 5-
Fluoro-2’-deoxycytidine. The inhibition by 5-F-CdR is mediated by the presence of
fluorine atom at C5 which prevents the covalent complex from releasing the enzyme.
D. Zebularine. The covalent complex persists with zebularine due to the absence of
an amino group at C4.
20
N
N
H
H O
N
H H
H
O
O
Enz
-S-Enz
N
N
H
H O
N
H H
H
O
O
Enz
S-Enz
S
R
H
3
C R'
N
N
H
H O
N
H H
CH
3
S-Enz
N
N O
N
H H
CH
3
H
-S-Enz
A.
B. C. D.
O
H O
H H
H H
N
N
N
NH
2
Enz
CH
3
O
O
O
H O
H H
H H
N
N
NH
2
Enz
CH
3
F
O
O O
H O
H H
H H
N
N Enz O
H
H
O
21
after the initial DNA methylation inhibition, which then leads to cell death and cell-
cycle arrest among other things. For that reason, epigenetic drugs have the
advantages of low dose regimen and decreased toxicity, but they also have the
disadvantage of having transient effects as the aberrant patterns may return with the
removal of the drug, allowing the malignant cell population to reappear. Recent work
has suggested that DNMT1 becomes a target for proteasomal degradation following
5-aza-CdR treatment (Ghoshal et al., 2005), although the incorporation of drug into
DNA followed by a covalent bond-formation seems to be the major pathway through
which the enzyme is inhibited (Schermelleh et al., 2005).
5-Aza-CR and 5-aza-CdR are two of many cytidine analogs with anticancer
properties (Pliml and Sorm, 1964; Sorm and Vesely, 1968) that can also induce
cellular differentiation and inhibit DNA methylation (Jones and Taylor, 1980; Taylor
and Jones, 1979). These agents are extremely potent in inhibiting DNA methylation
at micromolar concentration; however, their short half lives in aqueous solution (Lin
et al., 1981; Notari and DeYoung, 1975) complicate the delivery of these drugs. 5-
Aza-CR and 5-Aza-CdR have been widely studied for the treatment of hematologic
diseases; 5-Aza-CR and 5-Aza-CdR received FDA approval for the treatment of
myelodysplasia in 2004 (Kaminskas et al., 2005), and 5-Aza-CdR is now undergoing
phase III studies. For years, these drugs were escalated to maximum tolerated doses
(MTD), although in vitro experiments had shown that optimal methylation inhibition
occurred at low doses (Jones and Taylor, 1980). Recent clinical trials have confirmed
that low-dose exposures lead to greater responses and are associated with less
22
toxicity (Issa et al., 2004; Issa et al., 2005; Lubbert et al., 2004). Ongoing clinical
trials are exploring the use of low doses of DNA methyltransferase inhibitors for the
treatment of solid tumors including melanoma and cancers of breast, renal cell, colon,
and bladder (Aparicio et al., 2003). Many investigators have combined DNA
methylation inhibitors and HDAC inhibitors and shown synergistic tumor cell
growth inhibition and gene reexpression (Cameron et al., 1999; Rudek et al., 2005)
and it may be advantageous to use both drugs for the treatment of solid tumors
(Rudek et al., 2005)since treatment of DNA methylation inhibitors alone in solid
tumors have not been successful to date.
Dihydro-5-azacytidine (DHAC) is hydrolytically more stable (Beisler et al.,
1976; Beisler et al., 1977) and less cytotoxic (Presant et al., 1981; Stopper et al.,
1995) than 5-Aza-CR and has been shown to be an inhibitor of DNA methylation in
human lymphoid and leukemia cell lines as well as in tumor-bearing mice
(Antonsson et al., 1987; Jones and Taylor, 1980; Kees and Avramis, 1995; Powell
and Avramis, 1988). However, DHAC has received mixed reviews on its efficacy in
both phase I and II studies, leading to a discontinuation of clinical studies on this
compound (Curt et al., 1985). 5-F-CdR is another compound with antitumor and
demethylating properties (Eidinoff et al., 1959; Jones and Taylor, 1980). Although
the compound has proven to be useful in elucidating mechanism of inhibition of
DNMTs by cytosine analogs (Chen et al., 1991), 5-F-CdR is currently undergoing
phase I studies although its complicated metabolic activity in mammalian cells may
be problematic.
23
Zebularine or 1--D-ribofuranosyl-2(1H)-pyrimidinone is the most recent
addition to the list of demethylating agents in the family of nucleoside analogs. It
was first synthesized in 1961 and characterized as a potent inhibitor of cytidine
deaminase (Barchi et al., 1995; Frick et al., 1989; Kim et al., 1986; Laliberte et al.,
1992) with anticancer properties (Driscoll et al., 1991), however its demethylating
activity was not recognized until 2003 (Cheng et al., 2003). Zebularine is stable at
acidic and neutral pHs and in aqueous solution and has been shown to be less toxic
than the 5-aza-nucleosides in cultured cells making oral administration possible
(Cheng et al., 2003). However, zebularine’s near millimolar dose requirement
(Cheng et al., 2003), mutagenicity (although only demonstrated in E. coli to date
(Lee et al., 2004)), and limited bioavailability in rodents and primates (Holleran et al.,
2005) has kept it from swift integration into the clinic. Future work should focus on
the development of a zebularine pro-drug which will circumvent these metabolic
limitations.
Non-nucleoside analogs. Currently there are a handful of non-nucleoside analogs
that are known to inhibit DNA methylation and only a few have made it to clinical
trials, but active research in this field will probably lead to the introduction of more
compounds into this class in the near future. These small molecule inhibitors may be
more useful than the nucleoside analogs in the clinic, since they inhibit DNA
methylation by binding directly to the catalytic region of the enzyme, without
incorporation into DNA (Brueckner et al., 2005; Schuebel and Baylin, 2005). RG108
24
Table 1.2. DNA methylation inhibitors: non-nucleoside analogs.
Inhibitor Structure [Range] Clinical Trials
Non-Nucleoside Analogs
Hydralazine
N N
HN
H 2 N
µM I: Cervical
Procainamide
H 2 N
O
HN
N
µM Preclinical
EGCG
O HO
OH
O
H
H
O
OH
OH
OH
OH
OH
OH
µM Preclinical
Psammaplin A
N
H
N
O
HO
S S
H
N
N
OH
Br
OH
OH
Br
O
nM~µM Preclinical
MG98 N/A N/A
I: Advanced/meta-
static solid tumors
RG108
N
O
O
HO
H
NH
O
µM Preclinical
25
is a small molecule inhibitor designed specifically to fit into the catalytic pocket of
the human DNMT1 and render the enzyme inactive (Brueckner et al., 2005). MG98
is an antisense oligonucleotide of human DNMT1, which prevents the translation of
DNMT1 mRNA. However, MG98 demonstrated no antitumor activity in various
solid cancers and no dose-related effects have been observed in phase I studies
although a phase II study is underway (Davis et al., 2003; Stewart et al., 2003).
Naturally occurring DNA methylation inhibitors include psammaplins, a family of
bromotyrosine derivatives extracted from the sponge Pseudoceratina purpurea,
which have been shown to possess the ability to inhibit both DNA
methyltransferases and histone deacetylases (Pina et al., 2003) and EGCG ((-)-
epigallocatechin-3-gallate) or green tea extract (Fang et al., 2003), both of which
have not yet made it to the clinic as inhibitors of DNA methylation. Other drugs
include hydralazine, procainamide, and procaine(Alikhani-Koopaei et al., 2004;
Cornacchia et al., 1988; Lin et al., 2001; Segura-Pacheco et al., 2003; Villar-Garea et
al., 2003). Recently, a phase I study of hydralazine was conducted (Zambrano et al.,
2005), however it is controversial as to how competent hydralazine, procainamide,
and EGCG are in inhibiting DNA methylation; these drugs may not be as robust
inhibitors as the nucleoside analogs and may work better in some system than others
(Chuang et al., 2005).
26
Figure 1.4. Effects of HDAC inhibitors in human cancer. HDAC inhibitors
prevent hypomethylation of histones, which leads to chromatin remodeling,
transcriptional activity, and restoration of malignant cells to a more normal state.
Almost all HDAC inhibitors are known to induce the p21
WAF1/CIP1
gene expression,
which leads to the inhibition of the CyclinD/CDK4 complexes and cell-cycle arrest
and differentiation. HDAC inhibitors are also known to have anti-angiogenic effects
and promote apoptosis. Many pathways may be involved in promoting growth
inhibition, differentiation, apoptosis and anti-angiogenesis.
HDAC Inhibitors
Cell Cycle Arrest/
Differentiation
Apoptosis
p21
WAF1/CIP1
Anti-Angiogenesis
CyclinD
CDK4
27
HISTONE DEACETYLASE INHIBITORS
HDAC inhibitiors are divided into four groups: short-chain fatty acids,
hydroxamic acids, cyclic tetrapeptides, and benzamides (Table 2). However, the
development of molecules containing a novel or combination of functional groups to
improve potency as well as specificity is continuing. Each class of HDAC inhibitors
possesses different functional groups; these agents inhibit histone deacetylase
enzymes, which leads to the accumulation of acetylation in histones, followed by
changes in cellular processes that have become defective in cancerous cells (Fig 4).
The exact mechanism through which these drugs mediate anti-tumor activity
has not been elucidated, although there are many speculations as to which cellular
pathways are involved. One model suggests that hyperacetylation of histones
activates tumor suppressor genes and represses oncogenes. For example, HDAC
inhibitors have been shown to induce cyclin-dependent kinase (CDK) inhibitors,
such as p21, which are responsible for cell-cycle arrest in G1 and G2 phases and
subsequent cell differentiation (Rocchi et al., 2005). They can also activate the death
receptor-mediated and intrinsic apoptotic pathways (Nebbioso et al., 2005; Peart et
al., 2005) in which various factors are involved, including NF-B (Shetty et al.,
2005), c-JNK (Dai et al., 2005), and bcl-2 (Duan et al., 2005). Furthermore, they can
change the expression of genes involved in the inhibition of angiogenesis (Michaelis
et al., 2004) and metastasis (Joseph et al., 2004). Other possible mechanism include
hyperacetylation of histones leading to genomic instability, which ultimately triggers
28
Table 1.3. Histone Deacetylase inhibitors: short-chain fatty acids and
hydroxamic acid.
Inhibitor Structure [Range]
Clinical
Trials
Short-Chain Fatty Acids
Butyrate
OH
O
mM
I, II:
Colorectal
Valproic acid
O
OH
mM
I: AML,
leukemias
Hydroxamic Acids
m-Carboxy
cinnamic acid
bishydroxamic
acid (CBHA)
HO
N
H
N
H
OH
O O
µM Preclinical
Oxamflatin
S
NH
N
H
OH
O
O
O
µM Preclinical
PDX 101
H
N
S
H
N
O
OH
O O
µM I
Pyroxamide
N
H
N
N
H
OH
O
O
nM Preclinical
29
Table 1.3. Histone Deacetylase inhibitors: short-chain fatty acids and
hydroxamic acid, continued.
Inhibitor Structure [Range]
Clinical
Trials
Hydroxamic Acids
Scriptaid
N
O
O
H
N
OH
O
µM Preclinical
Suberoylanilide
hydroxamic acid (SAHA)
H
N
N
H
O
O
O
µM
I, II:
Hematologic
and solid
tumors
Trichostatin A (TSA)
N
N
H
O
O O
nM Preclinical
LBH589 N/A nM I
NVP-LAQ824
HN
N
O
H
OH
nM I
30
the cell-cycle checkpoint (Qiu et al., 2000). HDAC inhibitors also lead to acetylation
of non-histone proteins, including tumor suppressors and transcription factors such
as p53 (Gu and Roeder, 1997) and NF-B (Chen and Greene, 2003).
Short-chain fatty acids. While short-chain fatty acids (SCFAs) generally are not
very potent in inhibiting HDACs, requiring millimolar concentrations, they have
become a useful tool in studying the structure and mechanism of HDAC inhibitors.
Butyrate, first synthesized in 1949 (Stadtman and Barker, 1949), and valproic acid
(Gottlicher et al., 2001), an anti-epileptic drug, were the first known HDAC
combination with all-trans retinoic acid in elderly patients with acute myelogenous
leukemia (Raffoux et al., 2005). In combination with 5-Aza-CdR, it showed
promising results in in vitro experiments with various leukemic cell lines (Yang et al.,
2005). Valproic acid has been shown to inhibit HDAC1 and HDAC2 by decreasing
catalytic activity and proteasomal degradation, respectively (Kramer et al., 2003).
However, SCFAs are highly unspecific, cause pleiotropic effects on other enzymes
and have low bioavailability (Perrine et al., 1994), making them less desirable for
clinical use. Nevertheless, studies on SCFA should be continued as these compounds
are already established and widely used in the clinic, can be administered orally, and
have shown promising results (Raffoux et al., 2005; Yang et al., 2005). Trybutyrin, a
more stable analog of sodium butyrate, has been synthesized (Perrine et al., 1994)
and is reported to be anti-proliferative and anti-apoptotic (van de Mark et al., 2003).
31
Hydroxamic acids. Hydroxamates are potent inhibitors of HDACs, and are active at
micromolar to subnanomolar concentrations. Trichostatin A (TSA) derived from
Streptomyces was first shown to be a potent inducer of differentiation and cell-cycle
arrest, and later reported to possess anti-HDAC activity (Yoshida et al., 1990). TSA
was shown to act synergistically with the demethylating agent 5-Aza-CdR in a
mouse cancer model, where it inhibited tumor growth and promoted gene
reactivation (Cameron et al., 1999).
Suberoylanilide hydroxamic acid (SAHA) is a synthetic inducer of
differentiation in transformed cells and has yielded promising results when given
orally and intravenously in both solid and hematologic malignancies in phase I
studies (Kelly et al., 2003). It was also shown to be safe in a chronic oral treatment
and to be effective against advanced cancer (Kelly et al., 2005). SAHA inhibits
HDAC by binding to a zinc ion in the catalytic domain of the enzyme, thereby
preventing the deacetylation of histones (Finnin et al., 1999) and it is postulated that
other hydroxamates work in a similar fashion. Other hydroxamates and hydroxamic
acid-containing compounds with anti-proliferative and HDAC inhibitory properties
include m-carboxycinnamic acid bishydroxamide, oxamflatin, scriptaid, pyroxamide,
and PDX-101, all of which are in preclinical stage except for PDX-101, which is in a
phase I study (Monneret, 2005). LDH589 and NVP-LAQ824 are among many
HDAC inhibitors identified by Novartis in an archival screen for HDAC inhibitors;
they are known to cause hyperacetylation and induction of apoptosis. Currently both
compounds are undergoing phase I studies (Remiszewski, 2003).
32
Table 1.4. Histone deacetylase inhibitors: cyclic tetrapeptides.
Inhibitor Structure [Range]
Clinical
Trials
Cyclic Tetrapeptides.
Apicidin
N
H
NH
H
N
N
O
O
O
O O
N
OCH 3
nM Preclinical
Depsipeptide
(FK-228,
FR901228)
NH
NH
H
N
HN
O
O
O
O
O
O
S
S
µM
I,II: CLL,
AML, T-cell
lymphoma
TPX-HA
analogue
(CHAP)
NH
NH N
HN
O
O
O
O
H
N
OH
O
nM Preclinical
Trapoxin NH
NH N
HN
O
O
O
O
O
O
nM Preclinical
33
Cyclic tetrapeptides. Cyclic tetrapeptides also inhibit HDACs at very low
concentrations and it is thought that the epoxyketone group alkylates the catalytic
pocket of HDACs (Kijima et al., 1993). Depsipeptide, the best known HDAC
inhibitor from this group, has antitumor activity against chronic lymphocytic
leukemia, acute myelogenous leukemia, and other refractory neoplasms and is
currently undergoing a phase II study for the treatment of T-cell lymphoma (Byrd et
al., 2005; Marshall et al., 2002; Piekarz et al., 2001; Sandor et al., 2002). Trapoxin is
known for irreversible inhibition of HDACs, possibly by binding covalently to
HDACs via the epoxides (Remiszewski et al., 2003) but has not been used clinically
due to its instability and toxicity (Monneret, 2005). The-epoxyketone moiety is not
essential in inhibiting the enzyme as in the case for apicidin, a fungal metabolite with
antiprotozoal and HDAC-inhibitory activities (Singh et al., 2001), which led to the
synthesis of a hybrid compound, CHAP, a Trapoxin analog which replaces the
epoxyketone with a hydroxamate (Furumai et al., 2001). Other cyclic tetrapeptide
analogs containing functional groups such as trifluoromethyl and pentafluoroethyl
ketones, retrohydroxamate, and sulfhydryls, all directed at the zinc ion in the HDAC
catalytic domain, have been synthesized and have demonstrated their potent HDAC
inhibitory effects in vitro (Jose et al., 2004; Nishino et al., 2003; Nishino et al., 2004).
Benzamides. MS-275 and CI-994 are two of the most well-known synthetically
derived inhibitors of HDACs in this group. The benzamide group of MS-275 inhibits
HDACs by binding to the catalytic zinc ion but the mechanism of inhibition by CI-
34
Table 1.5. Histone deacetylase inhibitors: benzamides.
Inhibitor Structure [Range]
Clinical
Trials
Benzamides
CI-994 (N-
acetyl
dinaline)
O
HN
O
HN
H 2 N
µM
I, II: Solid
tumors
MS-275
N
O N
H
O
H
N
O
NH 2
µM
I, II: Solid
tumors and
lymphoma
35
994 is not known. MS-275 has shown antitumor activity in mouse xenograft models
when administered orally (Saito et al., 1999) and also has radiosensitizing properties
(Camphausen et al., 2004). Activation of the retinoic acid receptor2 by MS-275
has proven to be effective against renal cell carcinoma and prostate cancer cells
(Wang et al., 2005), and the drug is currently undergoing phase II studies. CI-994
has the advantage of possible oral administration and has been used in several phase
I studies, independently and in combination with other chemotherapeutic agents
(Pauer et al., 2004; Prakash et al., 2001; Undevia et al., 2004).
36
PERSPECTIVES
Epigenetic drugs, whether demethylating agent or HDAC inhibitor, target
aberrantly heterochromatic regions, leading to reactivation of tumor suppressor
genes and/or other genes critical for the normal function of cells (Yoo et al., 2004).
The use of these drugs can be therapeutic per se or in combination with other
therapeutic modalities, such as chemotherapy, immunotherapy, or radiotherapy. For
example, the reactivation of tumor suppressor genes and restoration of DNA repair
pathways by epigenetic drugs result in more chemosensitive cells, which can then be
targeted by another type of therapy. Epigenetic drugs may also help to alleviate the
resistance to other drugs by reactivating DNA repair genes such as MLH1 and
MGMT. Conventional chemotherapeutic drugs such as cisplatin have been used in
combination with Aza-CdR in patients with solid tumors. Although only moderately
successful, these strategies may prove to be an alternative to those who remain
resistant to other forms of therapy (Pohlmann et al., 2002; Schwartsmann et al.,
2000).
Coupling epigenetic drugs with immunotherapy is another way that these
drugs may prove beneficial. Among the genes reactivated by epigenetic drugs are a
group of genes called cancer/testis antigens (CTAs) that lead to increased
immunogenicity. It may be more advantageous to employ a combinatorial approach
of using epigenetic therapy with other types of therapy since demethylating agents
only have a transient effect on treated cells and abnormal methylation patterns return
37
with the removal of the drug. Therefore, epigenetic therapy is two-faceted in its use
in chemotherapy of cancer: first, it can be used to reactivate tumor suppressor genes
and to restore the normal function of cells, and second, it can be used in combination
with other drugs to increase the efficacy of existing therapies.
Epigenetic therapy may also be useful for chemopreventive approaches,
especially for those individuals who have been diagnosed with aberrant epigenetic
alterations but have not yet acquired neoplastic lesions. Epimutations, or aberrant
DNA methylation and histone modification patterns, are observed in individuals with
no history of malignancy (Holst et al., 2003) and may be used as an indicator of the
likelihood of developing cancer (Laird, 2003). If these epimutations are corrected
with DNA methylation and HDAC inhibitors, it may delay or completely prevent
tumorigenesis in these individuals. Having a detailed map of specific epigenetic
patterns in each tissue type in their normal and in cancerous states would make
detection of premalignant epimutations feasible, even from as little as a drop of
blood (Laird, 2003). Furthermore, comprehensive knowledge of the epigenome
would open up a new avenue for development of various drugs designed to target a
specific region of the genome in which an epimutation has occurred. However, the
lack of specificity is a disadvantage of current epigenetic drugs, and the development
of highly specific drugs targeting a subset of these epigenetic modifiers or a small
region of the genome are anticipated in the near future.
It will be important to identify and elucidate the exact role of the key players
involved in generating the epigenetic patterns. Although several DNMTs are known,
38
we still do not understand the precise function of each enzyme. Other members of
this class may remain to be identified, possibly including different isoforms of
DNMT3 (Goll and Bestor, 2004). Many histone acetylases and deacetylases have
been identified but specific inhibitors targeted for each individual deacetylase remain
to be discovered. The list of histone demethylases is far from complete – a new
histone demethylase has just been discovered and exciting results are anticipated
(Kubicek and Jenuwein, 2004). Only with the complete understanding of these
epigenetic modifiers will the development of the most effective therapies be possible.
With such knowledge of epigenetics at our disposal, we may have the full capacity to
not only treat but to prevent cancer.
39
OVERVIEW OF THESIS RESEARCH
As discussed above, aberrant hypermethylation of promoter regions of
regulatory genes and tumor suppressor genes have become an ideal target for
chemotherapeutic intervention of cancer due to their labile nature. Mechanism-based
inhibitors of DNA methylation such as 5-aza-CR and 5-aza-CdR are now routinely
used for the treatment of hematological malignancies. With the advent of more stable
and less toxic compounds, chemoprevention of cancer may be possible in the near
future. This thesis highlights the use of DNA methylation inhibitors as
chemotherapeutic and chemopreventive agents, suggesting that epigenetic therapy
may offer a promising therapeutic alternative to cancer patients.
Chapter 2 describes a thorough characterization of zebularine as a novel
inhibitor of DNA methylation. We identified key factors that facilitate the DNA
incorporation of zebularine in cultured cells. First, we found that naturally occurring
cytosine nucleoside can completely inhibit the incorporation of zebularine into DNA.
In addition, the amount of fetal bovine serum used in growth medium can
dramatically affect the incorporation rate of the drug. We also showed that folic acid
is essential for active cell division as well as driving the activation of zebularine. The
comparison of continuous zebularine treatment of normal fibroblasts and cancer cell
lines demonstrated that zebularine is preferentially active in cancer cells in terms of
DNMT depletion, hypomethylation of methylated loci, and global as well as
individual gene expression. This is due to variable rates of DNA incorporation of
40
zebularine and partly due to the differential activity of enzyme uridine/cytidine
kinase in these cells. Global gene expression profiles of normal and cancer cells after
zebularine treatment showed that only a handful of genes were affected, suggesting
that this drug is relatively non-toxic. Lastly, we obtained a group of marker genes
called cancer/testis antigens (CTAs) from microarray analysis of normal and cancer
cells, which may be useful targets of immunotherapy as well as providing important
clues regarding the drug resistance of cancer cells.
In Chapter 3, we screened a large panel of compounds for a zebularine
pronucleotide. Our initial approach began with various derivatives of zebularine and
cytidine analogs, which proved to be futile. We then attempted to bypass the
metabolic hurdles posed by uridine/cytidine kinase and ribonucleotide reductase by
delivering the monophosphate moiety of deoxy-zebularine. The deoxy-zebularine
monophosphate moiety containing a cycloSal protecting group was not active in T24
bladder cancer, HCT15 colon cancer, and Cf-Pac-1 pancreatic cancer cells. However,
the deoxy-zebularine monophosphate phosphoramidates were found to be more
potent inhibitors of DNA methylation than zebularine in Cf-Pac-1 pancreatic cancer
cells when supplemented with thymidine. In order to overcome the thymidine
requirement, we tested cyclic deoxy zebularine monophosphate dimers and TpZ
dinucleotides. These two classes of compounds activated the p16 gene more robustly
than zebularine in the presence of thymidine. Although we were not successful in
identifying a single agent with greater potency than zebularine, this study provided
41
us with invaluable information regarding the metabolism of zebularine and cellular
response to demethylating agents.
In Chapter 4, we synthesized and characterized short oligonucleotide DNA
methylation inhibitors and introduced a novel way of delivering nucleoside analogs
to cells. Traditionally, oligonucleotides that are therapeutically used have sequence
specificity that then renders the efficacy of the compound by their complementarity.
In our approach, we utilized the transient stability afforded by the longer chain
oligonucleotides to deliver the target compound while protecting it from enzymatic
degradation. We characterized a family of oligonucleotides containing one or more
molecules of 5-aza-CdR in the sequence which are comparable to the potency of 5-
aza-CdR to inhibit DNA methylation but are not subject to deamination by cytidine
deaminase. These oligonucleotides are most likely cleaved into individual
nucleosides and nucleotides once taken up into the cells and salvaged during DNA
synthesis. This approach may be most useful in clinical setting. In addition,
applications of short oligonucleotides may be employed in many existing therapies.
In the final chapter, we tested the chemopreventive property of zebularine
using a murine colon cancer model and examined the effects of chronic zebularine
administration in normal tissues. Zebularine was administered ad libitum in the
drinking water to APC
min/+
mice, which are prone to polyp formation in the small
intestine. After 113 days of zebularine administration, the majority of mice did not
exhibit any sign of toxicity, as measured by their body weight and the outward
appearance. Hematoxylin and eosin staining of intestines and liver of both gender
42
showed that the architecture of these tissues were normal in all groups, attesting to
the low level of toxicity by chronic zebularine treatment. The average number of
polyps in males remained unaffected, while the average polyp number decreased
from 58 to 1 in females. This result indicates that zebularine had prevented the
formation of polyps in a gender-specific manner. H&E staining of intestines showed
that there were microscopic lesions in the guts of female mice, although undetectable
with a dissecting microscope. This suggests that zebularine had slowed down the
polyp formation but did not prevent it completely. The gender-specific response to
zebularine may be attributed to differential activity of liver enzyme aldehyde oxidase
which converts zebularine into uridine. DNA methylation analysis of B1 elements
(SINEs) by pyrosequencing and of A repeats (LINEs) by Ms-SNuPE detected
demethylation of DNA from small and large intestines of treated female mice.
Microarray analysis of gene expression profile of colonic epithelia of female mice
showed that less than 5% of the entire genome was affected as a result of zebularine
treatment. Taken together, our data suggest that zebularine did not disturb the normal
structure and function of colon while causing a notable decrease in methylation level.
The low toxicity and high stability of zebularine shown here make it an attractive
candidate for cancer chemotherapeutic agent, as well as a potentially safe alternative
for chemoprevention of cancer.
Our studies have demonstrated the potential utility of DNA methylation
inhibitors in chemotherapy and chemoprevention of cancer. They have provided
insights in to the use of the novel compound, zebularine, in cultured cells, as well as
43
in mice. In addition, our studies have lead to the identification and characterization
of prodrugs with improved stability and potency, giving us invaluable information
with biological and clinical implications.
44
CHAPTER 2
MECHANISM OF ACTION OF ZEBULARINE
INTRODUCTION
Tumor suppressor genes are frequently silenced as a result of epimutation i.e.
aberrant DNA methylation and chromatin structural changes (Jones and Baylin,
2002). Since DNA methylation is reversible in nature, demethylating agents have
become promising candidates for treatment of human cancers in which there is
aberrant hypermethylation of tumor suppressors or genes critical for a normal
function (Yoo and Jones, 2006).
The most widely used inhibitors of DNA methylation are 5-azacytidine (5-
aza-CR) and 5-aza-2’-deoxycytidine (5-aza-CdR). These compounds were first
synthesized as cancer chemotherapeutic agents and later characterized as
demethylating agents (Jones and Taylor, 1980; Sorm et al., 1964). 5-Aza-CR is
activated by uridine/cytidine kinase (UCK) and incorporated into both RNA and
DNA. On the other hand, 5-aza-CdR is phosphorylated by cytidine kinase and is
incorporated only into DNA (Bouchard and Momparler, 1983). Once in the DNA,
both compounds form covalent complexes with DNA methyltransferases (DNMTs),
leading to the inhibition of active enzymes (Santi et al., 1983; Taylor and Jones,
1982). Both agents have been shown to be clinically effective for the treatment of
leukemia and myelodysplasia and have been approved by the FDA (Kaminskas et al.,
45
2005; Lubbert et al., 2004). However, 5-aza-CR and 5-aza-CdR are quite toxic and
are unstable enzymatically and in aqueous solution, making clinical application of
these drugs difficult.
Zebularine is a novel mechanism-based inhibitor of DNA methylation
characterized in our laboratory. Zebularine is a 2-(1H)-pyrimidinone ribonucleoside
which lacks the amino group on C-5 of the pyrimidine ring. It has been shown to be
stable in acidic and neutral solutions and is relatively less toxic compared to the 5-
aza-CR and 5-aza-CdR (Cheng et al., 2003; Cheng et al., 2004a). The mechanism of
action of zebularine is similar to those of 5-aza-CR and 5-aza-CdR. Once in the
DNA zebularine forms a covalent complex with DNMTs (Hurd et al., 1999; Zhou et
al., 2002). Zebularine has previously been shown to actively demethylate methylated
loci in T24 cells upon continuous treatment (Cheng et al., 2004a). We decided to
extend our study to a group of normal fibroblasts and various cancer cell lines and
compared the effects of continuous zebularine treatment on these cells at DNA, RNA
and protein levels. We also identified a set of marker genes that are consistently
affected by zebularine treatment in cancer cell lines but not in normal fibroblasts.
This set of marker genes may potentially be utilized to identify cancer patients who
would be good candidates for treatment with zebularine in combination with
immunotherapy.
46
MATERIALS AND METHODS
Cell lines and drug treatment. T24 (bladder transitional carcinoma cells), HCT15,
SW48, and HT-29 (colon carcinoma cells), Cf-Pac-1 (pancreatic carcinoma cells),
CALU-1 (lung carcinoma cells) and CCD-1070Sk (human normal fibroblasts) were
obtained from the American Type Culture Collection (Rockville, MD). PC3 (prostate
carcinoma) cells were kindly provided by Dr. Gerry Coetzee. LD419, LD98, and T-1
(human normal fibroblasts) were established in our laboratory. T24, SW48, HT-29,
LD419, LD98, and T-1 cells were cultured in McCoy’s 5A medium supplemented
with 10% heat-inactivated fetal calf serum (FCS), 100 units/ml penicillin, and 100
µg/ml streptomycin (Gibco/Life Technologies, Inc., Palo Alto, CA). HCT15 was
cultured in RPMI 1640 medium supplemented with 10% FCS,
penicillin/streptomycin, and 1x sodium pyruvate (Gibco/Life Technologies, Inc.).
Cf-Pac-1 was cultured in IMDM medium supplemented with 10% FCS,
penicillin/streptomycin, 1x glutamine (Gibco/Life Technologies, Inc.). CALU-1 was
cultured in McCoy’s 5A medium supplemented with 10% FCS,
penicillin/streptomycin, and 1x glutamine. PC3 was cultured in RPMI 1640 medium
supplemented with 5% FCS and penicillin/streptomycin. CCD-1070Sk was cultured
in MEM medium supplemented with 10% FCS and penicillin/streptomycin, 1x
sodium pyruvate (Gibco/Life Technologies, Inc.), and 1x MEM nonessential amino
acids (Gibco/Life Technologies, Inc.). All cultures were grown in a humidified
incubator at 37
o
C in 5% CO
2
.
47
All normal and cancer cell lines were plated (3 x 10
5
cells/10 cm dish) and
treated 24 h later with 10
-4
M zebularine continuously for 8 days. The medium was
changed every 3 days thereafter along with fresh zebularine. DNA, RNA and protein
lysates were harvested at the end of the treatment period for methylation, RT-PCR
and Western blot analyses, respectively.
Determination of doubling time. All cultured cells (3 x 10
5
cells/10 cm dish) were
plated and treated with 10
-4
M zebularine 24 h later continuously for 8 days, with
fresh zebularine and media changes every 3 days. The cell number/dish was counted
with a Z1 Coulter Particle Counter (Beckman Coulter Corporation, Hialeh, FL) every
2 to 3 days. Untreated cells were analyzed under similar conditions as a control. The
average cell number from two plates was determined, and the mean cell numbers
were plotted to define the cell population doubling times, where population doubling
PD = log (number of cells harvested/ number of cells seeded)/log 2. Initial drug
treatment was started 24 h after seeding.
Nucleic acid isolation. RNA was collected from T24 and HCT116 cells with the
RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol.
DNA was collected using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according
to the manufacturer’s protocol.
48
RT-PCR analysis. Total RNA (5µg) was reverse transcribed with Moloney murine
leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) and random primers
(Invitrogen, Carlsbad, CA). The reverse transcription was carried out in a total
volume of 50µl as previously described (Gonzalez-Zulueta et al., 1995). cDNA was
amplified with primers specific for either p16, p21, p27 or GAPDH. The reverse
transcription (RT)-PCR conditions were as follows: for p16, 94
o
C for 3 min, 28-30
cycles of 94
o
C for 1 min, 56
o
C for 30 s, 72
o
C for 40 s, and a final extension step at
72
o
C for 5 min; for p21, 94
o
C for 3 min, 18-23 cycles of 94
o
C for 1 min, 57
o
C for 1
min, 72
o
C for 1 min, and a final extension step at 72
o
C for 5 min; for p27, the
conditions were exactly the same as p21, except 26-28 cycles were used; for GAPDH,
94
o
C for 1 min, 19 cycles of 94
o
C for 1 min, 58
o
C for 30 s, 72
o
C for 45 s, and a final
extension step at 72
o
C for 2 min. The primer sequences are as follows: p16 sense, 5’-
AGC CTT CGG CTG ACT GGC TGG-3’; p16 antisense, 5’-CTG CCC ATC ATC
ATG ACC TGG A-3’; p21 sense, 5'-AGG GTG ACT TCG CCT GGG AGC-3'; p21
antisense, 5'-CAC ACA AAC TGA GAC TAA GGC AGA AGA TGT-3'; p27, sense,
5'-GCG CCT TTA ATT GGG GCT CCG GCT AA-3'; p27, antisense, 5'-GCT ACA
TCC AAC GCT TTT AGA GGC AGA TCA-3'; GAPDH sense, 5’-CAG CCG AGC
CAC ATC GCT CAG ACA-3’; and GAPDH antisense, 5’-TGA GGC TGT TGT
CAT ACT TCT C-3’. RT-PCR amplification reactions of each of the expressed
genes was performed with 200 ng cDNA, 10% dimethylsulfoxide (DMSO), 100µM
dNTPs, Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO), and 1µM primers.
49
All reactions were analyzed in the linear range of amplification. PCR products were
resolved on 1.5% agarose gels.
Quantitative RT-PCR analysis. The quantitation of mRNA levels was carried out
by a real-time fluorescence detection method as described previously (Cheng et al.,
2004a; Eads et al., 1999). All samples were normalized to the reference gene,
GAPDH. The primer and probe sequences are as follows: for p16, sense 5’-CTG
CCC AAC GCA CCG A-3’, probe 5’ 6-FAM –TGG ATC GGC CTC CGA CCG
TAA CT BHQ-1 3’, and antisense 5’-CGC TGC CCA TCA TCA TGA C-3’; for
SPANXA1, sense 5’-TGT GAT TCC AAC GAG GCC A-3’, probe 5’ 6-FAM-CGA
GAT GAT GCC GGA GAC CCC A BHQ-1 3’, and antisense 5’-GCG GGT CTG
AGT CCC CA-3’; for MAGEA1, sense 5’-GAA CCT GAC CCA GGC TCT GTG-3’,
probe 5’ 6-FAM-CAA GGT TTT CAG GGG ACA GGC CAA C BHQ-1 3’, and
antisense 5’-CCA CAG GCA GAT CTT CTC CTT C’-3’; for MAGEB2, sense 5’-
CGG CAG TCA AGC CAT CAT G-3’, probe 5’ 6-FAM-TCG TGG TCA GAA
GAG TAA GCT CCG TGC BHQ-1 3’, and antisense 5’-GCG GGT CTG AGT CCC
CA-3’; for GAGE, sense 5’-GCT GAT AGC CAG GAA CAG GG-3’, probe 5’ 6-
FAM-CAC CCA CAG ACT GGG TGT GAG TGT GA BHQ-1 3’, and antisense 5’-
CCT GCC CAT CAG GAC CAT C-3’; for XAGEA1, sense 5’-TCC CCA GAC
GGG ACC AG-3’, probe 5’ 5-FAM-AGA GGG ACG GCA TGA GCG ACA CAC
BHQ-1 3’, and antisense 5’-CTG GCT GTG TGG TTC TGT GTT T-3’; for GAPDH,
sense 5’-TGA AGG TCG GAG TCA ACG G-3’, probe 5’ 6-FAM –TTT GGT CGT
50
ATT GGG CGC CTG G BHQ-1 3’, and antisense 5’-AGA GTT AAA AGC AGC
CCT GGT G-3’. The conditions for real time RT-PCR are: 94ºC for 9 min followed
by 45 cycles at 94ºC for 15 s and 60ºC for 1 min.
cRNA preparation. The cRNA preparation was done by Drs. Thomas Thykjaer and
Torben Orntoft (Aarhus University Hospital, Denmark). Total RNA (10 μg) was
used as starting material for the cDNA preparation. The first and second strand
cDNA synthesis was performed using the SuperScript II System (Invitrogen Corp.,
Carlsbad, CA) according to the manufacturer’s instructions, except using an oligo-dT
primer containing a T7 RNA polymerase promoter site. Labeled cRNA was prepared
using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Life Sciences,
Inc., Farmingdale, NY). Biotin-labeled CTP and UTP (Enzo) were used in the
reaction, together with unlabeled triphosphates. Following the in vitro transcription
reaction, the unincorporated nucleotides were removed using RNeasy columns
(Qiagen).
Array hybridization and scanning. Array hybridization and scanning were done
by Drs. Thomas Thykjaer and Torben Orntoft (Aarhus University Hospital,
Denmark). Fifteen μg of cRNA was fragmented at 94
o
C for 35 min in a buffer
containing 40 mM Tris-acetate pH 8.1, 100 mM potassium acetate, 30 mM
magnesium acetate. Prior to hybridization, the fragmented cRNA in a 6x SSPE-T
hybridization buffer (1 M NaCl, 10 mM Tris pH 7.6, 0.005% Triton) was heated to
51
95
o
C for 5 min and subsequently to 45
o
C for 5 min before loading onto the
Affymetrix HG_U133A probe array cartridge. The probe array was then incubated
for 16 h at 45
o
C at constant rotation (60 rpm). The washing and staining procedure
was performed in the Affymetrix Fluidics Station. The probe array was exposed to
10 washes in 6x SSPE-T at 25
o
C, followed by 4 washes in 0.5x SSPE-T at 50
o
C. The
biotinylated cRNA was stained with a streptavidin-phycoerythrin conjugate [final
concentration of 2 mg/ml (Molecular Probes, Eugene, OR)] in 6x SSPE-T for 30 min
at 25
o
C, followed by 10 washes in 6x SSPE-T at 25
o
C. An antibody amplification
step was followed using normal goat IgG as blocking reagent [final concentration of
0.1 mg/ml, (Sigma-Aldrich)] and biotinylated anti-streptavidin goat antibody [final
concentration of 3 mg/ml, (Vector Laboratories, Burlingame, CA)]. This was
followed by a staining step with a streptavidin-phycoerythrin conjugate [final
concentration of 2 mg/ml (Molecular Probes)] in 6x SSPE-T for 30 min at 25
o
C and
10 washes in 6x SSPE-T at 25
o
C. The probe arrays were scanned at 560 nm using a
confocal laser-scanning microscope (Hewlett Packard GeneArray Scanner G2500A;
Affymetrix, Inc., Santa Clara, CA). The readings from the quantitative scanning
were analyzed by the Affymetrix Gene Expression Analysis Software. In our
analysis, genes induced or reduced 3-fold after drug treatment were categorized
into different groups.
Western blot analysis. Cell pellets were lysed in radioimmunoprecipitation (RIPA)
buffer containing 0.1% SDS, 0.5% nonidet P-40, and 0.5% sodium deoxycholate in
52
PBS and incubated on ice for 30 min. The lysates were centrifuged at 4ºC for 30 min
at 14,000 rpm. Supernatant was collected and stored at -80ºC. Approximately 60µg
of protein was electrophoresed on a Ready Gel Tris-HCl Gel, 4-15% linear gradient
(Bio-Rad Laboratories, Hercules, CA), transferred to a PVDF membrane using
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories,
Hercules, CA). The membrane was hybridized with antibodies against human
DNMT1 (H-300; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), human
DNMT3b (T-16; 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and
proliferating cell nuclear antigen (PCNA) (PC10; 1:500; Santa Cruz Biotechnology,
Santa Cruz, CA) in Tris-buffered saline-Tween (TBS-T) buffer (0.1M Tris, 1.5M
NaCl, and 1% Tween 20) with 5% nonfat dry milk overnight at 4ºC. The human
DNMT3a antibody was provided by Dr. Ye-Guang Hu (Shanghai, China). The
membranes were washed four times with TBS-T buffer at room temperature and
incubated with secondary antibodies for 1 hr at room temperature. Secondary
antibodies used were anti-rabbit-IgG-HRP for DNMT1 (1:7500; Santa Cruz
Biotechnology, Santa Cruz, CA), anti-goat-IgG HRP for DNMT3b (1:10000;
Calbiochem, San Diego, CA), and anti-mouse-IgG-HRP for PCNA (1:7500; Santa
Cruz Biotechnology, Santa Cruz, CA). The membranes were washed six times with
TBS-T at room temperature. Proteins were detected with the ECL Western Blotting
Detection Reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) and by
exposure to Kodak X-OMAT AR film (Rochester, NY).
53
Quantitation of DNA methylation. Genomic DNA (4µg) was treated with sodium
bisulfite as previously described (Cheng et al., 2004a). Methylation analysis was
performed using the methylation-sensitive single-nucleotide primer extension (Ms-
SNuPE) assay for p16 5’ region as previously described (Gonzalgo and Jones, 2002).
The PCR primers used are: for p16, sense 5’-TTT GAG GGA TAG GGT-3’ and
antisense 5’-TCT AAT AAC CAA CCA ACC CCT CC-3’; for D4Z4, sense 5’-GGG
TTG AGG GTT GGG TTT AT-3’ and antisense 5’-AAC TTA CAC CCT TCC CTA
CA-3’ and for M4-4, sense 5’-ATG GTT TGA GGG TTT AGA TTA GGT-3’ and
antisense 5’- ACA TCA AAA TAA ACT TCC TCT TAC CA-3’. An initial
denaturation at 94ºC for 3 min was followed by 94ºC for 45 s, annealing for 45 s,
72ºC for 45 s for 40 cycles. The annealing temperatures for each locus were: for p16,
65ºC, for D4Z4, 58 ºC and for M4-4, 56
o
C. Primers used for Ms-SNuPE analysis
were as follows: for p16, 5’-TTT TTT TGT TTG GAA AGA TAT-3’, 5’-TTT TAG
GGG TGT TAT ATT-3’, and 5’-GTA GAG TTT AGT T-3’; for D4Z4, 5’-TGA
GGG TTG GGT TTA TAG T-3’, TAT ATT TTT AGG TTT AGT TTT GTA A-3’,
5’-GTG GTT TAG GGA GTG GG-3’, and 5’-GAA AGG TTG GTT ATG T-3’; for
M4-4, 5’-GGG TTT AGA TTA GGT TTT TT-3’, 5’- GTA ATA AGG ATT ATT
TGA ATA G-3’, and 5’- TAA TAA TGT GGA TTT GTT TAA ATT-3’. Conditions
for primer extension were: 94ºC for 1 min, 50ºC for 30 s, and 72ºC for 20 s.
Radioactive incorporation assay. All cell lines were plated (2 x 10
5
cells/60-mm
dish) and treated 24 h later with 0.75µCi/mL 2-[
14
C]-Zebularine for 24 h. A
54
separate dish of untreated cells was used for each cell line to determine the total
number of cells per dish at the end of 24 h treatment with a Z1 Coulter Particle
Counter (Beckman Coulter Corporation, Hialeh, FL). Cells were washed three times
with PBS, trypsinized, suspended in 450µL of water, lysed with 0.3 M KOH, and
then incubated at 37˚C for 18 h to hydrolyze RNA. Protein and DNA were
precipitated with 90% trichloroacetic acid (TCA) on ice for 5 min, and the DNA
pellet was thoroughly washed with 5% TCA and hydrolysed in 100µL of 5% TCA
for 30 min. The samples were centrifuged and radioactivity in 50µL of supernatant
was counted in 10 mL of scintillation fluid (Research Products International Corp.,
Mt. Prospect, IL) in a Packard Tri-Carb 1600 TR Liquid Scintillation Spectrometer
(Downers Grove, IL).
Uridine/cytidine kinase assay. The uridine/cytidine kinase activity in normal
fibroblasts and cancer cells were measured using assays modified as described by
Luccioni et al (Luccioni et al., 1994).
55
RESULTS
The rate of DNA incorporation of zebularine in normal fibroblasts and cancer
cell lines.
Before comparing the differences in response to zebularine between a panel
of normal fibroblasts and a panel of cancer cell lines, we first sought to normalize the
conditions in different cell lines. All cancer cell lines that were available to us in the
laboratory were cultured in growth media containing 10% heat-inactivated fetal calf
serum (FCS), whereas the normal fibroblasts established in our laboratory, LD419
and LD98, were cultured in media containing 20% heat-inactivated FCS. In order to
exclude the contribution of different serum concentrations on zebularine treatment of
these cells, we conditioned the normal fibroblast cell lines, LD419, LD98, T-1, and
CCD-1070Sk to media containing either 10 or 20% FCS for two weeks. Following
the media conditioning, the rate of DNA incorporation of radiolabeled 2-[
14
C]-
zebularine was measured after 24 hr treatment in these cells. The rate of
incorporation of zebularine into DNA of LD419 cells nearly doubled in 10% serum
compared to the cells grown in medium containing 20% serum (Fig 2.1). Similarly,
the incorporation rate of zebularine was 3-6 fold greater in cells grown in 10% serum
than those grown in 20% serum (Fig 2.1), indicating that the rate of DNA
incorporation of zebularine is governed in part by the serum concentration in media.
We also compared the incorporation rate of zebularine into DNA of T24
bladder cancer cells and LD419 normal fibroblasts that were grown in media
56
Figure 2.1. Rate of DNA incorporation of zebularine in normal fibroblasts
cultured in 10 or 20% fetal bovine serum. Normal fibroblasts, LD419, LD98, T-1,
and CCD-1070Sk were conditioned to grow in medium containing either 10 or 20%
fetal bovine serum for 2 weeks. The rate of DNA incorporation of 2-[
14
C]-zebularine
in these cells was measured after 24 hr treatment.
0
40
80
120
160
LD419 LD98 T-1 CCD-1070Sk
CPM/10,000 cells/24hr
10% FCS
20% FCS
57
containing 20% of either dialysed or non-dialysed FCS. In both T24 and LD419 cells,
about twice as much zebularine was incorporated into the DNA of cells grown in
media containing dialysed serum than that grown in non-dialysed serum (Fig 2.2).
this suggests that there was an unknown factor in FCS which may hinder the
incorporation of zebularine into DNA. It was likely that there was a small amount of
natural nucleosides such as cytidine and thymidine in FCS that may potentially be a
hindrance to the phosphorylation of zebularine. We used increasing concentration of
cytidine to compete with zebularine incorporation into DNA in both non-dialysed
and dialysed conditions; 0.1% of cytidine was enough to inhibit completely the
incorporation of zebularine into DNA of T24 and LD419 cells regardless of the
serum condition (Fig 2.2). This is another indication that the incorporation of
zebularine into DNA is in part controlled by the concentration of serum, and more
importantly, the contents of serum. Particularly, there may be a minute amount of
cytidine in FCS, which may cause lower incorporation rate of zebularine as observed
with the cells grown in growth media with higher serum content (Fig 2.1). In
addition, since zebularine is a cytidine analog, it is a less preferred nucleoside than
cytidine and must compete with natural nucleosides for the DNA incorporation. As a
result of the above experiments, it became imperative that all cell lines are cultured
in media containing the same concentration of FCS. Since LD419 and LD98 were
the only two cell lines requiring 20% FCS and they proliferated well in media
58
Figure 2.2. The rate of DNA incorporation of 2-[
14
C]-zebularine in T24 and
LD419 cells grown in media containing 20% fetal calf serum. T24 bladder cancer
cells and LD419 fibroblasts were conditioned to grow media with both either non-
dialysed or dialysed 20% FCS. Cells were treated with 15μM 2-[
14
C]-zebularine and
increasing concentrations of cytidine for competition of incorporation for 24 hr and
the rate of incorporation of zebularine was measured.
0
50
100
150
200
250
300
CPM/10,000cells/24hr
in non-dialysed serum
in dialysed serum
Zeb + + + + + + + +
Cytidine - -
T24 LD419
59
containing 10% FCS, we decided to use media with 10% FCS for all cell lines in the
zebularine experiments.
Folic acid is essential for cell proliferation and DNA incorporation of zebularine.
To test whether the removal of folic acid from media would facilitate the
DNA incorporation of zebularine, we treated HCT15 colon cancer cells with
zebularine in the presence or absence of folate. Folate is converted into methylene-
tetrahydrofolate (MTHF) which is a cofactor essential for the conversion of
homocysteine to methionine by methionine synthase. Methionine is converted into S-
adenosylmethionine (Ado-Met) by SAM synthetase (Loenen, 2006). Ado-Met is a
methyl donor and a critical player for methylation by DNMTs. HCT15 cells are
normally grown in RPMI 1640 medium which contains 1mg/L of folate. We
reasoned that by restricting the amount of folate in the growth media, we would
lower the Ado-Met pool in the cells, which would in turn inhibit DNA methylation to
a greater extent than if zebularine had been used alone.
We cultured HCT15 cells in RPMI 1640 in the presence or absence of folate
followed by nine days of continuous zebularine treatment. The induction of gene
expression was analyzed using five different genes, namely, p16, SPANXA1,
MAGEA1, MAGEB2, and XAGE1. These genes are all known to be hypermethylated
at the 5’ end of the gene and silenced in HCT15 cells ((Gonzalez-Zulueta et al.,
1995) and Chapter 2). The cells that were grown in media containing folate showed
induction of all genes that were analyzed, but no or very little gene induction was
60
Figure 2.3. Effects of folate on the level of gene induction by zebularine in
HCT15 colon cancer cells. HCT15 cells were cultured in media with or without
folate and treated with 100μM zebularine continuously for 9 days. RNA was
collected at day 3 and 9 of treatment. The induction of gene expression in p16,
SPANXA1, MAGEA1, MAGEB2, and XAGE1 was measured by quantitative real time
RT-PCR.
0
0.003
0.006
0.009
0.012
0.015
0.018
Day 0 Day 3 Day 9 Day 0 Day 3 Day 9
+Folate -Folate
p16/GAPDH
0
0.004
0.008
0.012
0.016
0.02
Day 0 Day 3 Day 9 Day 0 Day 3 Day 9
+Folate -Folate
SPANXA1/GAPDH
0.000
0.004
0.008
0.012
0.016
Day 0 Day 3 Day 9 Day 0 Day 3 Day 9
+Folate -Folate
MAGEA1/GAPDH
0.00
0.01
0.02
0.03
0.04
0.05
Day 0 Day 3 Day 9 Day 0 Day 3 Day 9
+Folate -Folate
MAGEB2/GAPDH
0.000
0.003
0.006
0.009
0.012
Day 0 Day 3 Day 9 Day 0 Day 3 Day 9
+Folate -Folate
XAGEA1/GAPDH
61
observed in cells cultured in media without folate (Fig 2.3). We then added
increasing concentrations of folate up to 1mg/L to rescue the cells from the effect of
folate deficiency on gene induction by zebularine. The induction of gene expression
was observed with 0.1mg/L and 1mg/L of folate, but the level of expression was
generally lower with folate supplement (Fig 2.4). We measured the doubling time of
these cells in each condition before and after zebularine treatment and observed that
HCT15 cells did not divide at a normal rate when folate was removed from the
media (Table 2.1). The doubling time increased more than 200% after the removal of
folate. When folate was added to the media again, the growth rate of the cells did not
fully recover. The growth inhibitory effects of zebularine on these cells were similar
with the increase in doubling time, ranging from 49 to 66 hr but never reached the
level of cells cultured in normal media (76%), probably because the cells had
undergone extensive growth arrest due to folate deficiency (Table 2.1).
Contrary to our hypothesis, the results demonstrated that the lack of folate in
the growth media severely stunted the growth of the cells and as a result, prevented
the inhibition of DNA methylation and subsequent gene induction in these cells.
Therefore, by inhibiting cell division with folate deprivation, we are preventing the
incorporation of zebularine into DNA and the inhibition of methylation. Since folate
plays an essential role in the recycling of methyl groups in cells, it has a tremendous
impact in other cell cycle regulatory pathways besides DNA methylation (Jang et al.,
2005), and the exclusion of folate may not be the best way to maximize the
inhibitory potential of zebularine.
62
Figure 2.4. Effects of folate on the level of gene expression by zebularine in
HCT15 colon cancer cells. HCT15 cells were cultured in medium containing no
folate to which increasing concentrations of folate was added and treated with
100μM zebularine continuously for 6 days. The induction of gene expression in p16,
MAGEA1, MAGEB2, and XAGE1 was measured by quantitative real time RT-PCR.
0
0.003
0.006
0.009
Unt Zeb Unt Zeb Unt Zeb Unt Zeb Unt Zeb
0mg/L 0.01mg/L 0.1mg/L 1mg/L
+Folate -Folate
p16/GAPDH
0
0.002
0.004
0.006
0.008
Unt Zeb Unt Zeb Unt Zeb Unt Zeb Unt Zeb
0mg/L 0.01mg/L 0.1mg/L 1mg/L
+Folate -Folate
XAGEA1/GAPDH
0
0.001
0.002
0.003
Unt Zeb Unt Zeb Unt Zeb Unt Zeb Unt Zeb
0mg/L 0.01mg/L 0.1mg/L 1mg/L
+Folate -Folate
MAGEA1/GAPDH
0
0.007
0.014
0.021
0.028
Unt Zeb Unt Zeb Unt Zeb Unt Zeb Unt Zeb
0mg/L 0.01mg/L 0.1mg/L 1mg/L
+Folate -Folate
MAGEA1/GAPDH
63
Table 2.1. Effects of folic acid on the growth rate of HCT15 colon cancer cells.
HCT15 colon carcinoma cells were cultured in medium with or without folic acid
and treated with 100μM zebularine continuously for 6 days. Increasing
concentration of folic acid was added to the medium without folic acid.
Folate added
(mg/L)
Zebularine
Doubling time
(Hr)
- 21
+ Folate 0
+ 37
- 44
0
+ 73
- 37
0.01
+ 55
- 22
0.1
+ 36
- 28
-Folate
1
+ 42
64
Preferential inhibition of DNA methylation by zebularine in cancer cells.
It had previously been reported that zebularine is an effective inhibitor of
DNA methylation with lower toxicity and better stability than 5-aza-CdR. This leads
to the possibility that zebularine can be used for chronic treatment in cell lines as
well as oral administration in animal models of cancer (Cheng et al., 2003; Cheng et
al., 2004a). On the other hand, the non-specific effects of nucleoside analogs such as
zebularine toward normal and cancer cells in patients had become problematic for
clinical application of these drugs. Therefore, our main interest was to compare the
activity of zebularine in a wide range of cell lines and define its preferential
mechanism of action. We had learned from our previous work that 100µM dose of
continuous zebularine treatment was sufficient to inhibit DNA methylation and
induce the expression of hypermethylated tumor suppressor genes in T24 bladder
cancer cells (Cheng et al., 2004a). We therefore tested a panel of normal fibroblasts
(LD419, LD98, T-1, and CCD-1070Sk) and cancer cell lines (T24, HCT15, Cf-Pac-1,
SW48, HT29, PC3, Calu-1) that were available to us and subjected them to
continuous zebularine treatment for 8 days.
Earlier studies indicate that mechanidsm-based inhibitors of DNA
methylation induce demethylation by DNA incorporation of the said compound,
followed by a covalent trapping of extractable DNMT proteins in the cells (Cheng et
al., 2004a; Hurd et al., 1999; Santi et al., 1984; Taylor and Jones, 1982; Zhou et al.,
2002). We therefore began our comparison of zebularine by analyzing the protein
levels of DNMT1, -3a, and -3b in the normal and cancer cells before and after
65
Figure 2.5. Effects of zebularine on DNMT protein levels in normal fibroblasts
and cancer cells. Western blot analysis of DNMT1, DNMT 3a and 3b2/3 protein
levels after continuous 100μM zebularine treatment for 8 days in a panel of (A)
normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk) and (B) cancer cell lines
(T24 bladder cancer, HCT15, SW48, and HT-29 colon cancer, CFPAC-1 pancreatic
cancer, PC3 prostate cancer and CALU-1 lung cancer cells). Either DNMT3b2 or
3b3 represents the predominant isoform in that specific cell line. Cell lysates were
obtained from treated and untreated control cells and were analyzed by Western blot
analysis with specific antibodies for DNMT1, DNMT3a, DNMT3b, and PCNA
proteins.
DNMT1
DNMT3a
DNMT3b2
PCNA
Zebularine
Zebularine
DNMT1
DNMT3a
DNMT3b2
DNMT3b3
+ + + +
LD98 T-1 LD419
CCD-
1070Sk
PCNA
A
T24 HCT15 CFPAC-1
+
PC3
+ + + +
SW48 HT29
+
+
B
DNMT3b3
CALU-1
66
continuous zebularine treatment. The levels of extractable DNMT1 in all normal
fibroblasts and PC3 and Calu-1 cells decreased slightly, suggesting that the
methylation capacity was not completely lost in these cells. On the contrary,
zebularine caused complete depletion of DNMT1 in T24, HCT15, Cf-Pac-1, SW48,
and HT29 cells (Fig 2.5). The levels of DNMT3a and -3b2/3 were less consistently
affected in the entire cell lines tested, with a minor change in the extractable protein
levels (Fig 2.5). The mRNA levels of each DNMT proteins, as measured by RT-PCR,
remained unchanged after zebularine treatment (personal communication, Jonathan
Cheng), suggesting that the depletion of DNMT proteins was caused by the trapping
of the enzymes to DNA-incorporated zebularine, rather than by the inhibition of
transcription.
We then assayed the methylation levels of three loci, 5’ region of p16 gene,
D4Z4, and M4-4. The 5’ region of p16 gene is aberrantly hypermethylated in all the
cancer cell lines under investigation, while it is hypomethylated and expressed in
normal cell lines. D4Z4 is a subtelomeric repeat sequence, and M4-4 is a single copy
sequence located in a CpG island, previously characterized in our laboratory (Cheng
et al., 2004a; Kondo et al., 2000); both sequences are heavily methylated in all cell
lines. Methylation analysis by quantitative Ms-SNuPE showed that the methylation
levels in the normal fibroblasts remained unchanged or slightly affected at the three
loci after zebularine treatment. Demethylation was observed only in T24, HCT15,
Cf-Pac-1, SW48, and HT29, while the methylation levels in PC3 and Calu-1 cells
remained almost unchanged at all three loci (Fig 2.6). Consistent with the western
67
Figure 2.6. Effects of zebularine on DNA methylation in normal fibroblasts and
cancer cells. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk)
and cancer cell lines (T24 bladder cancer, HCT15, SW48, and HT-29 colon cancer,
CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells)
were either untreated or treated in the presence of 100μM zebularine continuously
for 8 days. Methylation status of the p16 5’ region (A), M4-4(B) and D4Z4(C) were
quantitated by Ms-SNuPE analysis. Methylation percentage represents the average of
three individual CpG sites in each region as assayed from two independent
experiments.
A.
0
20
40
60
80
100
LD98 T-1 LD419 CCD-
1070Sk
T24 HCT15 Cf-Pac-1 SW48 HT29 PC3 Calu-1
% Methylation
Untreated
Zebularine
B.
0
20
40
60
80
100
LD98 T-1 LD419 CCD-
1070Sk
T24 HCT15 Cf-Pac-1 SW48 HT29 PC3 Calu-1
% Methylation
Untreated
Zebularine
C.
0
20
40
60
80
100
LD98 T-1 LD419 CCD-
1070Sk
T24 HCT15 Cf-Pac-1 SW48 HT29 PC3 Calu-1
% Methylation
Untreated
Zebularine
68
blot analysis, the normal fibroblasts and PC3 and Calu-1 cell lines remained virtually
unaffected by zebularine treatment, while five out of seven cancer cell lines
displayed demethylation as well as depletion of extractable DNMT1 proteins.
Furthermore, the RT-PCR analysis of the expression of p16 gene indicated that the
level of gene expression remained unchanged in normal fibroblasts after zebularine
treatment, while the p16 expression was induced in all but PC3 and Calu-1 cell lines
(Fig 2.7). This is consistent with the observations made earlier. Our results also
suggested that while the majority of cancer cell lines tested were subject to
demethylation by zebularine treatment, some cancer cells and normal fibroblasts
were more resistant to changes incurred by the drug.
Preferential growth inhibition by zebularine in cancer cells.
The most prominent biological changes caused by zebularine treatment in
cancer cells, but not in normal fibroblasts, were the inhibition of growth and the
increase in doubling time of these cells. The increase in doubling time ranged from
12 to 21% in normal fibroblasts after zebularine treatment; however, the doubling
time increase from 22 to 68% in the cancer cell lines (Table 2.2). Interestingly, PC3
and Calu-1, which were not subject to demethylation, also exhibited substantial
growth inhibition, indicating that increased doubling time in these cells may be
caused by other unknown mechanisms besides demethylation. Since the DNA
incorporation of zebularine is seven times higher than the incorporation of DNA, the
effects of of zebularine in RNA cannot be overlooked (Ben-Kasus et al., 2005).
69
Figure 2.7. Effects of zebularine on gene expression in normal fibroblasts and
cancer cells. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk)
and cancer cell lines (T24 bladder cancer, HCT15, SW48, and HT-29 colon cancer,
CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells)
were either untreated or treated in the presence of 100μM zebularine continuously
for 8 days. Expression levels of p16, p21 and p27 mRNAs were determined by RT-
PCR analysis. GAPDH mRNA expression levels were measured to control for
relative cDNA input.
70
Table 2.2. Effects of zebularine on growth inhibition in normal fibroblasts and
cancer cells. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk)
and cancer cell lines (T24 bladder cancer, HCT15, SW48, and HT-29 colon cancer,
CFPAC-1 pancreatic cancer, PC3 prostate cancer and CALU-1 lung cancer cells)
were either untreated or treated in the presence of 100μM zebularine continuously
for 8 days. The effects of zebularine on cell growth were analyzed in these cell lines
by comparing doubling times before and after drug treatment. The percent increase
represents the increase in doubling time of cells after treatment as compared to the
untreated control cells.
Doubling time (Hr)
Cell lines
Zebularine
(-) (+)
Doubling time
increase (%)
LD98 38 46 21
T-1 58 68 17
LD419 38 43 13
CCD-1070Sk 58 65 12
T24 21 30 44
HCT15 26 37 41
Cf-Pac-1 38 51 32
SW48 24 39 63
HT29 25 42 68
PC3 39 59 53
Calu-1 32 42 33
71
The p21 (WAF1) and p27 (KIP1) genes are inhibitors of cyclin-CDK
complexes involved in G
1
and S phase progression (Gong et al., 2003; Tam et al.,
1997). To demonstrate the involvement of the genes in the observed growth
inhibition, we measured the expression of these genes by semi-quantitative RT-PCR
after zebularine treatment. In normal fibroblasts, the expression levels did not change
after the drug treatment (Fig 2.7a). On the other hand, growth inhibition of cancer
cells by zebularine was associated with the induction of the p21 mRNA levels but
not p27 mRNA levels, which remained unaffected after treatment (Fig 2.7b). The
growth inhibitory effects of zebularine on the seven cancer cell lines were p21-
dependent. In addition, the demethylation and the activation of p16 in T24, HCT15,
Cf-Pac-1, SW48, and HT29 cells may also have partially contributed to the growth
inhibitory effects in these cells. Overall, the cancer cells were more prone to the
growth inhibitory effects of zebularine than the normal fibroblasts and this was
found to be in part due to p21 and p16 induction.
Differential activity of uridine/cytidine kinase is responsible for the rate of DNA
incorporation of zebularine in cells.
The preferential effects of zebularine on these cells may be due to differential
incorporation of zebularine into DNA. The first step that leads to DNA incorporation
is the phosphorylation of zebularine into its monophosphate moiety by enzyme
uridine/cytidine kinase (UCK). The UCK activity levels in the cancer cell lines were
3- to 40-fold higher than that in the normal fibroblasts (Figure 2.8). The only
72
Figure 2.8. Level of incorporation of 2-[
14
C]-zebularine into DNA and level of
uridine/cytidine kinase activity in normal and cancer cells. Cells in logarithmic
phase were treated 24 h later with 2-[
14
C]-zebularine to measure the level of
incorporation. Twenty-four hours after the treatment, the cultures were harvested
and the radioactivity incorporated into DNA was determined. The incorporation
results are expressed as counts per minute per 10,000 cells (adjusted for population
doubling), as shown in black bars (). The levels of uridine/cytidine kinase activity
are expressed as pmol/min/mg of proteins in white bars (). Incorporation results
represent the average values of two or three separate experiments. Error bars
represent the standard deviation of three separate determinations.
U/C kinase activity (pmol/min/mg protein)
0
100
200
300
400
500
600
700
LD419 LD98 T-1 CCD-
1070Sk
T24 Calu-1 HCT15 Cf-Pac-1 PC3 SW48 HT29
CPM/10,000 cells/24 hrs
73
exception was Calu-1 cells, which had low UCK activity. We next measured
directly the rate of incorporation of radioactive zebularine into the DNA in each cell
line. Previous experiments confirmed that incorporation of 2-[
14
C]-zebularine was
almost completely inhibited by the presence of a 1% of non-radioactive cytidine,
suggesting that the initial activation of the drug is probably dependent on the activity
of UCK (Fig 2.2). Our results showed a similar trend to the UCK activity levels; the
cancer cells showed higher rates (2- to 50-fold) of incorporation than the normal
fibroblasts (Fig 2.8). These results were generally consistent with the fact that
zebularine had greater inhibitory effects and demethylating effects on cancer cell
lines than normal fibroblasts, except for PC3 and Calu-1 cells, which both have
moderate leveles of DNA incorporation, yet minimal effect on DNA methylation.
These latter results suggest that incorporation of the fraudulent base into DNA is not
sufficient to inhibit DNA methylation in itself and that there may be other factors
that dictate the final outcome.
Microarray analysis of zebularine treatment of normal fibroblasts and cancer
cells.
Next, we utilized an Affymetrix human gene chip containing over 13,000
genes for our analysis of changes in global gene expression profile of four normal
fibroblasts (LD419, LD98, T-1, and CCd-1070Sk) and three cancer cell lines (T24,
HCT15, and Cf-Pac-1) after continuous zebularine treatment in a collaborative work
with the Aarhus University Hospital in Denmark. The analysis showed that a
74
Table 2.3. Genes upregulated 2 fold in all cancer cell lines, but not in fibroblast cell lines eight days after
continuous zebularine treatment. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk) and cancer cell
lines (T24 bladder cancer, HCT15, Cf-Pac-1 pancreatic cancer) were either untreated or treated in the presence of 100μM
zebularine continuously for 8 days. RNA was subject to cRNA conversion and used in an expression gene chip array
analysis. Sample preparation and gene expression array analysis conducted by Drs. Thomas Thykjaer (Aarhus University
Hospital, Denmark).
Fibroblasts Cancer cells
LD98 LD419 T-1
CCD-
1070Sk
T24
Cf-
Pac-1
HCT15
Unigene
Gene
Symbol
Map
Location
FC FC FC FC FC FC FC
Hs.72879 MAGEA1 xq28 1.1 -2.8 1.0 -2.3 2.1 5.3 10.6
Hs.113824 MAGEB2 xp21.3 1.1 1.6 1.1 1.6 2.5 2.6 4.6
Hs.176661 GAGE3 xp11.4-p11.2 -1.4 -1.5 1.4 -1.3 6.1 4.0 7.0
Hs.272484 GAGE6 xp11.4-p11.2 1.2 -1.4 -1.9 -1.2 2.8 9.8 11.3
Hs.278606 GAGE7 xp11.2-p11.4 1.7 -3.2 1.4 -3.0 2.6 9.2 17.1
Hs.251677 GAGE7B xp11.4-p11.2 -4.9 1.2 1.4 -1.1 3.2 9.8 24.3
Hs.112208 XAGE1 xp11.23 -1.4 1.3 -1.2 1.1 7.5 7.5 48.5
Hs.334464 SPANXA1 xq27.1 -1.1 1.1 1.1 1.1 315.2 445.7 13.9
Hs.2962 S100P 4p16 -1.7 -1.6 1.4 -4.0 11.3 6.1 6.5
Hs.296323 SGK 6q23 -1.3 1.1 -1.6 1.3 2.1 2.8 2.6
Hs.79748 SLC3A2 11q13 -1.5 -1.1 -1.1 1.1 2.8 3.7 2.0
Hs.449863 ACACA 17q21 1.1 1.3 1.3 -1.4 10.6 2.3 2.0
FC, fold change.
75
common set of twelve genes was upregulated by2-fold after zebularine treatment
in all three cancer cell lines, but not in the normal fibroblasts (Table 2.3). Among the
twelve upregulated genes were a family of genes known as cancer/testis antigens
(CTAs) that are often expressed in the testis, ovaries and in various cancers
(Simpson et al., 2005). A total of 6 genes were downregulated in cancer cells but not
in normal fibroblasts (Table 2.4). Similarly, only one gene out of over 13,000 genes
was found to be upregulated or downregulated in all four normal fibroblasts (Table
2.5 and 2.6). The discrepancy between the RT-PCR results (Fig 2.7) and the
microarray results (Table 2.3) raised a concern for the validity of the global gene
expression profile analysis. The microarrays did not detect the upregulation of p16
and p21 found earlier. This is possibly due to technical issues related to gene chip
arrays, such as low signal intensities and the presence of multiple transcripts and
probes, which may not always correspond. However, the array results were in accord
with earlier work from our laboratory on the global effects of gene expression by 5-
aza-CdR in T24 and LD419 cell lines, showing that more gene expression changes
are found in cancer cells and not in normal fibroblasts (Liang et al., 2002b). In
addition, the activation of CTAs in cancer cells suggests the utilization of zebularine
in the upregulation of these antigens which may become a target for immunotherapy
(Gillespie et al., 1998; Karpf et al., 2004; Weber et al., 1987). Furthermore, a very
small percentage of genes were affected globally (20 out of 13,000), indicating that
zebularine treatment did not cause a dramatic change in the gene expression profile
in these cells.
76
Table 2.4. Genes downregulated2 fold in all cancer cell lines, but not in fibroblast cell lines eight days after
continuous zebularine treatment. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk) and cancer cell
lines (T24 bladder cancer, HCT15, Cf-Pac-1 pancreatic cancer) were either untreated or treated in the presence of 100μM
zebularine continuously for 8 days. RNA was subject to cRNA conversion and used in an expression gene chip array
analysis. Sample preparation and gene expression array analysis conducted by Drs. Thomas Thykjaer (Aarhus University
Hospital, Denmark).
Fibroblasts Cancer cells
LD98 LD419 T-1
CCD-
1070Sk
T24
Cf-Pac-
1
HCT15
Unigene
Gene
Symbol
Map
Location
FC FC FC FC FC FC FC
Hs.99816 CTNNBIP1 1p36.22 1.1 1.2 -1.1 -1.1 -2.3 -2.6 -2.5
Hs.1690 HBP17 4p16-p15 -2.6 -1.1 1.3 2.3 -8.6 -5.7 -4.0
Hs.79069 CCNG2 4q21.21 -1.3 -1.1 1.1 2.1 -3.2 -2.0 -2.3
Hs.2794 SCNN1A 12p13 -1.2 -1.9 1.0 -1.4 -2.0 -2.5 -3.7
Hs.348350 DHRS1 14q11.2 -1.1 1.0 -1.2 1.0 -2.0 -2.0 -2.8
Hs.3797 RAB26 16p13.3 -1.3 1.4 1.1 -1.7 -5.3 -3.7 -4.9
FC, fold change.
77
Table 2.5. Genes upregulated 2 fold in all fibroblast cell lines, but not in cancer cell lines eight days after
continuous zebularine treatment. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk) and cancer cell
lines (T24 bladder cancer, HCT15, Cf-Pac-1 pancreatic cancer) were either untreated or treated in the presence of 100μM
zebularine continuously for 8 days. RNA was subject to cRNA conversion and used in an expression gene chip array
analysis. Sample preparation and gene expression array analysis conducted by Drs. Thomas Thykjaer (Aarhus University
Hospital, Denmark).
Fibroblasts Cancer cells
LD98 LD419 T-1
CCD-
1070Sk
T24
Cf-
Pac-1
HCT15
Unigene
Gene
Symbol
Map
Location
FC FC FC FC FC FC FC
Hs.119689 CGA 6q12-q21 4.3 2.1 2.8 5.7 -1.3 1.7 1.5
FC, fold change.
78
Table 2.6. Genes downregulated2 fold in all fibroblast cell lines, but not in cancer cell lines eight days after
continous zebularine treatment. A panel of normal fibroblasts (LD98, T-1, LD419, and CCD-1070Sk) and cancer cell
lines (T24 bladder cancer, HCT15, Cf-Pac-1 pancreatic cancer) were either untreated or treated in the presence of 100μM
zebularine continuously for 8 days. RNA was subject to cRNA conversion and used in an expression gene chip array
analysis. Sample preparation and gene expression array analysis conducted by Drs. Thomas Thykjaer (Aarhus University
Hospital, Denmark).
Fibroblasts Cancer cells
LD98 LD419 T-1
CCD-
1070Sk
T24
Cf-
Pac-1
HCT15
Unigene
Gene
Symbol
Map
Location
FC FC FC FC FC FC FC
Hs.29423 COLEC12
18pter-
p11.3
-2.0 -3.0
-
13.9
-2.0 1.5 1.2 -1.3
FC, fold change.
79
Identification and establishment of marker genes for zebularine response by
cancer cells.
Cancer testis antigens (CTAs) are expressed in testis, ovaries, and in some
cancers but not in other normal tissues (Simpson et al., 2005). Most CTAs are found
on the X chromosome (Chomez et al., 2001; Lucas et al., 1998; Muscatelli et al.,
1995). Although the role of these antigens has not been determined yet, they may
serve as useful targets for when immunotherapy and epigenetic therapy are combined.
Zebularine can be used to upregulate these antigens in cancer cells, thereby making
the cancer cells with low or no expression of these antigens better targets of
immunotherapy. In addition, these antigens can be used to gauge the extent of
zebularine response in cancer cells, since they seem to be induced in cancer cells but
not in normal cells. Therefore, we were inclined to extend our study to verify that the
CTAs were indeed upregulated by zebularine treatment in the cancer cells by real
time RT-PCR.
We chose SPANXA1, MAGEA1, MAGEB2, and XAGE1 for our study; all four
antigens are located on the X chromosome (Fig 2.9). MAGEB2 and XAGE1 have
CpG islands at the 5’ regions, whereas SPANXA1 and MAGEA1 have CpG-poor
promoter regions (Fig 2.10), as determined according to the criteria set by Takai and
Jones (Takai and Jones, 2002). The same RNA that was used for the microarray
analysis were reverse transcribed into cDNA for the real time PCR analysis to ensure
a direct comparison may be made between the two analytical methods. In the normal
fibroblasts, LD419, LD98, T-1, and CCD-1070Sk, no expression of the CTAs were
80
Figure 2.9. Position of cancer-testis antigens on X chromosome. Cancer-testis
antigens, SPANXA1, MAGEA1, MAGEB2, and XAGE1 are located on the X
chromosome. MAGEB2 and XAGE1 contain a CpG island at the 5’-end while the 5’-
ends of SPANXA1 and MAGEA1 are CpG-poor.
81
Figure 2.10. CpG content and map of cancer-testis antigens. The GC content,
ratio of observed to expected CpG content, the length of CpG island, and number of
CpG sites are given for the following genes. CpG maps of cancer-testis antigens are
shown with their transcriptional start site in the center. The upward arrows indicate
the CpG sites at which the DNA methylation level was measured by Ms-SNuPE. The
red bars indicate the location of CpG islands for MAGEB2 and XAGE1. For
SPANXA1 and MAGEA1, the red bars indicate smaller CpG-dense regions which do
not quite meet the criteria set by Takai and Jones (2004).
82
Figure 2.11. Effects of zebularine on the expression cancer-testis antigens in
normal fibroblasts and cancer cell lines. LD419, LD98, T-1, and CCD-1070Sk
normal fibroblasts and T24 bladder, HCT15 colon, and Cf-Pac-1 pancreatic cancer
cells were treated with 100μM zebularine continuously for 8 days and RNA was
collected for real time RT-PCR analysis of SPANXA1(A), MAGEA1(B),
MAGEB2(C), and XAGE1(D).
A.
B.
C.
D.
0.00
0.05
0.10
0.15
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
SPANXA1/GAPDH
Untreated
Zeb
0.000
0.001
0.002
0.003
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
MAGEA1/GAPDH
0.000
0.005
0.010
0.015
0.020
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
MAGEB2/GAPDH
0.0000
0.0010
0.0020
0.0030
0.0040
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
XAGE1/GAPDH
83
observed before or after zebularine treatment (Fig 2.11). Consistent with the
microarray analysis, the induction of CTAs were observed in T24, Cf-Pac-1, and
HCT15 cancer cells after zebularine treatment but not in untreated cells (Fig 2.11).
These results validated the microarray results as well as identified a family of genes
that are extremely sensitive to zebularine treatment in cancer cells but not in normal
cells.
To determine whether the induction of CTAs is regulated by promoter
methylation, we also analyzed the methylation status of the promoter regions of these
four genes (Fig 2.10). The methylation of normal fibroblasts remained similar before
and after zebularine treatment, consistent with the real time PCR results (Fig 2.12).
Regardless of whether the promoter contained a CpG island or not, the expression of
all four antigens seems to be inversely related to the promoter methylation in cancer
cells; demethylation of the promoter regions was observed for all four antigens after
zebularine treatment, which may be responsible for the induction of gene expression
(Fig 2.12).
84
Figure 2.12. Effects of zebularine on the expression cancer-testis antigens in
normal fibroblasts and cancer cell lines. LD419, LD98, T-1, and CCD-1070Sk
normal fibroblasts and T24 bladder, HCT15 colon, and Cf-Pac-1 pancreatic cancer
cells were treated with 100μM zebularine continuously for 8 days and DNA was
collected for methylation analysis of promoter regions of SPANXA1(A),
MAGEA1(B), MAGEB2(C), and XAGE1(D) by Ms-SNuPE.
A.
B.
C.
D.
0
20
40
60
80
100
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
% Methylation
Untreated
Zeb
0
20
40
60
80
100
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
% Methylation
0
20
40
60
80
100
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
% Methylation
0
20
40
60
80
100
LD98 LD419 T-1 CCD-
1070Sk
T24 Cf-Pac-1 HCT15
% Methylation
85
Heterogeneous induction of cancer/testis antigens by zebularine.
It would be of interest to observe how the methylation level is changed when
cells are treated chronically with a demethylating agent. In addition, when patients
are treated with demethylating agent for a prolonged period, it would be desirable to
have the entire population of malignant cells respond to the drug at the same time;
however, some cancer cells acquire resistance to the treatment and may persist after
a chronic treatment. Therefore, we utilized the fact that zebularine has low toxicity
which make long-term administration possible to monitor the degree of
demethylation in these cells by examining the CTAs.
T24 bladder cancer cells were treated with low-dose zebularine continuously
for 40 days during which the methylation level and expression of cancer/testis
antigens were observed. In the untreated cells, very little or no expression of CTAs
was observed (Fig 2.13a, c, e, g). During the course of the treatment, the induction of
all four antigens was seen; while the peak was seen around day 27 of treatment, the
levels of gene expression had a tendency to fluctuate for the remaining treatment
period. In addition, the untreated cells displayed the highest levels of methylation at
the promoter regions of each antigen, and demethylation ranging from 15 to 30%
was seen at all four loci (Fig 2.14a, c, e, g). While the levels of gene expression
fluctuated, the levels of methylation remained quite static after the initial
demethylation. This could be due to a number of factors, including the possibility
that DNA methylation may control the expression of these antigens. Furthermore, the
bulk of demethylation occurred in the first 12 days of treatment and the methylation
86
Figure 2.13. Effects of chronic zebularine treatment on the expression of cancer-
testis antigens. T24 bladder cancer cells were treated with 100μM zebularine
continuously for 40 days. Individual clones were obtained after chronic zebularine
treatment and RNA extracted for real time RT-PCR analysis.
A. B.
B.
C. D.
E. F.
G. H.
0
0.05
0.1
0.15
Unt d12 d27 d31 d37 d40
SPANXA1/GAPDH
0
0.0007
0.0014
0.0021
0.0028
0.0035
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
MAGEA1/GAPDH
0.00
0.02
0.04
0.06
0.08
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
MAGEB2/GAPDH
0
0.005
0.01
0.015
0.02
0.025
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
XAGE1/GAPDH
0
0.05
0.1
0.15
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
SPANXA1/GAPDH
0
0.0007
0.0014
0.0021
0.0028
0.0035
Unt d12 d27 d31 d37 d40
MAGEA1/GAPDH
0.00
0.02
0.04
0.06
0.08
Unt d12 d27 d31 d37 d40
MAGEB2/GAPDH
0.000
0.005
0.010
0.015
0.020
0.025
Unt d12 d27 d31 d37 d40
XAGE1/GAPDH
87
Figure 2.14. Effects of chronic zebularine treatment on DNA methylation. T24
bladder cancer cells were treated with 100μM zebularine continuously for 40 days.
Individual clones were obtained after chronic zebularine treatment and DNA
extracted for methylation analysis by Ms-SNuPE.
A. B.
C. D.
E. F.
G. H.
0
20
40
60
80
100
Unt d12 d27 d31 d37 d40
% Methylation
0
20
40
60
80
100
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
% Methylatio n
0
20
40
60
80
100
Unt d12 d27 d31 d37 d40
%Methylation
0
20
40
60
80
100
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
% M e th y la tio n
0
20
40
60
80
100
Unt d12 d27 d31 d37 d40
% Methylation
0
20
40
60
80
100
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
% M e th y la tio n
0
20
40
60
80
100
Unt d12 d27 d31 d37 d40
% Methylation
0
20
40
60
80
100
Cl1 Cl2 Cl3 Cl4 Cl5 Cl6 Cl7 Cl8
% M e th y la tio n
N/D N/D
N/D N/D
88
level decreased only slightly afterwards, demonstrating that the chronic treatment of
zebularine does not cause a complete demethylation, and the cells may have a
mechanism which allowsthem to retain a residual level of methylation for their
survival. In fact, this is consistent with works showing the essential role of DNMTs
in development and maintenance of normal function of cultured cells as well as
animals (Kaneda et al., 2004; Liang et al., 2002a; Okano et al., 1999).
To answer our second question, individual clones were seeded and cultured
for clonal expansion after 40 days of low-dose zebularine treatment, from which
gene expression and methylation analysis studies were performed. Interestingly, the
expression pattern of the CTAs varied between clones as well as between the
antigens. For example, clone 7 displayed low level expressions of SPANXA1 and
MAGEB2 and high levels of expression of MAGEA1 and XAGE1. Likewise, clone 8
had intermediate levels of expression of SPANXA1, MAGEA1, and MAGEB2 and
very low expression of XAGE1 (Fig 2.13b, d, f, h). Looking at clones 1 through 8,
the expression of each antigen varied greatly between the clones as well, displaying
heterogeneous pattern of gene induction. Furthermore, the methylation levels of each
clone did not correlate well with the degree of expression. For example, clones 1, 2,
and 3 have similar levels of methylation at the SPANXA1 promoter, while the
expression of SPANXA1 in these clones vary from very high in clone 1 to low in
clone 3 (Fig 2.13b and 2.14b). Zebularine may demethylate different loci at different
rates, hence yielding the mixed demethylation pattern seen in Fig 2.14. This may be
important when considering clinical application of zebularine for cancer therapy
89
since chronic treatment of zebularine may still leave behind a population of
malignant cells, which may lead to clonal expansion and pose as a threat. There may
be a mechanism, which zebularine cannot bypass and is necessary for the survival of
these clones.
Taken together, chronic treatment of T24 cells with zebularine resulted in
heterogeneous upregulation of CTAs in individual clones and decrease in
methylation levels; however, the methylation status and expression levels do not
always agree with each other at the clonal level and other mechanisms may be
involved.
90
DISCUSSION
We have discovered an important property of zebularine, which may have
huge impact on the clinical application of the drug. It is of obvious concern that a
therapeutic drug is selective toward the target cells and not toward the surrounding
normal tissues. Our findings suggest that zebularine may be selective toward cancer
cells compared to normal fibroblasts, in terms of growth inhibition, demethylation,
reactivation of genes, depletions of DNMTs and incorporation into DNA.
Our first agenda was to determine a standardized growth condition for the
cultured normal fibroblasts and cancer cells. Preliminary studies indicated that the
incorporation of zebularine is affected by a variety of factors such as serum
concentration, presence of natural nucleosides (i.e. cytidine), and concentration of
folate in growth medium. Our results showed that the use of standardized media for
the normal and cancer cells was critical for the accurate measurement of the rate of
zebularine incorporation as well as its secondary effects such as demethylation,
depletion of DNMTs and growth inhibition. In addition, we acquired a better
understanding of the mechanism of action of zebularine as a hypomethylating agent.
The preferential activity of zebularine may be explained in part by the
differential incorporation of zebularine into the DNA. The activation of zebularine
requires the phosphorylation by uridine/cytidine kinase (UCK) into the
monophosphate, followed by further phosphorylation and subsequent incorporation
into RNA and DNA. The UCK is generally thought to be the rate limiting step of
91
zebularine metabolism, which was strongly supported by the incorporation data. The
rate of incorporation of zebularine was much higher in all cancer cell lines tested
compared to the normal fibroblasts, although the effects of demethylation was
observed in five out of seven cancer cell lines. This suggests that perhaps the
threshold level required to induce methylation and gene expression changes for each
cell line may be different. Interestingly, the UCK activity levels in the normal
fibroblast cell line, CCD-1070Sk, is similar to the level in the Calu-1 cells and
neither cell lines showed decrease in methylation. However, the level of zebularine
incorporation in the Calu-1 cells are much higher than that in the CCD-1070Sk cells,
further strengthening the previous statement that each cell line may have a different
threshold level of response.
The differential depletion of DNMTs by zebularine in the normal and cancer
cell lines suggests that the rate of metabolism of zebularine may be differential in
different cell lines or that there may be differential levels of enzyme present in each
cell lines or it may be a combination of these two possibilities. The levels of DNMT1,
DNMT3a, and DNMT3b2/3 were less affected in normal cells, consistent with the
incorporation data. It is interesting to note that a complete depletion of DNTM1 and
partial depletion of DNMT3a and DNMT3b2/3 were seen in the cancer cells that
responded to zebularine in terms of demethylation. Unpublished work from our
laboratory regarding the position of DNMT enzymes with respect to nucleosomes
provided important clues as to how this may be occurring. DNMT1, whose main job
is to methylate the newly synthesized DNA, is primarily found at the replication fork
92
during DNA synthesis. Therefore they are immediately trapped by zebularine which
is incorporated into the new NDA strand and cause a complete depletion of the
enzyme. On the other hand, DNMT3a and DNMT3b2/3 are associated with
nucleosomes and are responsible for completing the methylation unfinished by
DNMT1 and as a result, are only partly depleted by zebularine since most of the drug
is occupied with DNMT1. This model supports well the results described in this
chapter as well as previous work published in our laboratory that zebularine and 5-
aza-CdR display preferential depletion of DNMTs (Cheng et al., 2004a; Velicescu et
al., 2002).
Our microarray results demonstrated the feasibility of a combinatorial
approach involving immunotherapy for the treatment of human cancers. Indeed,
early studies (Simcik et al., 1989) showed that 5-aza-CR could induce the formation
of strongly immunogenic variants of mouse cells, providing support for this concept.
While the MAGE family of antigens have been identified as useful targets of
immunotherapy, only a subset of cancer express these antigens on their cell surface.
Our findings show that the cancer cells are extremely sensitive to CTA induction by
zebularine but not the normal fibroblasts, which make the combinatorial approach
attractive.
Although we do not have a full explanation as to why the methylation levels
do not correlate with the expression profile of the antigens in the zebularine-treated
clones, we interpret that while the methylation levels remain static after the initial
demethylation, the expression of these genes is quite dynamic. Similar observations
93
were made in the mass culture. Here, the methylation levels did not change while the
gene expression, potentially under the control of several mechanisms, fluctuated.
This is reflected again when we studied the individual clones. The expression levels
varied greatly from clone to clone and from gene to gene, while the methylation
levels in all clones were similar from each other after treatment.
Since the majority of clones had no or low level of expression of the antigens,
and only a few clones had high levels of expression, it is possible that the majority of
cells under chronic treatment are perhaps resistant to zebularine. It is possible that
the individual clones may have different degree of resistance toward zebularine,
hence responding in a heterogeneous manner as seen with the induction of CTAs
(Fig 2.13). While it is widely accepted that methylation is the primary mechanism
responsible for silencing CTAs, no clear correlation was found between methylation
and gene expression in hematological malignancies (De Smet et al., 1999; Sigalotti
et al., 2002; Suyama et al., 2002). In the MAGEA1 gene, a single CpG dinucleotide
in the promoter region has been identified to mediate protein-DNA interaction and
transcriptional silencing, suggesting that the general methylation status may not be
responsible for the induction of the gene (Zhang et al., 2004). Additionally,
hypomethylating agents such as zebularine and 5-aza-CdR may reverse the promoter
hypermethylation, but the presence of transcriptional activators necessary for the
activation of the target promoters is also required in order to activate a silenced gene
(Karpf et al., 2004).
94
One caveat to our finding was the comparison of normal fibroblasts to and
epithelially-derived cancer cells. Virally transformed or spontaneous immortalized
normal epithelial cells were treated with zebularine, but these cells did not survive
the treatment and died within a couple of days of treatment. Also, the use of normal
epithelial cell lines such as HMECs and HUVECs were tried but yielded ambiguous
results. It was imperative that zebularine effects on normal tissue be studied using
animal models, since we were not successful with the in vitro experiments. Chapter 5
deals with the effects of chronic zebularine administration in the normal tissues of
mice. Our in vitro findings suggest that zebularine, in addition to being chemically
stable and non-toxic, is selective in its demethylating activity to cancer cells, thereby
making it a promising candidate for epigenetic therapy.
95
CHAPTER 3
ZEBULARINE PRODRUGS
INTRODUCTION
Zebularine (1--D-ribofuranosyl-2(1H)-pyrimidinone) is a cytidine analog
that has recently been characterized as a mechanism-based DNA methylation
inhibitor. The chemical properties of zebularine have been extensively studied,
demonstrating that it is an exceptionally stable compound in aqueous solution in a
wide range of pH from acidic to neutral thus allowing oral delivery of the drug
possible. In addition, a comparison of zebularine to 5-azacytidine (5-aza-CR), a
potent demethylating agent, demonstrated that it is much less toxic than the latter.
This makes zebularine ideally suited for further development into a
chemotherapeutic drug. Its mechanism of action is well-defined by works showing
that zebularine forms a covalent complex with DNA methyltransferases (Hurd et al.,
1999; Zhou et al., 2002). Zebularine was originally synthesized as an inhibitor of
cytidine deaminase. Therefore it is not subject to enzymatic degradation unlike 5-
aza-CR, resulting in longer half-life of the drug in vivo (Kim et al., 1986). Most
importantly, zebularine has been shown repeatedly to be an effective demethylating
agent with an antitumor property in animal models as well as against human cancer
cell lines (Cheng et al., 2003; Cheng et al., 2004b; Herranz et al., 2005).
96
Zebularine works by a mechanism similar to that of 5-aza-CR to inhibit DNA
methylation. It is phosphorylated by uridine/cytidine kinase and is eventually
incorporated into RNA and/or DNA. The zebularine diphosphate molecule can
undergo two distinct fates: it may continue to get incorporated into the triphophate
and end up in RNA or it may undergo reduction by ribonucleotide educates, and
become 2’-deoxy zebularine diphosphate which is then incorporated into DNA. Only
when zebularine is incorporated into the DNA, can it form the DNA methylation-
inhibitory covalent complexes with DNA methyltransferases. It is currently
postulated that the phosphorylation by uridine/cytidine kinase is the rate limiting step
for DNA incorporation of zebularine. Furthermore, the rate of RNA incorporation of
zebularine is about seven-fold higher than that for the DNA incorporation, indicating
that only a small portion of the drug is used effectively against DNA methylation
(Ben-Kasus et al., 2005). These are the reasons why zebularine is over 10-fold and
100-fold less potent than 5-aza-CR and 5-aza-CdR in terms of inhibiting DNA
methylation (unpublished data).
In an attempt to make zebularine a more potent inhibitor of DNA methylation,
we introduced a series of nucleoside analogs and zebularine derivatives. In our first
attempt, we synthesized ribonucleoside analogs that have structural similarities to
zebularine with slight modifications in the pyrimidine ring. We then tested a number
of zebularine monophosphate pronucleotides whose lypophilic property would
facilitate the intermembrane crossing of the target compound, which then would
spontaneously or enzymatically be metabolized to either yield zebularine or 2’-deoxy
97
zebularine monophosphate. With the use of 2’-deoxy monophosphate moiety of
zebularine, we would bypass the rate-limiting uridine/cytidine kinase and the
ribonucleotide reductase step, allowing more zebularine to be incorporated into DNA.
Lastly, we tested cyclic 2’-deoxy zebularine monophosphate dimers which utilizes a
temperature-sensitive processing of the lypophilic protection group at 37ºC, followed
by an endogenous phosphodiesterase-1 cleavage to yield 2’-deoxy zebularine
monophosphate. TpZ dinucleotide was used to demonstrate that the 2’-deoxy
zebularine monophosphate is an alternate method of delivering the nucleoside
analogs for DNA incorporation. These compounds provided us an opportunity to
learn about the mechanism of action of the drug and suggested possible approaches
that may be taken in the future for the development of other demethylating agents.
98
MATERIALS AND METHODS
Cell lines and drug treatment. T24 bladder carcinoma cells and HCT116 colon
carcinoma cells were obtained from the American Type Culture Collection
(Rockville, MD) and cultured in McCoy’s 5A medium supplemented with 10% heat-
inactivated fetal calf serum (FCS), 100 units/ml penicillin, and 100µg/ml
streptomycin (Invitrogen, Carlsbad, CA). Cf-Pac-1 pancreatic carcinoma cells also
from American Type Culture Collection were cultured in IMDM medium
(Gibco/Life Technologies Inc., Palo Alto, CA) supplemented with 10% heat-
inactivated FCS, 100 units/ml penicillin, and 100µg/ml streptomycin (Invitrogen,
Carlsbad, CA). All cells were cultured in a humidified incubator at 37ºC in 5% CO
2
.
Zebularine (obtained from Dr. Victor E. Marquez at NCI at Frederick) and 5-aza-
CdR (Sigma-Aldrich, St. Louis, MO) were dissolved in PBS. Zebularine prodrugs
were synthesized by collaborators, Victor E. Marquez (NCI at Frederick, Frederick,
MD) and Chris McGuigan (Cardiff University, Wales, UK), dissolved in PBS or
ethanol, depending on solubility, and store at -80ºC. Cells were seeded (3x10
5
cells/10cm dish) and treated with various compounds after 24 hr. The medium was
changed every 2-3 days after the initial treatment and supplemented with a fresh dose
of drug.
Determination of doubling time. The cell number/dish was counted with a Z1
Coulter Particle Counter (Beckman Coulter Corporation, Hialeh, FL) every 2 to 3
99
days. Untreated cells were analyzed under similar conditions as a control. The
average cell number from two plates was determined, and the mean cell numbers
were plotted to define the cell population doubling times, where population doubling
PD = log (number of cells harvested/ number of cells seeded)/log 2. Initial drug
treatment was started 24 h after seeding.
Nucleic acid isolation. RNA was collected from T24 and HCT116 cells with the
RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol.
DNA was collected using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according
to the manufacturer’s protocol.
Quantitative RT-PCR. Total RNA (5µg) was reverse transcribed with Moloney
murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) and random
primers (Invitrogen, Carlsbad, CA). The reverse transcription was carried out in a
total volume of 50µl as previously described (Gonzalez-Zulueta et al., 1995). The
quantitation of mRNA levels was carried out by a real-time fluorescence detection
method as described previously (Cheng et al., 2004a; Eads et al., 1999). All samples
were normalized to the reference gene, GAPDH. The primer and probe sequences are
as follows: for p16, sense 5’-CTG CCC AAC GCA CCG A-3’, probe 5’ 6-FAM –
TGG ATC GGC CTC CGA CCG TAA CT BHQ-1 3’, and antisense 5’-CGC TGC
CCA TCA TCA TGA C-3’; for SPANXA1, sense 5’-TGT GAT TCC AAC GAG
100
GCC A-3’, probe 5’ 6-FAM-CGA GAT GAT GCC GGA GAC CCC A BHQ-1 3’,
and antisense 5’-GCG GGT CTG AGT CCC CA-3’; for MAGEA1, sense 5’-GAA
CCT GAC CCA GGC TCT GTG-3’, probe 5’ 6-FAM-CAA GGT TTT CAG GGG
ACA GGC CAA C BHQ-1 3’, and antisense 5’-CCA CAG GCA GAT CTT CTC
CTT C’-3’; for MAGEB2, sense 5’-CGG CAG TCA AGC CAT CAT G-3’, probe 5’
6-FAM-TCG TGG TCA GAA GAG TAA GCT CCG TGC BHQ-1 3’, and antisense
5’-GCG GGT CTG AGT CCC CA-3’; for GAGE, sense 5’-GCT GAT AGC CAG
GAA CAG GG-3’, probe 5’ 6-FAM-CAC CCA CAG ACT GGG TGT GAG TGT
GA BHQ-1 3’, and antisense 5’-CCT GCC CAT CAG GAC CAT C-3’; for XAGEA1,
sense 5’-TCC CCA GAC GGG ACC AG-3’, probe 5’ 5-FAM-AGA GGG ACG
GCA TGA GCG ACA CAC BHQ-1 3’, and antisense 5’-CTG GCT GTG TGG TTC
TGT GTT T-3’; for GAPDH, sense 5’-TGA AGG TCG GAG TCA ACG G-3’,
probe 5’ 6-FAM –TTT GGT CGT ATT GGG CGC CTG G BHQ-1 3’, and antisense
5’-AGA GTT AAA AGC AGC CCT GGT G-3’. The conditions for real time RT-
PCR are: 94ºC for 9 min followed by 45 cycles at 94ºC for 15 s and 60ºC for 1 min.
Quantitation of DNA methylation. Genomic DNA (4µg) was treated with sodium
bisulfite as previously described (Cheng et al., 2004a). Methylation analysis was
performed using the methylation-sensitive single-nucleotide primer extension (Ms-
SNuPE) assay for p16 5’ region as previously described (Gonzalgo and Jones, 2002).
The PCR primers used are: for p16, sense 5’-TTT GAG GGA TAG GGT-3’ and
antisense 5’-TCT AAT AAC CAA CCA ACC CCT CC-3’; for MAGEA1, sense 5’-
101
GTT TAT TTT TAT TTT TAT TTA GGT AGG A-3 and antisense 5’-TTA CCT
CCT CAC AAA ACC TAA A-3’; for MAGEB2, sense 5’-TTG AGG GAG GTG
GGG GTA TTG T-3’ and antisense 5’-CTT CAA TTT ACA CTC AAA ATC CTC
ACC T-3’; for D4Z4, sense 5’-GGG TTG AGG GTT GGG TTT AT-3’ and
antisense 5’-AAC TTA CAC CCT TCC CTA CA-3’. An initial denaturation at 94ºC
for 3 min was followed by 94ºC for 45 s, annealing for 45 s, 72ºC for 45 s for 40
cycles. The annealing temperatures for each locus are: for p16, 65ºC, for MAGEA1,
53ºC, for MAGEB2, 62 ºC, and for D4Z4, 58 ºC. Primers used for Ms-SNuPE
analysis are as follows: for p16, 5’-TTT TTT TGT TTG GAA AGA TAT-3’, 5’-TTT
TAG GGG TGT TAT ATT-3’, and 5’-GTA GAG TTT AGT T-3’; for MAGEA1, 5’-
AGG TTT TTA TTT TGA GGG A-3’, 5’-TGG GGT AGA GAG AAG-3’, and 5’-
TTT TAT TTT TAT TTA GGT AGG ATT-3’; for MAGEB2, 5’-ATT GTT TGG
AGG TTG G-3’, 5’-GAG GAT TTT TAG TGA AGA-3’, and 5’-GAT GTG GTT
TAT TTT GAT TTT-3’; for D4Z4, 5’-TGA GGG TTG GGT TTA TAG T-3’, TAT
ATT TTT AGG TTT AGT TTT GTA A-3’, 5’-GTG GTT TAG GGA GTG GG-3’,
and 5’-GAA AGG TTG GTT ATG T-3’. Conditions for primer extension were: 94ºC
for 1 min, 50ºC for 30 s, and 72ºC for 20 s.
102
RESULTS
Nucleoside analog approach.
The first approach we took in the identification and characterization of DNA
methylation inhibitors that are more potent and stable than the existing drugs
available to us was to test nucleoside analogs that contained different functional
groups on the pyrimidine ring of the cytosine base (Table 3.1). Our standard protocol
for determining a bona fide DNA methylation inhibitor is to first look for the
decrease in the rate of growth of cells as measured by doubling time and then to
measure the induction of p16 gene or cancer/testis antigens after 8 or 9 days of
continuous treatment in cancer cells. Methylation-silenced p16 is induced in cancer
cells treated with demethylating agents such as 5-aza-CdR or zebularine (Cheng et
al., 2003; Gonzalez-Zulueta et al., 1995) and cancer-testis antigens (CTAs) are
sensitive markers of methylation changes (Chapter 2). The reexpression of p16 gene
which is a cell-cycle regulator would slow down the growth rate of the cells, which
would be reflected in the doubling time of the cells under treatment. Zebularine
treatment of various cancer cell lines have been shown to exhibit increases in their
doubling time by 30 to 60% (Cheng et al., 2004a). In this study, T24 cells were
treated with 4-methylzebularine, 3-deaza-5-aza-2’-deoxycytidine, NSC740467, and
NSC737452. Cf-Pac-1 cells were treated with NSC737451 and NSC737453 at
concentration ranging from 1 to 100µM. These compounds were provided by Dr.
Victor Marquez at National Cancer Institute at Frederick (Frederick, Maryland).
103
Table 3.1. Cytidine analogs; candidate DNA methylation inhibitors.
Compound Structure MW (g/mol) Formula p16 induction
4-Methylzebularine
N
CH 3
O
N
O
OH OH
H H
H H
HO 242.23 C
10
H
14
N
2
O
5
No
2’-Deoxy-3-deaza-5-azacytidine
N
NH 2
O
N
O
OH
HO 227.22 C
9
H
13
N
3
O
4
No
NSC740467
N
O
N
O
OH OH
H H
H H
HO
O 2 N
257.20 C
9
H
11
N
3
O
6
No
NSC737451
N
O
N
O
OH OH
H H
H H
HO
236.22 C
11
H
12
N
2
O
4
No
NSC737452
N
O
N
O
OH OH
H H
H H
HO
NC
237.21 C
10
H
11
N
3
O
4
No
NSC737453
N
O
N
O
OH OH
H H
H H
HO
H 3 CO
O
270.24 C
11
H
14
N
2
O
6
No
104
Table 3.2. Effects of zebularine prodrugs on the growth rate of cancer cells.
Percent increase in doubling time of cells treated with derivatives of zebularine
continuously for 8 days is shown.
Compound Concentration % Increase
Untreated 0
0.1% Ethanol 6
100μM Zebularine 33
3-Deaza-5-aza-CdR 1μM 6
10μM 6
4-Methylzebularine 1μM 0
10μM 6
100μM 17
NSC740467 1μM 17
10μM 6
100μM 11
NSC737451 1μM -7
10μM -7
NSC737452 1μM 6
10μM 6
100μM 6
NSC737453 1μM 9
10μM -2
100μM -7
Note: T24 bladder carcinoma cells were used for all compounds tested, with the
exception of NSC737451 and NSC737453 for which Cf-Pac-1 were used.
105
Figure 3.1. Effects of 4-methylzebularine on gene expression in T24 cells. T24
cells were treated with 4-methylzebularine continuously for 9 days, after which RNA
was collected and the expression level of p16, SPANXA1, MAGEA1, MAGEB2,
GAGE, and XAGE1 were measured by real time RT-PCR. No induction of genes was
observed at the concentrations tested.
0.000
0.001
0.002
0.003
0.004
Untreated 100μM Zeb 1μM 10μM 100μM
p16/GAPDH
0.00
0.02
0.04
0.06
0.08
0.10
Untreated 100μM Zeb 1μM 10μM 100μM
SPANXA1/GAPDH
0.000
0.002
0.004
0.006
0.008
0.010
Untreated 100μM Zeb 1μM 10μM 100μM
MAGEA1/GAPDH
0.000
0.005
0.010
0.015
0.020
0.025
0.030
Untreated 100μM Zeb 1μM 10μM 100μM
MAGEB2/GAPDH
0.00
0.01
0.02
0.03
0.04
Untreated 100μM Zeb 1μM 10μM 100μM
GAGE/GAPDH
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
Untreated 100μM Zeb 1μM 10μM 100μM
XAGE1/GAPDH
106
Figure 3.2. Effects of 3-deaza-5-aza-2’-deoxycytidine on p16 expression in T24
cells. T24 cells were treated with 3-deaza-5-aza-2’-deoxycytidine continuously for 9
days at which point RNA was collected and expression level of p16 gene was
measured by real time RT-PCR. No induction of p16 expression was observed at the
concentrations tested.
0
0.001
0.002
0.003
0.004
Untreated 1μM 5-aza-CdR 1μM 10μM
3-deaza-5-aza-CdR
p16/GAPDH
107
Figure 3.3. Effects of NSC740467 and NSC737452 on p16 expression in T24 cells.
T24 cells were treated with NSC740467 or NSC737452 continuously for 9 days at
which point RNA was collected and expression level of p16 gene was measured by
real time RT-PCR. No induction of p16 expression was observed at the
concentrations tested.
0
0.01
0.02
0.03
0.04
Untreated
100μM
Zeb
1μM
10μM
100μM
1μM
10μM
100μM
NSC740467 NSC737452
p16/GAPDH
108
Figure 3.4. Effects of NSC737451 and NSC737453 on p16 expression in Cf-Pac-1
cells. Cf-Pac-1 cells were treated with NSC737451 or NSC737453 continuously for
9 days at which point RNA was collected and expression level of p16 gene was
measured by real time RT-PCR. No induction of p16 expression was observed at the
concentrations tested.
0.000
0.001
0.002
0.003
0.004
0.005
Untreated
100uM
Zeb
1μM
10μM
100μM
1μM
10μM
100μM
NSC737451 NSC737453
p16/GAPDH
109
After 8 days of treatment with 100µM zebularine, a 33% increase in the rate
of growth was noted as measured by doubling time in T24 cells (Table 3.2). In
addition, induction of p16 and CTAs was seen by real time RT-PCR (Fig 3.1).
However, minimal growth inhibition was observed in T24 and Cf-Pac-1 cancer cells
treated with the nucleoside analogs (Table 3.2). Likewise, no induction of p16 and
CTAs was observed after 4-methylzebularine, 3-deaza-5-aza-2’-deoxycytidine,
NSC740467, and NSC737452 treatment of T24 cells (Fig 3.1, 3.2, 3.3) and after
NSC737451 and NSC737453 treatment of Cf-Pac-1 cells (Fig 3.4). These results
demonstrated that nucleoside analogs that resemble zebularine in their chemical
structures are not good inhibitors of DNA methylation as shown by a negligible
change in the growth rate and the absence of gene induction after the drug treatment.
cycloSal prodrug approach.
We next tested a family of nucleoside analogs containing a cyclosaligenyl
(cycloSal) phosphotriester, named CME-1 through CME-5, which was also provided
by Dr. Marquez from NCI at Frederick (Table 3.3). The cycloSal phosphotriester
approach is a potential vehicle for the delivery of monophosphate nucleosides, which
has been tested for the delivery of nucleoside and nucleotide antiviral and anticancer
agents such as d4T and FUdR (Lorey and Meier, 1999; Meier et al., 1998). These
five compounds all contain a cycloSal phosphotriester monophosphate at the 5’ end
of the nucleoside. CME-4 and CME-5 have a 5-POM
110
Table 3.3. Cytidine analogs; candidate DNA methylation inhibitors.
Compound Structure MW (g/mol) Formula
p16
induction
3-Methyl—cycloSal-2’-
deoxy-2’--fluoro-
zebularine-
monophosphate;
CME-1
N
O
N
O
OH
F
O P
O
O O 412.31 C
17
H
18
FN
2
O
7
P No
3-Methyl-cycloSal-2’-
deoxyzebularine-
monophosphate;
CME-2
N
O
N
O
OH
O P
O
O O 394.32 C
17
H
19
N
2
O
7
P No
3-Methyl-cycloSal-2’,3’-
dideoxy-m5K-
monophosphate;
CME-3
N
O
N
O
O P
O
O O
392.34 C
18
H
21
N
2
O
6
P No
5-POM-
oxycarbonylethyl-
cycloSal-2’-deoxy-m5K-
monophosphate;
CME-4
N
O
N
O
OH
O P
O
O O
O O
O O
580.52 C
26
H
33
N
2
O
11
P No
5-POM-
oxycarbonylethyl-
cycloSal-2’,3’-dideoxy-
m5K-monophosphate;
CME-5
N
O
N
O
O P
O
O O
O O
O O
564.52 C
26
H
33
N
2
O
10
P No
111
Table 3.4. Effects of zebularine prodrugs on the growth rate of cancer cells.
Percent increase in doubling time of cells treated with derivatives of zebularine
continuously for 8 days is shown.
Compound Concentration % Increase
Untreated 0
0.1% Ethanol 6
100μM Zebularine 33
CME-1 10μM 6
100μM 56
CME-2 10μM 6
100μM 11
CME-3 10μM 33
CME-4 10μM 6
CME-5 10μM 6
112
Figure 3.5. Effects of CME-1 through CME-5 on p16 expression in T24 cells.
T24 cells were treated with CME-1 through CME-5 continuously for 9 days at which
point RNA was collected and expression level of p16 gene was measured by real
time RT-PCR. No induction of p16 expression was observed at the concentrations
tested.
0
0.002
0.004
0.006
0.008
Untreated
0.1%
EtOH
100μM
Zeb
10μM
100μM
10μM
100μM
10μM
10μM
10μM
CME-1 CME-2 CME-3 CME-4 CME-5
p16/GAPDH
113
oxycarbonylethyl group in addition to the cycloSal group, which is a combination of
two functional groups. The main goal of this approach is to mask the negative charge
of the zebularine monophosphate to facilitate the 5’ monophosphate uptake into cells.
Once in the cells, the protection groups are cleaved either spontaneously or
enzymatically to yield a free 2’-deoxy zebularine monophosphate. This approach
will allow us to bypass several enzymatic steps that are either slowing down
zebularine metabolism or depleting the concentration of zebularine. All five
compounds caused retardation of growth of T24 cells to various degrees after nine
days of continuous treatment. CME-1 inhibited growth up to 56% after 100µM
treatment and CME-3 caused a 33% increase in doubling time with 10µM treatment,
while the others caused a minimal increase in doubling time of T24 cells (Table 3.4).
Unfortunately, real time RT-PCR analysis showed that there was no induction of p16
gene after treatment by these compounds (Fig 3.5), suggesting that the growth
inhibition was caused by a mechanism other than DNA methylation inhibition and
the subsequent cell-cycle arrest. Although we were not successful with the cycloSal
approach, we decided to pursue other pronucleotide approaches whereby a
zebularine monophosphate is protected with functional groups at the 5’ end to allow
facile transport of the prodrug through the cell membrane.
114
Table 3.5. Zebularine phosphoramidite prodrugs.
Compound Structure MW (g/mol) Formula p16 induction
2’-Zeb-dCR
phosphoramidite
N
O
N
O
OH
O P O
NH
O
CH H
3
C
O H
3
CO
453.00 C
19
H
24
N
3
O
8
P No
2’-F-Zeb-dCR
phosphoramidite
N
O
N
O
OH
F
O P O
NH
O
CH H
3
C
O H
3
CO
471.37
C
19
H
23
FN
3
O
8
P
No
115
Table 3.5. Zebularine phosphoramidite prodrugs, continued.
Compound Structure MW (g/mol) Formula p16 induction
CPF190
O
OH
O
N
N
O
P O
NH
O
O
O
OH
545.48 C
25
H
28
N
3
O
8
P No
CPF191
O
OH
O
N
N
O
P O
NH
O
O
O
529.48 C
25
H
28
N
3
O
7
P No
CPF192
O
OH
O
N
N
O
P O
NH
O
O
O
F
547.47
C
25
H
27
FN
3
O
7
P
No
CPF208
O
OH
O
N
N
O
P O
NH
O
CO 2 Et
Cl
501.85
C
20
H
25
ClN
3
O
8
P
Yes
CPF209
O
O
O
N
N
O
P O
NH
O
CO 2 Et
Cl
P O
NH
O
CO 2 Et
Cl
791.51
C
31
H
38
Cl
2
N
4
O
12
P
2
Yes
116
Table 3.5. Zebularine phosphoramidite prodrugs, continued.
Compound Structure MW (g/mol) Formula p16 induction
CPF213
O
OH
O
N
N
O
P O
NH
O
O
O
543.51 C
26
H
30
N
3
O
8
P Yes
CPF214
O
O
O
N
N
O
P
H
N
O
O
P O
NH
O
O
O
O
O
998.95
C
53
H
452
N
4
O
1
2
P
2
No
CPF242
O
O
O
N
N
O
P O
NH
O
O
O
P
O
O
NH
O
O
890.26
C
43
H
49
N
4
O
11
P
3
Yes
117
Figure 3.6. Effects of 2’-zebularine deoxycytidine phosphoramidite and 2’-F-
zebularine deoxycytidine phosphoarmidite on p16 expression in T24 cells. T24
cells were treated with 2’-zeb-CdR phosphoramidite (A) or 2’-F-zeb-CdR
phosphoramidite (B) continuously for 9 days after which RNA was collected and the
level of p16 expression was measured by real time RT-PCR. No induction of genes
was observed at the concentrations tested.
A.
0.0000
0.0001
0.0002
0.0003
0.0004
Untreated 100μM Zeb 1μM 10μM 100μM
p16/GAPDH
B.
0.00000
0.00005
0.00010
0.00015
Untreated 100μM Zeb 1μM 10μM 100μM
p16/GAPDH
118
Figure 3.7. Effects of 2’-zebularine deoxycytidine phosphoramidite p16
expression in Cf-Pac-1 cells. Cf-Pac-1 cells were treated with 2’-zeb-CdR
phosphoramidite continuously for 9 days after which RNA was collected and the
level of p16 expression was measured by real time RT-PCR. No induction of genes
was observed at the concentrations tested.
0
0.001
0.002
0.003
0.004
0.005
Untreated 100uM Zeb 1uM 10uM 100uM
p16/GAPDH
119
Phosphoramidate prodrug approach.
We then tested a series of zebularine deoxycytidine phosphoramidates for
their ability to inhibit DNA methylation in various cancer cell lines (Table 3.5).
Zebularine deoxycytidine phosphoramidate and 2’-F-Zeb-dCR phosphoramidate
were synthesized by Dr. Marquez. For the synthesis of CPF series, deoxyzebularine
was provided by Dr. Marquez and the final prodrugs were generated by Dr.
Christopher McGuigan from Cardiff University (Cardiff, UK). This approach is
similar to the cycloSal approach, except that the cycloSal phosphotriester group is
replaced with a phosphoramidate group. Once in the cells, the phosphoramidate is
cleaved by phosphatases and esterases to give 2’-deoxy zebularine monophosphate
which should be further phosphorylated into the 5’-triphosphate and promptly
incorporated into DNA.
T24 cells were treated with 2’-zeb-dCR phosphoramidate and 2’-F-zeb-dCR
phosphoramidate continuously for nine days. No induction of the p16 gene was
observed at 1, 10, and 100µM concentrations indicating that these two compounds
cannot inhibit DNA methylation in T24 cells (Fig 3.6). Zeb-2’-dCR
phosphoramidate was used to treat Cf-Pac-1 pancreatic cancer cells but failed to
induce the p16 gene in these cells as well (Fig 3.7). Since these two
phosphoramidates were not able to induce p16 after nine days of treatment, they
were omitted from further study.
We then continued our study with the first generation CPF pronucleotides,
CPF190-CPF192 (Table 3.5). These compounds were used to treat T24 bladder,
120
Figure 3.8. Effects of CPF190-192 on p16 expression in cancer cells. T24 bladder
(A), HCT15 colon (B), and Cf-Pac-1 pancreatic (C) cancer cells were treated with
CPF190-192 zebularine phosphoramidite continuously for 9 days. RNA was
collected and the level of p16 expression was measured by real time RT-PCR. No
induction of p16 was detected at the concentrations tested.
A.
0.000
0.001
0.002
0.003
0.004
Untreated
100μM
Zeb
1uM
10uM
100uM
1uM
10uM
100uM
1uM
10uM
100uM
CPF190 CPF191 CPF192
p16/GAPDH
B.
0.000
0.001
0.002
0.003
Untreated
0.1%
EtOH
100μM
Zeb
10μM
100μM
10μM
100μM
10μM
100μM
CPF190 CPF191 CPF192
p16/GAPDH
C.
0
0.01
0.02
0.03
Untreated
0.1% EtOH
100μM Zeb
100μM Thy
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
CPF190 CPF190 + Thy CPF191 CPF191 + Thy CPF192 CPF192 + Thy
p16/GAPDH
*Thy, thymidine.
121
Table 3.6. Effects of zebularine phosphoramidite prodrugs on the growth rate
of T24 bladder cancer cells. Percent increase in doubling time of T24 bladder
carcinoma cells treated with zebularine phosphoramidite continuously for 9 days is
shown.
Compound Concentration
Doubling time
increase (%)
Untreated 0
0.1% Ethanol 0
100μM Zebebularine 70
100μM Thymidine 15
CPF190 1μM 0
10μM -5
100μM 0
CPF191 1μM -5
10μM 5
100μM 10
CPF192 1μM 5
10μM 10
100μM 60
CPF208 1μM 0
10μM 0
20μM 0
CPF209 1μM 0
10μM 5
20μM 15
CPF213 1μM 10
10μM 10
100μM 10
CPF213 + Thymidine 1μM 15
10μM 20
100μM 50
CPF214 1μM 15
10μM 70
CPF214 + Thymidine 1μM 20
10μM 110
122
Table 3.7. Effects of zebularine phosphoramidite prodrugs on the growth rate
of HCT15 colon cancer cells. Percent increase in doubling time of HCT15 colon
carcinoma cells treated with zebularine phosphoramidite continuously for 9 days is
shown.
Compound Concentration
Doubling time
increase (%)
Untreated 0
0.1% Ethanol 9
100μM Zebebularine 45
100μM Thymidine 27
CPF190 10μM 23
100μM 14
CPF191 10μM 18
100μM 105
CPF192 10μM 18
100μM 50
CPF213 10μM 36
100μM 27
CPF213 + Thymidine 10μM 36
100μM 50
CPF242 1μM 32
10μM 36
CPF242 + Thymidine 1μM 23
10μM 59
123
HCT15 colon, and Cf-Pac-1 pancreatic cancer cells for nine days. In all three cell
lines, no gene induction was observed after treatment and these compounds were not
further pursued (Fig 3.8). Interestingly, CPF192 in T24 cells and CPF191 in HCT15
cells caused retardation of cell growth at 100µM concentration (Tables 3.6 and 3.7),
indicating that these compounds may be slowing down the cell cycle progression via
an unknown mechanism in these cells.
The second generation pronucleotides, CFP208 and CPF209 were
subsequently tested in T24 and Cf-Pac-1 cells. In T24 cells, 1, 10, and 20µM
concentrations did not induce the expression of p16 gene (Fig 3.9a). Likewise, the
growth rate of T24 cells was not affected by CPF208 and CPF209 treatment (Table
3.6). When these compounds were used to treat Cf-Pac-1 cells, 100µM thymidine
was also included. The rationale for including thymidine in the treatment was to
provide for a pool of thymine monophosphate to the cells and thereby allow the
DNA synthesis to progress without cell-cycle arrest. The 2’-deoxy monophosphate
analog of zebularine is known to inhibit thymidylate synthase (TS), an enzyme
responsible for converting dUMP into dTMP in a salvage pathway (Votruba et al.,
1973). By inhibiting TS, 2’-deoxy zebularine monophosphate may be slowing down
the DNA synthesis which is essential for inhibition of DNA methylation. We
therefore supplemented the cells with 100µM thymidine to allow cell cycle to
progress without interference from the inhibition of TS. Surprisingly, induction of
p16 expression was observed when Cf-Pac-1 cells were treated with 100µM CPF208
or CPF209 in the presence of 100µM thymidine (Fig 3.9b). Furthermore, CPF208
124
Figure 3.9. Effects of CPF208 and CPF209 on p16 expression in cancer cells.
T24 bladder (A) and Cf-Pac-1 pancreatic (B) cancer cells were treated with CPF208
or CPF209 zebularine phosphoramidite continuously for 9 days. RNA was collected
and the level of p16 expression was measured by real time RT-PCR. CPF208 and
CPF209 induced p16 expression in the presence of 100μM thymidine in Cf-Pac-1
cells but not in T24 cells.
A.
0
0.000004
0.000008
0.000012
0.000016
Untreated
0.1%
EtOH
100μM
Zeb
1μM
10μM
20μM
1μM
10μM
20μM
CPF208 CPF209
p16/GAPDH
B.
0.00
0.03
0.06
0.09
0.12
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
100μM
CPF208 CPF208 + Thy CPF209 CPF209 + Thy
p16/GAPDH
*Thy, thymidine.
125
and CPF209 induced more robust expression of p16 than 100µM zebularine given
that the cells were treated for the same duration of time (Fig 3.9b).
Subsequently, the third generation pronucleotides from Dr. McGuigan,
CPF213, CPF214, and CPF242, were tested in T24, HCT15, and Cf-Pac-1 cells. To
summarize the results, CPF213 and CPF214 did not induce p16 expression in T24
cells, regardless of the presence of thymidine (Fig 3.10a). CPF213 and CPF242 did
not induce p16 expression in HCT15 cells as well 100µM zebularine at the
concentrations tested although 100µM CPF213 and 10µM CPF242 in the presence of
thymidine showed a slight increase in p16 expression (Fig 3.10b). Remarkably,
100µM CPF213 and 10µM CPF242 induced more robust expression of p16 than
100µM zebularine in Cf-Pac-1 cells in the presence of thymidine (Fig 3.11) which
was complemented by the inhibition of cell growth (Table 3.8).
To confirm that these phosphoramidates were causing hypomethylation in
these cells, we analyzed DNA methylation status of Cf-Pac-1 cells after treatment by
CPF208, CPF209, CPF213, or CPF242 at 4 different loci by Ms-SNuPE. The regions
analyzed were 5’ region of p16, MAGEA1, and MAGEB2 and a repetitive element
called D4Z4. All four loci are hypermethylated and have previously been shown to
lose DNA methylation after zebularine treatment ((Cheng et al., 2004a) and Chapter
2). Table 3.9 summarizes the DNA methylation status at these four loci. All four
compounds tested showed demethylation with 100µM dose with the exception of
CPF242, which was active at 10µM concentration. Also, these drugs worked best in
Cf-Pac-1 pancreatic cancer cells and marginally in HCT15 colon cancer cells.
126
Figure 3.10. Effects of CPF213, CPF214 and CPF242 on p16 expression in
cancer cells. T24 bladder (A) and HCT15 colon (B) cancer cells were treated with
CPF213, CPF214, and CPF242 zebularine phosphoramidite continuously for 9 days.
RNA was collected and the level of p16 expression was measured by real time RT-
PCR. Induction of p16 was observed with CPF213 or CPF242 treated HCT15 in the
presence of thymidine.
A.
0
0.002
0.004
0.006
0.008
0.01
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
100μM
1μM
10μM
100μM
1μM
10μM
1μM
10μM
CPF213 CPF213 + Thy CPF214 CPF214 +
Thy
p16/GAPDH
B.
0
0.05
0.1
0.15
0.2
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
10μM
100μM
10μM
100μM
1μM
10μM
1μM
10μM
CPF213 CPF213 +
Thy
CPF242 CPF242 +
Thy
p16/GAPDH
*Thy, thymidine.
127
Figure 3.11. Effects of CPF213, CPF214 and CPF242 on p16 expression in Cf-
Pac-1 pancreatic cancer cells. Cf-Pac-1 cells were treated with CPF213, CPF214,
or CPF242 zebularine phosphoramidite continuously for 9 days. RNA was collected
and the level of p16 expression was measured by real time RT-PCR. Induction of
p16 was observed with CPF213 or CPF242 treated cells in the presence of thymidine.
A.
0
0.01
0.02
0.03
0.04
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
100μM
1μM
10μM
100μM
1μM
3μM
10μM
1μM
3μM
10μM
CPF213 CPF213 + Thy CPF214 CPF214 + Thy
p16/GADH
B.
0
0.01
0.02
0.03
0.04
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
1μM
10μM
CPF242 CPF242 + Thy
p16/GAPDH
*Thy, thymidine.
128
Table 3.8. Effects of zebularine phosphoramidite prodrugs on the growth rate
of Cf-Pac-1 pancreatic cancer cells. Percent increase in doubling time of Cf-Pac-
1 pancreatic carcinoma cells treated with zebularine phosphoramidite continuously
for 9 days is shown.
Compound Concentration Doubling time
increase (%)
Untreated 0
0.1% Ethanol 23
100μM Zebebularine 103
100μM Thymidine 0
CPF190 1μM 18
10μM 38
100μM 31
CPF190 + Thymidine 1μM 28
10μM 33
100μM 54
CPF191 1μM 18
10μM 26
100μM 33
CPF191 + Thymidine 1μM 18
10μM 15
100μM 23
CPF192 1μM 49
10μM 23
100μM 49
CPF192 + Thymidine 1μM 26
10μM 36
100μM 56
CPF208 1μM 13
10μM 18
100μM 23
CPF208 + Thymidine 1μM 36
10μM 49
100μM 54
CPF209 1μM 15
10μM 13
100μM 115
CPF209 + Thymidine 1μM 51
10μM 51
129
Table 3.8. Effects of zebularine phosphoramidite prodrugs on the growth rate
of Cf-Pac-1 pancreatic cancer cells, continued.
Compound Concentration % Increase
CPF213 1μM -13
10μM -13
100μM 0
CPF213 + Thymidine 1μM 0
10μM 10
100μM 67
CPF214 1μM -15
3μM -13
10μM 10
CPF214 + Thymidine 1μM 5
3μM 13
10μM 51
CPF242 1μM 51
10μM 149
CPF242 + Thymidine 1μM 72
10μM 208
130
Table 3.9. DNA methylation analysis of Cf-Pac-1 cells treated with zebularine
phosphoramidites. Cf-Pac-1 cells were treated with CPF208, CPF209, CPF213,
or CPF242 continuously for 9 days and DNA was collected. DNA methylation
status of 4 different loci in percent methylation was analyzed by quantitative Ms-
SNuPE.
Treatment Concentrations
p16
exon1
MAGEA1 MAGEB2 D4Z4
Untreated 95 75 82 87
0.1% Ethanol 100 69 53 90
100μM Zebularine 84 51 66 63
100μM Thymidine 100 72 78 89
1 μM 96 66 75 88
10 μM 95 72 77 90
CPF208
100 μM 94 70 76 88
1 μM 93 75 78 88
10 μM 89 68 73 87
CPF208 +
100μM
Thymidine
100 μM 78 49 63 65
1 μM 96 78 82 91
CPF209
10 μM 95 81 82 88
1 μM 94 79 81 86 CPF209 +
100μM
Thymidine
10 μM 88 61 71 72
CPF213 100 μM 100 69 N/D 89
CPF213 +
100μM
Thymidine
100 μM 71 52 N/D 62
1 μM 100 71 N/D 89
CPF242
10 μM 100 71 N/D 85
1 μM 91 59 N/D 73 CPF242 +
100μM
Thymidine
10 μM 75 43 N/D 57
131
The most potent DNA methylation inhibitor of the group as measured by the degree
of demethylation in Cf-Pac-1 cells is CPF242, which was at least 10-fold more
potent than the other phosphoramidates and zebularine in terms of concentration.
Since thymidine is known to block DNA synthesis at high concentrations, we
then tested to see if lower concentrations of thymidine may be used to aid the
demethylating activity of the prodrug, CPF213. Cf-Pac-1 cells were treated with
100µM CPF213 and varying concentrations of thymidine. The induction of p16 gene
was observed only at 100µM thymidine and not at the lower concentrations. This
indicates that these cells must be supplemented with a 1:1 ratio of thymidine to
zebularine prodrug (Fig 3.12). Replacement of 100µM thymidine with 100µM
uridine was tested, which was also incapable of p16 induction (Fig 3.12).
Cyclic deoxyzebularine monophosphate dimer approach.
In order to overcome the thymidine requirement, we synthesized a cyclic 2’-
deoxy zebularine monophosphate-zebularine monophosphate dimer (d(ZpZ)) and a
cyclic 2’-deoxyzebularine-thymidine monophosphate dimer (d(ZpT)) (Table 3.10).
The protecting groups on the 5’ phosphate group of the cyclic dimers that facilitate
the uptake of the zebularine monophosphate moiety are released at 37ºC. The
deprotected dimer is further processed by phosphodiesterase-1 (PDE-1) to yield
either two free zebularine monophosphate molecules or one deoxy zebularine
monophosphate and one dTMP in cells (Fig 3.13). We reasoned that in the case of
zebularine-thymidine dimer, the supplemental dose of thymidine would not be
132
Figure 3.12. Effects of thymidine and uridine on CPF213 induction of p16
expression in Cf-Pac-1 pancreatic cancer cells. Cf-Pac-1 cells were treated with
CPF213 zebularine phosphoramidite continuously for 9 days in the presence of
varying concentrations of thymidine. RNA was collected and the level of p16
expression was measured by real time RT-PCR. Induction of p16 was observed with
CPF213 only in the presence of 100μM thymidine.
A.
0
0.01
0.02
0.03
0.04
Untreated
0.1%
EtOH
100μM
Zeb
100μM
CPF213
1μM
10μM
100μM
1μM
10μM
100μM
100μM
100μM +
CPF213
Thymidine Thymidine +
CPF213
Uridine
p16/GAPDH
133
Figure 3.13. Mechanism of action of cyclic deoxyzebularine monophosphate dimer. The cyclic dimer containing two
zebularine monophosphate moieties releases its protecting group at 37ºC and is further processed by phosphodiesterase-1
(PDE-1) to yield two free zebularine monophosphate molecules in cells.
O P
O
O
O
N OHC
O
N
N
O
O N
N
O
O
P O O
O
N CHO
37degC
O P
O
HO
O
O
N
N
O
O
N
N
O
O
P O OH
O
PDE-1
O P
OH
HO
O
O
N
N
O
OH
2
134
Table 3.10. Cyclic deoxyzebularine monophosphate dimers.
Compound Structure
MW
(g/mol)
Formula
p16
induction
Deoxyzebularine-thymidine
cyclic phosphate dimer;
Cyclic-d(ZpT)
O P
O
O
O
N OHC
O
N
NH
O
O N
N
O
O
P O O
O
N CHO
O
748.57 C
27
H
38
N
6
O
15
P
2
Yes
Deoxyzebularine cyclic
phosphate dimer;
Cyclic-d(ZpZ)
O P
O
O
O
N OHC
O
N
N
O
O N
N
O
O
P O O
O
N CHO
718.54 C
26
H
36
N
6
O
14
P
2
Yes
135
necessary to activate the p16 gene but that the d(ZpZ) would only be active in the
presence of thymidine. We tested the zebularine-zebularine in T24 and Cf-Pac-1
cells in the presence and absence of thymidine and observed that p16 induction was
observed at 100µM dimer with thymidine in Cf-Pac-1 cells (Fig 3.14). The inhibition
of growth also indicated that the dimer was active at 100µM with thymidine but not
in T24 cells (Table 3.11). We then tested the d(ZpT) dimer in Cf-Pac-1 cells with or
without thymidine. Surprisingly, the d(ZpT) dimer was only active in the presence of
thymidine as demonstrated by both growth inhibition (Table 3.12) and p16 induction
(Fig 3.15). This data suggests that the release of 2’-deoxy zebularine monophosphate
and dTMP is not sufficient to aid zebularine monophosphate to get incorporated into
the DNA. Although we have yet to determine the role of thymidine in the activation
of zebularine monophosphate, it appears that thymidine, and not thymidine
monophosphate, is the essential component of the zebularine inhibition of DNA
methylation. In addition, both dimers in the presence of thymidine did not induce
p16 as robustly as zebularine did (Fig 3.11 and 3.12), suggesting that perhaps the
deprotection and cleavage step may slow down the metabolism of zebularine as a
whole, hence weakening the DNA methylation inhibition potential.
Zebularine dinucleotide approach.
To demonstrate that the 2’-deoxy zebularine monophosphate moiety is
activated in the presence of thymidine and not thymidine monophosphate, we treated
T24 bladder cancer and Cf-Pac-1 pancreatic cancer cells with TpZ dinucleotide.
136
Figure 3.14. Effects of cyclic d(ZpZ) monophosphate dimer on p16 expression in
T24 bladder and Cf-Pac-1 pancreatic cancer cells. T24 and Cf-Pac-1 cells were
treated with cyclic deoxyzebularine monophosphate continuously for 8 days and
RNA collected for p16 expression analysis by real time RT-PCR.
A.
0
0.002
0.004
0.006
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
100μM
1μM
10μM
100μM
cyclic d(ZpZ) cyclic d(ZpZ) + Thy
p16/GAPDH
B.
0
0.002
0.004
0.006
0.008
Untreated
0.1%
EtOH
100μM
Zeb
100μM
Thy
1μM
10μM
100μM
1μM
10μM
100μM
cyclic d(ZpZ) cyclic d(ZpZ) + Thy
p16/GAPDH
*Thy, thymidine.
137
Table 3.11. Effects of cyclic deoxyzebularine monophosphate dimer on the
growth rate of T24 bladder and Cf-Pac-1 pancreatic cancer cells. Percent
increase in doubling time of T24 and Cf-Pac-1 cells treated with cyclic zeb-CdR-
monophosphate continuously for 8 days is shown.
Compound Concentration
T24 doubling
time increase
(%)
Cf-Pac-1
doubling time
increase (%)
Untreated 0 0
0.1% Ethanol 0 -15
100μM Zebularine 39 69
100μM Thymidine 0 -10
1μM 0 -5
10μM 0 13
Cyclic d(ZpZ)
100μM 0 15
1μM 4 0
10μM 4 13
Cyclic d(ZpZ)
+ Thy
100μM 13 46
138
Table 3.12. Effects of cyclic d(ZpT) monophosphate dimer on the growth rate
of Cf-Pac-1 pancreatic cancer cells. Percent increase in doubling time of Cf-Pac-
1 cells treated with cyclic zeb-CdR-monophosphate continuously for 8 days is
shown.
Compound Concentration
Cf-Pac-1
doubling time
increase (%)
Untreated 0
0.1% Ethanol -3
100μM Zebularine 274
100μM Thymidine 23
1μM 6
10μM 13
Cyclic d(ZpT)
100μM 16
1μM 29
10μM 35
Cyclic d(ZpT)
+ Thy
100μM 55
139
Figure 3.15. Effects of cyclic d(ZpT) monophosphate dimer on p16 expression in
Cf-Pac-1 pancreatic cancer cells. Cf-Pac-1 cells were treated with cyclic deoxy-
(ZpT) monophosphate continuously for 8 days and RNA collected for p16
expression analysis by real time RT-PCR.
A.
0.000
0.001
0.002
0.003
0.004
Untreated
0.1%
EtOH
100µM
Zeb
100µM
Thy
1µM
10µM
100µM
1µM
10µM
100µM
cyclic d(ZpT) cyclic d(ZpT) + Thy
p16/GAPDH
*Thy, thymidine.
140
Figure 3.16. Effects of TpZ dinucleotide on p16 expression in T24 bladder
cancer and Cf-Pac-1 pancreatic cancer cells. T24 and Cf-Pac-1 cells were treated
with TpZ dinucleotide continuously for 8 days and RNA collected for p16
expression analysis by real time RT-PCR.
A.
0
0.01
0.02
0.03
0.04
0.05
0.06
Untreated 1µM 5-aza-
CdR
100µM zeb 100µM Thy 1µM 10µM 100µM1µM 10µM 100µM
TpZ TpZ + Thy
p16/GAPDH
B.
0
0.03
0.06
0.09
0.12
0.15
Untreated 100μM
Zeb
100μM
Thy
1μM 10μM 100μM 1μM 10μM
TpZ TpZ + Thy
p16/GAPDH
141
Dinucleotides containing 5-aza-CdR are potent inhibitors of DNA methylation
(Chapter 4). Dinucleotides are cleaved into a nucleoside and a nucleotide, leaving the
phosphate group at the 5’-end of the second sugar ring. Therefore, TpZ dinucleotide
would yield one molecule of thymidine and one molecule of 2’-deoxy zebularine
monophosphate. Upon treatment with TpZ, a marginal induction of p16 was
observed in T24 cells, but only in the presence of thymidine (Fig 3.16a). Remarkably,
when Cf-Pac-1 cells were treated with TpZ alone or with thymidine, upregulation of
the p16 gene was seen at all doses tested (Fig 3.16b). Treatment of TpZ and
thymidine together showed more robust expression of p16 than zebularine alone,
suggesting that perhaps a higher dose of thymidine may make the pronucleotides
stronger inhibitor of methylation. Our results demonstrated that the inhibition of
methylation by these mechanism-base inhibitors does indeed occur via DNA
incorporation. Furthere, the use of the monophosphate moiety demonstrates that
zebularine suffer from inefficient enzymatic pathways which can be overcome.
142
DISCUSSION
Zebularine is an effective inhibitor of DNA methylation capable inducing the
expression of methylation-silenced genes upon treatment. It has many properties that
are advantageous as a chemotherapeutic agent, including its stability in aqueous
solution and low pHs as well as its low toxicity in animals and cultured cells (Cheng
et al., 2003; Cheng et al., 2004a). However, zebularine must undergo many
metabolic steps before DNA incorporation, which subsequently weakens the
demethylating effect of the drug. A number of nucleoside analogs and zebularine
derivatives were tested in search of a demethylating agent more potent and efficient
than zebularine. We tested several different families of compounds, including
ribonucleoside analogs, cycloSal 2’-deoxy zebularine monophosphate, 2’-deoxy
zebularine phosphoramidates, cyclic 2’-deoxy zebularine dimers, and zebularine
dinucleotides. Zebularine phosphoramidates and TpZ dinucleotides were the only
compounds capable of inducing gene expression greater than that by zebularine.
Therefore, we will focus our discussion on these two groups.
The zebularine phosphoramidates, CPF208, CPF209, CPF213 and CPF 242,
were active in Cf-Pac-1 pancreatic cancer cells and marginally active in HCT15
colon cancer cells but not in T24 bladder cancer cells. The cell line specificity of the
phosphoramidates may be due to the availability and activity of the enzyme
responsible for processing these compounds to give a zebularine monophosphate
moiety. Since all compounds that were used for the treatment are pronucleotides that
143
must first be processed to yield the active drug and not all cell lines may have the
same level or activity of the necessary enzymes, it is likely that Cf-Pac-1 cells had
the sufficient level of enzyme activity for the activation of the phosphoramidates but
not the T24 and HCT15 cells.
The phosphoramidates were treated either in the presence or absence of
thymidine and were not able to induce the expression of p16 gene in the absence of
thymidine. Thymidine was used to prevent the TS inhibition by the 2’-deoxy analog
of zebularine monophosphate (Votruba et al., 1973). Since zebularine depends solely
on the synthesis of new DNA strand to inhibit DNA methylation and the inhibition
of TS would block DNA synthesis due to the shortage of thymidine monophosphate,
it was imperative that the TS inhibition was prevented or that the low level of
thymidine monophosphate was supplemented with an outside source.
Although these compounds demonstrated conceptually that the treatment
with deoxy zebularine monophosphate inhibits DNA methylation, it is not clinically
applicable. It would be highly desirable to have a single drug rather than a
combination of nucleosides which may potentially influence the normal metabolic
function tremendously in patients. In order to circumvent the thymidine requirement,
we utilized a couple of cyclic dimers: d(ZpZ) and d(ZpT). These cyclic dimers
would allow us eliminate the need for a separate dose of thymidine since the release
of zebularine monophosphate and thymidine monophosphate would emerge together
as the prodrug is activated in the cells. However, the d(ZpT) dimers were not active
unless thymidine was used together, indicating that thymidine monophosphate was
144
not sufficient but that thymidine is necessary for the prodrug activation. It is possible
that the inhibition of TS is not rescued with thymidine monophosphate via another
mechanism that does not involve the TS enzyme.
Another way to abrogate the thymidine requirement is to prevent the
inhibition of TS by zebularine monophosphate. HDAC inhibitors, trichostatin A
(TSA) and suberoylanilide hydroxamic acid (SAHA), upregulate the expression of
TS (personal communication, Robert Ladner), and these drugs modulate epigenetic
events synergistically when used together with DNA methylation inhibitors
(Cameron et al., 1999). The use of HDAC inhibitors may upregulate the expression
of TS, eliminating the need for thymidine rescue. At the same time, HDAC inhibitors
may aid the zebularine moiety to upregulate the tumor suppressor genes
synergistically. This may be a good way to not only test the involvement of TS in the
metabolism of 2’-deoxy zebularine monophosphate moiety but to test if the
zebularine prodrug can be used in combination with HDAC inhibitors to obtain
greater therapeutic effect. Our preliminary experiment combining CPF213 and 4-
phenylbutyric acid (PBA) caused a weaker upregulation of p16 gene (data not
shown) than when CPF213 and thymidine were used. PBA seems to cause cell-cycle
arrest at the concentrations being used which may be diminishing the activity of the
zebularine moiety. Also, PBA is not a specific inhibitor of HDACs which is why
there was a weaker response and perhaps a more specific and potent HDAC inhibitor
such as SAHA which fits into the catalytic pocket of HDACs may be more effective
in confirming our hypothesis (Finnin et al., 1999).
145
In summary, CPF208, CPF209, CPF213, and CPF242 induced the expression
of the methylation-silenced p16 gene in Cf-Pac-1 pancreatic cancer cells in the
presence of 100μM thymidine, and CPF213 and CPF242 were additionally found to
increase a marginal levels of p16 expression in HCT15 colon cancer cells. The TpZ
dinucleotide demonstrated conceptually that the delivery of a monophosphate moiety
is possible with the use of short oligonucleotides as elaborated in Chapter 4. All
compounds were also capable of inducing much greater expression of the p16 gene
than zebularine, indicating that although they are cell line-specific and require the
presence of another nucleoside, these drugs may pose a promising start for the
zebularine pronucleotide.
146
CHAPTER 4
SHORT OLIGONUCLEOTIDE DNA METHYLATION
INHIBITORS
INTRODUCTION
Aberrations in DNA methylation are frequently observed in various types of
cancer (Costello and Plass, 2001; Jones and Baylin, 2002; Jones and Laird, 1999).
Several tumor suppressor genes or cancer-related genes acquire de novo DNA
methylation in promoter or regulatory regions leading to inactivation, contributing to
tumorigenesis (Gonzalez-Zulueta et al., 1995; Greger et al., 1989; Herman et al.,
1994; Sakai et al., 1991; Zhang et al., 1993). DNA hypermethylation can be reversed
by demethylating agents and crucial cellular functions reestablished in the cells. In
recent years, the use of DNA methylation inhibitors has become a promising
alternative to patients with myelodysplastic syndrome (MDS) and hematological
malignancies (Kaminskas et al., 2005; Yoo and Jones, 2006).
The most widely known examples of DNA methylation inhibitors are 5-
azacytidine (5-aza-CR) and 5-aza-2’-deoxycytidine (5-aza-CdR); both drugs were
initially synthesized as anticancer agents and were later shown to inhibit DNA
methylation (Jones and Taylor, 1980; Sorm et al., 1964; Sorm and Vesely, 1968).
The clinical use of nucleotides rather than nucleosides is essentially impossible due
to the negative charge on the phosphate group, which prevents effective cellular
uptake. Thus, nucleoside analogs, which are taken up intracellularly and
147
phosphorylated to their respective mono-, di-, and triphosphates before incorporation
into replicating DNA, leading to covalent trapping of DNA methyltransferases
(DNMTs), are used (Taylor and Jones, 1982). 5-Aza-CR and 5-aza-CdR are
powerful demethylating agents, nevertheless, they have a number of drawbacks. The
aza pyrimidine ring is unstable in aqueous solution making it difficult to administer
and is quite toxic both in vitro and in vivo (Beisler, 1978). Furthermore, the drugs
have transient effects and DNA is gradually remethylated after removal of the drug
(Cheng et al., 2004a). Yet another problem arises due to cytidine deaminase (CDA)
which renders the drugs inactive by converting them into 5-azauridine compounds.
In our attempts to synthesize more stable and potent inhibitors of DNA
methylation, we found that short oligonucleotides containing an azapyrimidine
effectively inhibit DNA methylation in living cells. Here, we focus on S110, a 5’-
AzapG-3’ dinucleotide, whose aqueous stability and toxicity are quite similar to that
of 5-aza-CdR but is protected from deamination by CDA. The demethylating activity
seems to require incorporation of the azapyrimidine into DNA presumably after
degradation of the oligonucleotide by phosphodiesterases and is not limited to a
dinucleotide but is seen in tri- and tetranucleotides as well, demonstrating that short
oligonucleotides are effective prodrugs for delivery of inhibitors of DNA
methylation. The utilization of short oligonucleotides as nucleoside drug delivery
vehicles which provide protection against enzymatic degradation might have
application for delivery of other nucleoside drugs to cells.
148
MATERIALS AND METHODS
Synthesis of oligonucleotides containing 5-aza-CdR
Dinucleotides, trinucleotides and tetranucleotides containing 5-aza-CdR were
synthesized by standard procedures with modifications to increase coupling times,
different oxidizing agents, and use of phenoxyacetyl decitabine phosphoramidate,
instead of phenoxyacetyl cytidine phosphoramidate. A polystyrene based solid
support with loading of 240 μmol/g of dG(pac) or D(pac) was used (Beaucage and
Caruthers, 1981; McBride and Caruthers, 1983). All synthesis and purification were
carried out by Dr. Pasit Phiasivongsa (SuperGen Inc., Pleasanton, CA).
Briefly, synthesis of S53, 5’-GpAza-3’ dinucleotide, is described here.
Amersham ÄKTA Oligopilot 10 system was loaded with a protected decitabine-
linked CPG solid support (phenoxyacetyl protection of amino function) and coupled
with 2-2.5 equivalents of tert-butyl phenoxyacetyl 2’-deoxyguanosine
phosphoramidate in presence of 60% of 0.3 M benzylthiotetrazole activator in
acetonitrile for 2.5 min. The CPG solid support containing protected S53 was treated
with 20ml of 50 mM K
2
CO
3
in methanol for 1 hr and 20 min. The coupled product
was oxidized with 2 M tert-butylhydroperoxide in dry acetonitrile prepared by
dissolving tert-butylhydroperoxide in 80% tert-butylperoxide for 5 min. The
dimethoxy trityl protective group was removed with 3% dichloroacetic acid in
toluene. The CPG solid support was washed with dry methanol; the filtrate was
neutralized by addition of 2ml 1 M acetic acid in methanol. The solution was
149
concentrated by rotary evaporation; the residue was taken up in 200 mM
triethylammonium acetate, pH 6.9, washed with 500µl 50% aqueous acetonitrile, and
filtered through a syringe filter. S53 was subsequently purified to 95% purity by the
ÄKTA Explorer 100 HPLC with a Gemini C18 preparative column (Phenomenex),
250x21.2 mm, 10µm with guard column (Phenomenex), 50x21.2mm, 10µm, with 50
mM triethylammonium acetate (pH 7) in MilliQ water (Mobile Phase A) and 80%
acetonitrile in MilliQ water (Mobile Phase B), with 2% to 20/25% Mobile Phase B in
column volumes. The ESI-MS (-ve) of triethylammonium salt of S53 exhibited m/z
556.1 [M-H]
-
and 1113.1 for [2M-H]
–
, and the calculated exact mass for the neutral
compound C
18
H
24
N
9
O
10
P is 557.14.
Cytidine deaminase assay. Recombinant human CDA was prepared as described in
Vincenzetti et al (Vincenzetti et al., 2003), and CDA assay was performed by Dr.
Chunlin Chen (SuperGen Inc., Pleasanton, CA). Purified CDA (0.09-0.1 unit) was
incubated with 0.2mM 5-aza-CdR or S110 in 3ml 0.1M Tris-HCl, pH 7.5 at 38°C.
Percent substrate remaining was measured after 45 and 90 min of incubation by a
Waters 2695 HPLC system with a 996 photodiode array detector (Waters
Corporation, Milford, MA).
Determination of nucleic acid stability. The absorbance of 5-aza-2’-deoxycytidine
(5-aza-CdR) and S110 was measured with a Beckman DU Series 600 UV/visible
Spectrophotometer (Beckman Coulter Inc., Fullerton, CA). The wavelengths of
150
maximum absorbance of 5-aza-CdR and S110 were 241nm and 245nm, respectively.
The initial absorbance was measured immediately after 5-aza-CdR and S110 were
dissolved in PBS, and both compounds were incubated at 37ºC except when
absorbance reading was taken.
Cell lines and drug treatment. T24 bladder carcinoma cells and HCT116 colon
carcinoma cells were obtained from the American Type Culture Collection
(Rockville, MD) and cultured in McCoy’s 5A medium supplemented with 10% heat-
inactivated fetal calf serum (FCS), 100 units/ml penicillin, and 100µg/ml
streptomycin (Invitrogen, Carlsbad, CA) in a humidified incubator at 37ºC in 5%
CO
2
. Cells were plated (1.5 x 10
5
cells/60mm dish) and treated 24 hr later with 5-
aza-2’-deoxycytidine (Sigma-Aldrich, St. Louis, MO) or S110 continuously for 6
days. Each compound was dissolved in PBS. The medium was changed 3 days after
the initial treatment and supplemented with a fresh dose of 5-aza-CdR or S110.
Determination of cytotoxicity. A colony formation assay was used to compare
cytotoxicity of 5-aza-CdR and S110 as previously described (Cheng et al., 2003).
Briefly, T24 cells were plated at a low density (100 cells/60mm dish) and treated
with varying concentrations of 5-aza-CdR and S110. Colonies were allowed to form
for 10-14 days, fixed with methanol, and stained with 10% Giemsa. The number of
colonies from an untreated control plate was used to calculate the plating efficiency
151
in percent at each concentration. Triplicate dishes were used and error bars are
represented by one standard deviation of the mean.
Nucleic acid isolation. RNA was collected from T24 and HCT116 cells with the
RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol.
DNA was collected using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according
to the manufacturer’s protocol.
Quantitative RT-PCR analysis. Total RNA (5µg) was reverse transcribed with
Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA) and
random primers (Invitrogen, Carlsbad, CA). The reverse transcription was carried
out in a total volume of 50µl as previously described (Gonzalez-Zulueta et al., 1995).
The quantitation of mRNA levels was carried out by a real-time fluorescence
detection method as described previously (Cheng et al., 2004a; Eads et al., 1999). All
samples were normalized to the reference gene, GAPDH. The primer and probe
sequences are as follows: for p16, sense 5’-CTG CCC AAC GCA CCG A-3’, probe
5’ 6-FAM –TGG ATC GGC CTC CGA CCG TAA CT BHQ-1 3’, and antisense 5’-
CGC TGC CCA TCA TCA TGA C-3’; for GAPDH, sense 5’-TGA AGG TCG GAG
TCA ACG G-3’, probe 5’ 6-FAM –TTT GGT CGT ATT GGG CGC CTG G BHQ-1
3’, and antisense 5’-AGA GTT AAA AGC AGC CCT GGT G-3’. The conditions for
real time RT-PCR are: 94ºC for 9 min followed by 45 cycles at 94ºC for 15 s and
60ºC for 1 min.
152
Western blot analysis. Cell pellets were lysed in radioimmunoprecipitation (RIPA)
buffer containing 0.1% SDS, 0.5% nonidet P-40, and 0.5% sodium deoxycholate in
PBS and incubated on ice for 30 min. The lysates were centrifuged at 4ºC for 30 min
at 14,000 rpm. The supernatant was collected and stored at -80ºC. Approximately
60µg of protein was electrophoresed on a Ready Gel Tris-HCl Gel, 4-15% linear
gradient (Bio-Rad Laboratories, Hercules, CA) and transferred to a PVDF membrane
using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories,
Hercules, CA). The membrane was hybridized with antibodies against human
DNMT1 (H-300; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA), p16 (N-20;
1:500; Santa Cruz Biotechnology, Santa Cruz, CA), and proliferating cell nuclear
antigen (PCNA) (PC10; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) in Tris-
buffered saline-Tween (TBS-T) buffer (0.1M Tris, 1.5M NaCl, and 1% Tween 20)
with 5% nonfat dry milk overnight at 4ºC. The membranes were washed four times
with TBS-T buffer at room temperature and incubated with secondary antibodies for
1 hr at room temperature. Secondary antibodies used were anti-rabbit-IgG-HRP for
DNMT1 and p16 (1:7500; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-
mouse-IgG-HRP for PCNA (1:7500; Santa Cruz Biotechnology, Santa Cruz, CA).
The membranes were washed six times with TBS-T at room temperature. Proteins
were detected with the ECL Western Blotting Detection Reagents (GE Healthcare
Bio-Sciences Corp., Piscataway, NJ) and by exposure to Kodak X-OMAT AR film
(Rochester, NY).
153
Quantitation of DNA methylation. Genomic DNA (4µg) was treated with sodium
bisulfite as previously described (Cheng et al., 2004a). Methylation analysis was
performed using the methylation-sensitive single-nucleotide primer extension (Ms-
SNuPE) assay for p16 5’ region as previously described (Gonzalgo and Jones, 2002).
The PCR primers used were: sense 5’-TTT GAG GGA TAG GGT-3’ and antisense
5’-TCT AAT AAC CAA CCA ACC CCT CC-3’. An initial denaturation at 94ºC for
3 min was followed by 94ºC for 45 s, 62ºC for 45 s, 72ºC for 45 s for 40 cycles.
Primers used for Ms-SNuPE analysis were: 5’-TTT TTT TGT TTG GAA AGA
TAT-3’, 5’-TTT TAG GGG TGT TAT ATT-3’, and 5’-GTA GAG TTT AGT T-
3’.Conditions for primer extension were: 94ºC for 1 min, 50ºC for 30 s, and 72ºC for
20 s.
In vitro hemimethylation assay. An intronic region of p16 was amplified using
human genomic DNA as a substrate and a primer set with a methylated CpG to yield
a hemimethylated CpG site. The amplicon (1ng) was treated with M.Sss1 or human
DNMT1 (New England Biolab, Ipswich, MA) in 50μl of reaction buffers, as
recommended by the manufacturer, for 30 min at 37°C, in the presence of S110 of
various concentrations. The reaction was stopped by heat inactivation of enzymes.
Bisulfite converted DNA was amplified by 5’-CTC TTA CCA TCC TCT T-3’ and
5’-GAG TTA TAT TTA TGT GAT TAT TTT-3’, and Ms-SNuPE assay was done
154
using 5’-TTT TAA AAT TTT GTT AAT AGT TTG AAT T-3’ as a primer. This
assay was performed by Dr. Shinwu Jeong in the lab.
Sucrose density gradient ultracentrifugation. Nuclei were prepared according to
the procedure described in Gal-Yam et al (Gal-Yam et al., 2006). Briefly, cells were
trypsinized and washed once with PBS. The cells were then resuspended in ice-cold
RSB buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl
2
) containing
Complete Mini proteinase inhibitors (Roche, Palo Alto, CA) and kept on ice for 10
min before dounce homogenization with 0.5% NP-40 to break up cell membranes.
Nuclei were washed twice with RSB plus proteinase inhibitors without the detergent.
Purified nuclei (1x10
8
) were resuspended in 1 ml of RSB containing 0.25M sucrose,
3mM CaCl
2
, and 100μM PMSF, and digested with MNase (Worthington
Biochemical Corporation, Lakewood, NJ) for 15 min at 37°C, and then the reaction
was stopped with EDTA/EGTA (up to 10mM). After microcentrifugation at 5,000
rpm for 5 min, the nuclear pellet was resuspended in 0.3 ml of the buffer (10mM
Tris-HCl, pH 7.4, 10mM NaCl) containing 5mM EDTA/EGTA, gently rocked for
1hr at 4°C, and followed by microcentrifugation to obtain soluble nucleosomes,
which were then fractionated through a sucrose density gradient solution (5-25%
sucrose, 10mM Tris-HCl, pH 7.4, 0.25mM EDTA, 300mM NaCl) at 30,000 rpm for
16 hr at 4°C. Fractions were taken from the top of the centrifuge tube into 16 aliquots
and subject to Western blot analysis.
155
RESULTS
Reexpression of p16 in T24 bladder cancer cells by short oligonucleotides.
We treated T24 cells with varying concentrations of 5-aza-CdR analogs,
oligonucleotides containing the 5-azapyrimidine ring, and quinoline derivatives for 6
days to test their abilities to induce the expression of the methylation-silenced p16
gene in T24 bladder carcinoma cells (Table 4.1). These compounds were synthesized
by SuperGen Inc.(Pleasanton, CA) to identify and characterize potent and stable
inhibitors of DNA methylation. Induction of p16 is an indirect yet straightforward
method to detect inhibition of DNA methylation since demethylation of the 5’ CpG
island of p16 results in reexpression of the gene (Gonzalez-Zulueta et al., 1995;
Merlo et al., 1995). All short chain nucleotides tested, regardless of their chain
lengths, were able to induce robust p16 expression with the exception of S52R and
S112, which induced expression to lesser extents (Table 4.2). S52R, a
phosphorothioate derivative of S110 (Table 4.1), is less prone to cleavage by
phosphodiesterases and caused a small induction of p16 at high concentration,
suggesting that the cleavage of the oligonucleotides into nucleotides and nucleosides
is a determining factor in inducing gene expression. S112 is an S110 derivative with
a hexaethylene glycol phosphate linker at the 5’ end (Table 4.1), which is subject to
cleavage in cells. This extra cleavage requirement probably slows down the ability of
S112 to induce the p16 gene and thus explains the weaker activity of the compound.
Reexpression of p16 gene and demethylation of 5’ end of p16 gene was observed in
156
Table 4.1. 5-Aza-2’-deoxycytidine analogs and quinoline derivatives from
SuperGen. List of compounds used to treat T24 bladder cancer cells continuously
for 6 days at varying concentrations to measure the induction of p16 gene by real-
time RT-PCR.
Compound Structure
MW
(g/mol)
Formula
p16
induction
S2
O
OH
HO
N
N
N
OCH 3
O
243.22
C
9
H
13
N
3
O
5
-
S3
O
OH
HO
N
N
N
NH 2
O
243.23
C
9
H
14
N
4
O
4
-
S8
O
OH
HO
N
N
N
NH
O
243.23
C
9
H
14
N
4
O
4
-
S9
O
OH
HO
N
N
N
NH 2
O
F
246.20
C
8
H
11
FN
4
O
4
-
S16
O
OH
HO
N
N
N
N
S
272.32
C
10
H
16
N
4
O
3
S
-
157
Table 4.1. 5-Aza-2’-deoxycytidine analogs and quinoline derivatives from
SuperGen, continued.
Compound Structure
MW
(g/mol)
Formula
p16
induction
S17
O
OH
HO
N
N
N
N
O
256.26
C
10
H
16
N
4
O
4
-
S52
NH
N
N
O
NH 2
N
O
OH
O
N N
NH 2
O N
O
O
P S
HO
O
-
+NH(CH 2 CH 3 ) 3
674.67
C
24
H
41
N
9
O
9
PS
+
S53
N
O
OH
O
N
O
O
P O
HO
O
-
+NH(CH
2
CH
3
)
3
NN
NH
2
O
NH
N
N
O
NH
2
658.60
C
24
H
51
N
9
O
10
P
+++
S54
GpAza*pG
2+NH(CH
2
CH
3
)
3
1089.00 - +++
S55
AzapGpAzapG
3+NH(CH
2
CH
3
)
3
1480.36 - +++
S56
pGpAzapAzapG
3+NH(CH
2
CH
3
)
3
1480.36 - +++
*Aza, 5-aza-2’-deoxycytidine.
158
Table 4.1. 5-Aza-2’-deoxycytidine analogs and quinoline derivatives from
SuperGen, continued.
Compound Structure
MW
(g/mol)
Formula
p16
induction
S110
NH
N
N
O
NH 2
N
O
OH
O
NN
NH 2
O N
O
O
P O
HO
ONa
579.39
C
18
H
23
N
9
NaO
10
P
+++
S111
NH
N
N
O
NH 2
N
O
OH
O
N
NH 2
O N
O
O
P O
HO
O
-
+NH(CH 2 CH 3 ) 3
657.61
C
24
H
42
N
11
O
10
P
-
S112
NH
N
N
O
NH 2
N
O
OH
O
NN
NH 2
O N
O
O
P O
O
O
-
+NH(CH 2 CH 3 ) 3
+NH(CH 2 CH 3 ) 3
P
O - O
O
HOH 2 CH 2 C(CH 2 CH 2 O) 5
1104.09
C
36
H
81
N
11
O
19
P
++
S161
O
OH
HO
N
N
NH 2
O
227.22
C
9
H
13
N
3
O
4
-
S162
O
OH
HO
N
N O
212.20
C
9
H
12
N
2
O
4
-
S168 N/A 1480.36 N/A -
159
Table 4.1. 5-Aza-2’-deoxycytidine analogs and quinoline derivatives from
SuperGen, continued.
Compound Structure
MW
(g/mol)
Formula
p16
induction
S170
O
OH
HO
N
N O
NH 2
OH
243.22
C
9
H
11
N
3
O
5
-
S171
O
OH
HO
N
N O
OH
228.20
C
9
H
10
N
2
O
5
-
S172
O
OH
HO
N O
211.21
C
10
H
13
N
O
4
-
S173 N/A 227.22 N/A -
S1015
N
HN
H
N
O
N
H
N
+
Me
518.45
C
28
H
25
Cl
2
N
5
O
-
S1017
N
HN
H
N
O
N
H
N
+
Me
H 2 N
533.46
C
28
H
28
Cl
2
N
6
O
-
S1020
N
HN
N
H
Me 2 N
O
H
N
NN
Me
NH 2
541.06
C
29
H
29
Cl
N
8
O
-
S1021
N
HN
N
H
O 2 N
O
H
N
NN
Me
NH 2
542.99
C
27
H
23
Cl
N
8
O
3
-
160
Table 4.1. 5-Aza-2’-deoxycytidine analogs and quinoline derivatives from
SuperGen, continued.
Compound Structure
MW
(g/mol)
Formula
p16
induction
S1022
N
HN
N
H
Me 2 N
O
N
H
N
+
Me
561.52
C
30
H
30
Cl
2
N
6
O
-
S1027
N
HN
N
H
O
H
N
N N
CH 3
NH 2
497.98
C
27
H
24
Cl
N
7
O
-
S1028
N
HN
N
H
O
O 2 N
N
N NH 2
NH 2
Me
519.02
C
25
H
23
Cl
N
8
O
-
S1030
N
HN
H
N
Me 2 N
N
H
N
N
O
Me
NH 2
541.06
C
29
H
29
Cl
N
8
O
-
S1039
N
HN
H
N
H
N
NN
CH 3
NH 2
O
H 2 N
549.45
C
27
H
26
Cl
2
N
8
O
-
S1045
N
HN
H
N
N
H
N
N
O
NH 2
NH 2
535.43
C
26
H
24
Cl
2
N
8
O
-
161
Table 4.2. Short oligonucleotide DNA methylation inhibitors. List of short
oligonucleotides that inhibit DNA methylation and induce p16 expression in T24
cells after 6 days of continuous treatment. Dose indicates the lowest concentration at
which the induction of p16 expression is observed.
*
Aza, 5-aza-2’deoxycytidine;
**
HEG, hexaethylene glycol phosphate linker.
Compound Name Structure Dose p16 Induction
Decitabine
S110
S53
S54
S55
S56
S52R
S112
5-Aza-CdR
*
AzapG
GpAza
GpAzapG
AzapGpAzpG
pGpAzapAzapG
AzapsG
**
HEGpAzapG
1μM
1μM
1μM
1μM
1μM
1μM
100μM
100μM
+++
+++
+++
+++
+++
+++
+
++
162
Figure 4.1. Re-expression of p16 and inhibition of DNA methylation by S54, S55,
and S56 in T24 bladder cancer cells. T24 bladder cancer cells were treated with
1µM 5-aza-CdR or varying concentrations of S54, S55, and S56 continuously for 6
days. Induction of p16 gene expression was analyzed by real time RT-PCR (A), and
demethylation at D4Z4 repetitive element was measured by quantitative Ms-SNuPE
(B).
A.
0.000
0.005
0.010
0.015
0.020
0.025
Untreated 1μM 5-
Aza-CdR
0.1μM 1μM 10μM 0.1μM 1μM 10μM 0.1μM 1μM
S54 (GpAzapG) S55 (AzapGpAzapG) S56 (pGpAzapAzapG)
p16/GAPDH
B.
0
20
40
60
80
100
Untreated
1μM 5-Aza-
CdR
0.1μM
1μM
10μM
0.1μM
1μM
10μM
0.1μM
1μM
10μM
S54 (GpAzapG) S55 (AzapGpAzapG) S56 (pGpAzapAzapG)
% DNA methylation
163
cells treated with the short oligonucleotides containing 5-azapyrimidine moiety,
demonstrating that indeed, these compounds are bona fide inhibitors of DNA
methylation (Fig 4.1 and 4.2). From here on, we focused our detailed
characterization on S110 (Table 4.1) as an example of short oligonucleotide
demethylating agents.
Effects of S110 on DNA methylation and p16 gene expression in T24 and
HCT116 cells.
We extended on the initial screen performed in Table 1 to determine the dose
dependency of S110 using T24 bladder and HCT116 colon cancer cells. First, we
assessed global methylation status by determining the methylation level of long
interspersed nucleotide element-1 (LINE-1) sequences. Repetitive DNA elements,
such as LINE-1 retrotransposable elements, serve as a useful marker of genome-wide
methylation changes and have previously been shown to be demethylated upon
treatment with 5-aza-CdR (Yang et al., 2004). In both T24 and HCT116 cells, the
decrease in the level of methylation was dose-dependent and comparable for 5-aza-
CdR and S110 after 0.1μM and 1μM treatment (Fig 4.2a and b). At 10μM
concentrations, only a small decrease in methylation was noted, probably due to side
effects of high drug concentrations as we observed previously (Jones and Taylor,
1980). In fact, 10μM treatment may be too cytotoxic for effective demethylation to
take place as the plating efficiency of T24 cells indicates (Fig 4.8). It is well-
established that its
164
Figure 4.2. Inhibition of DNA methylation and re-expression of p16 and by S110
in cancer cells. T24 bladder and HCT116 colon cancer cells were treated with
varying concentrations of 5-aza-CdR or S110 continuously for 6 days. Levels of
methylation were measured by quantitative Ms-SNuPE: LINE-1 methylation (A) T24
cells and (B) HCT116 cells, p16 promoter (C) T24 cells and (D) HCT116 cells.
Induction of p16 gene expression was analyzed by real time RT-PCR ((A) T24 cells
and (B) HCT116 cells). Error bars represent the standard deviation of the mean from
three independent experiments.
A. B.
0
20
40
60
80
100
Untreated 0.1μM 1μM 10μM
% DNA methylation
0
20
40
60
80
100
Untreated 0.1μM 1μM 10μM
% DNA methylation
C. D.
0
20
40
60
80
100
Untreated 0.1μM 1μM 10μM
% DNA methylation
0
20
40
60
80
100
Untreated 0.1μM 1μM 10μM
% DNA methylation
E. F.
0.00
0.04
0.08
0.12
Untreated 0.1μM 1μM 10μM
p16/GAPDH
0
0.04
0.08
0.12
Untreated 0.1μM 1μM 10μM
p16/GAPDH
165
cytotoxic dose is not ideal for optimal epigenetic therapy, since these drugs inhibit
DNA methylation best at low doses in cell lines as well as in the clinic (Flatau et al.,
1984; Issa et al., 2004).
We then analyzed the DNA methylation status of exon 1 of the p16 gene in
these cells after 6-day treatment by quantitative Ms-SNuPE. In untreated T24 cells,
the 3 CpG sites under analysis are located in the first exon of the gene and are almost
always fully methylated. The methylation level decreased with increasing
concentrations of 5-aza-CdR or S110 in T24 cells (Fig 4.2c) and HCT116 cells (Fig
4.2d) with the greatest demethylation seen at 1μM 5-aza-CdR.
Next, we measured the expression of p16 in both cancer cell lines. Untreated
T24 bladder carcinoma cells do not express p16 and dose-dependent increases in p16
expression were observed after 6 days of continuous treatment with 5-aza-CdR or
S110 (Fig 4.2e). After HCT116 colorectal carcinoma cells were treated for 6 days, a
dose dependent increase in p16 expression was observed with S110 while the highest
p16 expression was seen at 1μM dose of 5-aza-CdR (Fig 4.2f). In addition, T24 and
HCT116 cells treated with either agent for 3 days also showed a dose-dependent
increase in the level of p16 protein (Fig 4.3), demonstrating the competence of S110
to inhibit DNA methylation and induce p16 at both mRNA and protein levels as well
as 5-aza-CdR. Thus, S110 is able to inhibit DNA methylation at 5’ region and induce
the expression of p16 gene in T24 and HCT116 cells at concentrations comparable to
5-aza-CdR, and the induction of p16 expression by both agents correlated with the
demethylation at the 5’ end region of the gene in both cell lines.
166
Figure 4.3. Effects of S110 treatment on DNMT1 and p16 protein levels in
cancer cells. T24 (A) bladder cancer and HCT116 (B) colon cancer cells were
treated with 5-aza-CdR or S110 for three days. Whole cell lysates were collected and
separated on a polyacrylamide gel and blotted against specific antibodies for human
DNMT1, p16, and PCNA.
A.
B.
5-Aza-CdR S110
DNMT1
p16
PCNA
5-Aza-CdR S110
DNMT1
p16
PCNA
0 0.1 1 10 0.1 1 10 μM
0 0.1 1 10 0.1 1 10 μM
167
Depletion of DNMT1 levels by S110.
To provide more clues to the mechanism of action of S110, DNMT1 protein
levels in T24 and HCT116 cells treated with the compound were analyzed by
Western blot analysis (Fig 4.3a and b). A number of studies has provided evidence
that incorporation of the azapyrimidine ring into DNA is necessary for the drug to
inhibit DNA methylation (Santi et al., 1983; Taylor and Jones, 1979; Taylor and
Jones, 1982). Work by Hurd et al (Hurd et al., 1999) showed a covalent bond
formation between a DNA methyltransferase and zebularine-incorporated
oligodeoxynucleotide, strengthening the notion that DNA incorporation is an
essential step toward inhibition of methylation by mechanism-based inhibitors. It is
probable then that S110 works via a similar mechanism and causes a depletion of
extractable DNMT1 in cells. Indeed, partial and complete depletion of DNMT1
protein was observed in T24 and HCT116 cells treated with 5-aza-CdR for 3 days
(Fig 4.3a and b). Similarly, in S110-treated T24 and HCT116 cells, partial depletion
of the enzyme was seen at the 1μM dose and a trace of DNMT1 remained after
10μM treatment. Our results suggest that S110 causes depletion of extractable
DNMT1 in cells and may work by a similar mechanism as that of 5-aza-CdR in
inhibition of DNA methylation.
168
Figure 4.4. Inhibition of DNMT1 in vitro by S110. A fragment of double-stranded
DNA containing a hemimethylated CpG site (1ng) was incubated with M.Sss1 or
recombinant human DNMT1 in the presence of S110 for 30 min at 37°C. Heat
inactivation of the enzyme was followed by bisulfite conversion and DNA
methylation analysis by quantitative Ms-SNuPE.
0
20
40
60
80
100
-Enz PBS 100 500 -Enz PBS 1 10 100 500
M.Sss1 DNMT1
% CpG methylation
S110 (μM)
169
Inhibition of DNMT1 in vitro by S110 and fractionation of DNMT1 in nuclear
extracts using sucrose density gradient ultracentrifugation.
To validate the notion that S110 works via DNA incorporation after
phosphodiesterase cleavage, we next determined whether the dinucleotide could
inhibit DNA methyltransferases in a cell-free hemimethylation assay. We tested the
ability of S110 to inhibit a bacterial methylase, M.Sss1, or human DNMT1 activity
to methylate a double-stranded DNA containing a hemimethylated CpG site. Figure
4.4 shows that both M.Sss1 and DNMT1 were able to methylate DNA even in the
presence of high concentrations of S110. This result provided important clues to the
mechanism of S110 by showing that the dinucleotide cannot inhibit DNMT1 directly.
Although Figure 4.4 showed that the dinucleotide cannot directly inhibit
DNA methylation, we still lacked direct evidence that S110 metabolite had to be
incorporated into DNA after cleavage. We therefore extracted nuclei from 5-aza-
CdR or S110-treated HCT116 cells, collected soluble nucleosomes and size
fractionated the nucleosomes by sucrose density gradient centrifugation to determine
whether the drugs would trap DNMT1 on chromatin (Fig 4.5a and b). Under the salt
concentrations used (300mM NaCl), DNMT1 was not associated with nucleosomes
and the majority of DNMT1 protein in untreated HCT116 cells was not bound to
chromatin (Fig 4.5b). After 5-aza-CdR or S110 treatment, the distribution of
DNMT1 changed dramatically and although the majority of the enzyme was present
in the low molecular portion of the gradient (fractions 4 and 5), it was also found in
fractions 6-16 along with high molecular weight DNA (Fig 4.5b). We interpret this
170
Figure 4.5. Fractionation of DNMT1 in nuclear extracts using sucrose density
gradient ultracentrifugation. HCT116 cells were plated and treated with 1μM 5-
aza-CdR or 1μM S110 for 24 hr. Cells were harvested and lysed for nuclei extraction.
All nuclei were subject to partial micrococal nuclease digestion, which yielded DNA
fragments of various sizes. The supernatant containing DNA was separated by
sucrose gradient centrifugation overnight at 4ºC, and 16 fractions were collected, 1
being the topmost fraction and 16, the bottom fraction. Absorbance of each fraction
was measured at = 260 nm prior to TCA precipitation (A). Protein was precipitated
from each fraction with trichloroacetic acid (TCA) and analyzed by Western blot
analysis using specific antibodies to human DNMT1 and histone H3 (B). Filled
diamonds represent untreated cells; filled squares, cells treated with 5-aza-CdR; and
filled triangles, S110-treated cells. Histone H3 is a loading control for the presence
of chromatin.
H3
Un
Az
S11
DNMT1
Unt
Aza
S110
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
A.
B.
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Abs
260
Untreated
5-Aza-CdR
S110
171
result to show that the incorporation of 5-aza-CdR or the hydrolyzed product of S110
caused DNMT1 enzyme to bind very strongly to the DNA in chromatin. Our
experiment strongly suggests that S110 works through a similar mechanism as 5-aza-
CdR after its cleavage by phosphodiesterases and that both 5-aza-CdR and S110 by-
products are incorporated into DNA where they covalently interact with DNMTs. It
is most probable that the S110 dinucleotide is cleaved into nucleotides and
nucleosides since incorporation of a dinucleotide or oligonucleotide is not known to
occur during DNA synthesis.
Reverse dinucleotide and phosphorothioate analogues of S110.
To gain further insight as to how the S110 dinucleotide inhibits DNA
methylation, we utilized S110 analogues, namely, S53, which is a reverse
dinucleotide of S110, and S52R and S52S, which are two optical isomers of
phosphorothioate analogs of S110 (Table 4.1). S53 would allow us to determine
whether the sequence of the dinucleotide plays a role in inhibiting DNA methylation.
In addition, the phosphorothioate analogs would help us determine whether a free 5-
aza-CdR is released from the dinucleotide or if S110 directly inhibits DNA
methylation. The phosphate backbones of phosphorothioate analogs, S52R and S52S,
are not readily cleaved, hence retaining the dinucleotide structure for longer period.
T24 cells were treated continuously for 6 days with these compounds at varying
concentrations. A dose-dependent induction of p16 and demethylation of p16 exon 1
were seen in T24 cells treated with S53 while the expression and the DNA
172
Figure 4.6. Re-expression of p16 and inhibition of DNA methylation by S53 but
not by S52 in T24 cancer cells. (A) T24 cells were treated with 5-aza-CdR, S53, a
reverse dinucleotide of S110, S52S and S52R, the phosphorothioate analogs of S110
at varying concentrations continuously for 6 days and harvested for RNA.
Quantitative real time RT-PCR analysis of p16 normalized against GAPDH is shown.
(B) T24 cells treated with 5-aza-CdR, S53, S52S, and S52R were subject to DNA
methylation analysis at 5’ region of p16 gene by Ms-SNuPE. The level of DNA
methylation shown in percent is an average of three CpG sites in exon 1 of p16 gene.
A.
0.00
0.02
0.04
0.06
Untreated
0.1μM
1μM
10μM
0.1μM
1μM
10μM
1μM
10μM
1μM
10μM
5-Aza-CdR S53 S52S S52R
p16/GAPDH
B.
0
20
40
60
80
100
Untreated
0.1μM
1μM
10μM
0.1μM
1μM
10μM
1μM
10μM
1μM
10μM
5-Aza-CdR S53 S52S S52R
% DNA Methylation
173
Figure 4.7. Comparison of stability of 5-aza-2’-deoxycytidine and S110. The
initial absorbance was measured immediately after the compounds were dissolved in
PBS. Both compounds were incubated at 37ºC and the absorbance of each compound
at its respective wavelength of maximum absorbance was measured over time. The
decrease in absorbance was expressed as percent initial absorbance.
0
20
40
60
80
100
0 30 60 90 120 150 180 210
Time (Hr)
% Initial Absorbance
5-Aza-CdR
S110
174
methylation status at the 5’ end region of p16 remained unaffected in cells treated
with S52R and S52S (Fig 4.6a and b). Overall, this suggests that the order of bases in
the dinucleotide does not play a crucial role in inhibiting DNA methylation, but the
cleavage of the dinucleotide is critical in rendering the compound active, suggesting
once again that the dinucleotide does not work as a whole and has to be cleaved into
individual nucleotides and nucleosides.
Stability of 5-aza-2’-deoxycytidne and S110 at 37ºC in neutral aqueous solution.
A drawback of 5-aza-CdR is its short half-life in aqueous solution due to
rapid hydrolysis of the azapyrimidine. To compare the stability of S110 to that of 5-
aza-CdR, we measured the absorbance of 5-aza-CdR and S110 at their respective
wavelengths of maximum absorbance (
max
) over time at 37ºC in phosphate-buffered
saline solution at pH 7.4. The absorbance of 5-aza-CdR showed a sharp decay in the
first 30 hr of incubation with a half-life of about 20 hr (Fig 4.7). The half-life of
S110 was 21 hr with 65% of its initial absorbance remaining for as long as 200 hr
(Fig 4.7). This residual absorbance at 245nm is attributed to the guanidine ring in the
dinucleotide. Therefore, whether the 5-azacytosine ring was in a single nucleotide or
a dinucleotide, the stability of the compound was not affected and the half-lives of
these two compounds in aqueous solution at 37ºC remained the same.
175
Figure 4.8. Comparison of cytotoxicity of 5-aza-2’-deoxycytidine and S110 in
T24 bladder cancer cells. Cytotoxicity was compared by measuring the average
plating efficiency. T24 cells were plated at a low density and treated with 5-aza-CdR
or S110 24 hr later. Number of colonies from a control or untreated dish was
expressed as 100% and the treated dishes as percent untreated. Data represent
triplicate dishes and error bars represent one standard deviation of the mean.
5-Aza-CdR S110
0 0.1 0.2 1 10 0.1 0.2 1 10 μM
0
20
40
60
80
100
120
Average plating efficiency (%)
176
Figure 4.9. Comparison of rate of enzymatic degradation of 5-aza-2’-
deoxycytidine and S110 by cytidine deaminase (CDA). Enzymatic degradation of
5-aza-CdR and S110 by recombinant human CDA was compared. Recombinant
CDA (0.1 unit) was incubated with 5-aza-CdR or S110 (0.2mM) at 38°C and percent
substrate remaining was determined by HPLC. Filled diamonds represent S110 with
CDA; filled squares represent S110 alone; filled circles represent 5-aza-CdR with
CDA; and filled triangles represent 5-aza-CdR alone.
0
20
40
60
80
100
0 20 40 60 80 100
Time (min)
% Substrate Remaining
S110-CDA
S110+CDA
5-Aza-CdR-CDA
5-Aza-CdR+CDA
177
Cytotoxicity of 5-aza-CdR and S110 in T24 cells.
We then compared the cytotoxicities of 5-aza-CdR and S110 in T24 bladder
carcinoma cells by measuring effects of the compounds on the plating efficiency of
T24 cells. 5-Aza-CdR decreased the plating efficiency in a dose-dependent manner
with no colonies forming at 10μM concentration (Fig 4.8). S110 was slightly less
toxic than 5-aza-CdR at the doses tested. Similar results were obtained for HCT116
cells (data not shown). Thus, the cytotoxicity of S110 as measured by plating
efficiency is quite similar to 5-aza-CdR in T24 cells.
Enzymatic degradation of 5-aza-CdR and S110 by human cytidine deaminase
(CDA).
Decitabine or 5-aza-CdR is quickly catabolized into 5-azadeoxyuridine by
CDA in vivo (Chabot et al., 1983). We incubated either 5-aza-CdR or S110 with
human recombinant CDA at 38ºC and measured the amount of substrate remaining
over time by HPLC (Fig 4.9). A slow decrease in the total amount of 5-aza-CdR in
the absence of CDA (filled triangles) over time is indicative of hydrolysis of the 5-
azapyrimidine ring in water. A rapid decrease in 5-aza-CdR was observed in the first
45 min of incubation with human CDA and only about 2% of the total substrate
remained after 90 min (Fig 4.9, filled circles). In contrast, S110 was subject to
hydrolytic cleavage as found earlier (Fig 4.7) but was resistant to enzymatic
degradation by CDA; the majority of S110 remained in the solution after 90 min
incubation with CDA (Fig 4.9, filled diamonds). Incubation of S110 in human
178
plasma showed similar results and the dinucleotide persisted with little enzymatic
degradation (data not shown). Although the dinucleotide structure cannot prevent the
hydrolysis of the 5-azacytosine ring, it may greatly lengthen the half-life of the drug
by protecting it from deamination, increasing the efficacy of the drug in patients as a
result.
179
DISCUSSION
Short oligonucleotides were synthesized to test whether the stability of 5-aza-
CdR, a cancer chemotherapeutic agent, could be improved without compromising its
potency to inhibit DNA methylation. Comparison of the S110 dinucleotide
containing 5-aza-CdR to 5-aza-CdR showed that S110 works via a similar
mechanism of action to the parent compound to inhibit DNA methylation, induce
expression of the p16 tumor suppressor gene, and inhibit tumor cell growth.
Characterization of S110 demonstrated that the stability in aqueous solution and
cytotoxicity is still comparable to that of 5-aza-CdR; however, a major improvement
was accomplished in terms of resistance to enzymatic deamination. Deamination of
5-aza-CdR by CDA rapidly depletes the plasma level of the drug, resulting in low
bioavailability which has been frequently pointed out as one of the major drawbacks
of 5-aza-CdR (Chabot et al., 1983). S110 dinucleotide is resistant to CDA
deamination, probably due to the enzyme’s substrate specificity, which may
potentially increase the half-life of the drug, enhance bioavailability, and make the
drug more efficacious. Lowering the dose requirement may also reduce the toxicity
in patients as well as diminishing common side effects of 5-aza-CdR, such as
myelosuppression.
Tri- and tetranucleotides containing one or more 5-aza-CdR residues in the
chain showed demethylating activity as well, demonstrating that the cleavage of
these oligonucleotides is extremely efficient. Additionally, the hydrolytic cleavage of
180
the 5-azacytosine ring may presumably be improved in the longer chain
oligonucleotides, further improving stability. In practice, this may be very
advantageous in terms of broadening the applications of epigenetic therapy to
patients with solid tumors.
Oligonucleotides have been widely studied as chemotherapeutics, and in
most cases, the base sequence plays a pivotal role in giving them therapeutic effects.
For example, oligonucleotides with CpG motifs activate toll-like receptors and elicit
immune responses (Krieg, 2006). Antisense morpholino oligonucleotides and
siRNAs all utilize sequence specificities of endogenous molecules to inhibit either
transcription or translation of target genes (Iversen, 2001; Tafech et al., 2006).
Unlike the existing approaches, the short oligonucleotides we have developed do not
require any sequence specificities and simply provide protection against enzymatic
degradation before the DNA incorporation of the active moiety following
endonuclease and/or phosphodiesterase cleavage. Our findings suggest that once
these prodrugs are delivered to the target site and taken up intracellularly, they are
cleaved and metabolized to induce effects of similar levels to the parent molecule.
This concept eliminates the need for chemical modifications such as lipid
esterification and complex delivery vehicles that are commonly required for
prodrugs.
Although it is most likely that these oligonucleotides are cleaved and
incorporated into DNA in order to inhibit DNA methylation, it is still not clear as to
whether the cleavage occurs extracellularly or intracellularly. It is not likely that the
181
oligonucleotides are processed into 5-aza-CdR or its nucleotide by
deoxyribonucleases in plasma before entering the cell because there is a little or
weak exonuclease activity in plasma and endonucleases are known to fragment DNA
into larger pieces, thereby protecting the short oligonucleotides from degradation
(Barry et al., 1999; Bernardi and Griffe, 1964; Tidd and Warenius, 1989).
Furthermore, CpG oligodeoxynucleotides must retain the CpG motifs in order to
generate immune response, which provides evidence that short oligonucleotides are
not subject to nuclease degradation extracellularly (Krieg et al., 1995). It is probable
then that the short oligonucleotide DNA methylation inhibitors retain their structure
when they enter the cell and are only cleaved in the cells.
Oligonucleotides are thought to internalize themselves into cells through both
passive and active mechanisms (Krieg, 2006). Much effort has been devoted to
expedite such uptake with liposomal encapsulation and peptide mediated delivery of
these oligonucleotides (Gait, 2003; Gursel et al., 2001). Several membrane-bound
proteins and receptors were found to interact with oligonucleotides and facilitate
their cellular uptake (Benimetskaya et al., 1997; Geselowitz and Neckers, 1992;
Geselowitz and Neckers, 1995; Yakubov et al., 1989). There are also speculations
about cell surface channel proteins that conduct nucleic acids and oligonucleotides
(Hanss et al., 1998). Other transmembrane proteins are thought to cleave
phosphodiester and phosphosulfate bonds at the cell surface (Yano et al., 2003).
There are therefore many possible mechanisms by which short oligonucleotides may
enter the cell.
182
Once the oligonucleotides are internalized, they are probably cleaved into
individual nucleotides and nucleosides. The most likely candidates for the cleavage
of oligonucleotides are the 11 distinct subfamilies of phosphodiesterases which are
abundant in all cell types (Menniti et al., 2006). Phosphodiesterases catalyze the
hydrolysis of 3’ ester bonds of cyclic AMP and cyclic GMP into their respective
monophosphate moeities, leaving a phosphate group at the 5’ end (Beavo and
Brunton, 2002; Williams, 2004). It is probable then that these enzymes, when they
process the oligonucleotides, also leave the phosphate group on the 5’ end of the
nucleoside in the sequence, hence yielding both nucleotides and nucleosides.
However, it is not clear from preliminary data whether the phosphodiesterases are
the main player involved in processing these inhibitors, and further work is necessary
to assess their role in oligonucleotide metabolism.
Regardless of the mechanism, our data demonstrate that these short
oligonucleotides are quite efficient in entering the cell and inhibiting DNA
methylation. Di-, tri-, or tetranucleotides containing one or more 5-aza-CdR residues
are as effective as 5-aza-CdR in inducing p16 expression. Considerable improvement
was made regarding the half-life of the drug by preventing the deamination reaction
by CDA. We have therefore introduced a novel drug delivery system in which the
oligonucleotides are not restricted to their sequences for therapeutic potential and
provide protection against enzymes involved in the drug metabolism. This may find
numerous applications in delivering 5’ phosphates to cells. In addition, other
nucleoside drugs that are subject to enzymatic degradation may benefit by utilizing
183
this delivery method. The true merits of these short oligonucleotide DNA
methylation inhibitors may only be realized in a clinical setting.
184
CHAPTER 5
CANCER CHEMOPREVENTION BY ZEBULARINE
INTRODUCTION
Colorectal cancer is the second most prevalent cancer in the United States
(Krishnan et al., 2000). It is widely accepted that the development of colon cancer is
a multi-step process, each of which occurs as a result of a specific genetic event
(Fearon and Vogelstein, 1990). Recent advances in epigenetics have led us to believe
that aberrant DNA methylation and histone modifications play an important role in
colorectal tumorigenesis. Epigenetic changes have been noted in normal tissues,
preinvasive lesions or high-risk tissues, potentially serving as targets of
chemoprevention (Crawford et al., 2004; Holm et al., 2005; Holst et al., 2003;
Kopelovich et al., 2003). Therefore, epigenetic intervention using pharmacologic
inhibitors to completely abrogate or delay the process of colorectal carcinogenesis
may be feasible. In fact, the use of a wide range of compounds such as NSAIDS,
estrogen and demethylating agents in murine intestinal cancer models demonstrated
that cancer chemoprevention is possible (Calle et al., 1995; Laird et al., 1995;
Oshima et al., 1996). DNA methylation inhibitors are of particular interest as a
chemoprevention agent. Several studies demonstrated that the modulation of
epigenetics and/or DNA methylation prevents tumorigenesis (Belinsky et al., 2003;
Eads et al., 2002; Laird et al., 1995).
185
Zebularine is a novel inhibitor of DNA methylation which has been shown to
have anti-cancer property in vivo and in vitro (Cheng et al., 2004a; Cheng et al.,
2004b). Unlike 5-azacytidine (5-aza-CR) and 5-aza-2’-deoxycytidine (5-aza-CdR),
which are chemically labile and quite toxic, zebularine is stable and non-toxic,
making it possible to formulate a safe, oral chemotherapeutic agent. Here, we
explored the chemopreventive property of zebularine in a murine colon cancer model.
First, we tested to see if zebularine can be administered chronically, and then we
examined if zebularine can prevent or delay the process of tumor progression.
Apc
min/+
(Min) mice were treated with zebularine orally for a prolonged period. Min
mice have a nonsense mutation of the Apc gene which leads to the development of
multiple adenomas in the intestines along with other phenotypes such as anemia
(Moser et al., 1990; Su et al., 1992). Examination of the mice showed that there was
no adverse effect on the growth rate and structural integrity of tissues of these mice
and the level of DNA methylation in all organs was unaffected with the exception of
small and large intestines. Demethylation and prevention of polyps were seen in
females only. Taken together, these results suggest that zebularine may potentially be
used as a surrogate of 5-aza-CR and 5-aza-CdR in epigenetic therapy.
186
MATERIALS AND METHODS
Aminal care and drug treatment. C57BL/6 female and C57/BL/6 Apc
Min/+
male
mice were purchased from the Jackson Laboratories (Bar Harbor, Maine) and were
maintained in the facilities at Zilkha Neurogenetic Institute. The wild type C57BL/6
female mice were crossed with C57/BL/6 Apc
Min/+
male mice. Mice were allowed to
drink water containing 3% sucrose and zebularine (0.2mg/ml) ad libitum starting at
day 7 from birth until they were 120 ± 3 days old at which point the mice were
sacrificed. The weights of mice were monitored by weighing each mouse weekly on
a Mettler balance.
Preparation of colonic epithelial cells and polyp analysis. The entire
gastrointestinal tract was removed from just below the stomach through rectum
immediately after the sacrifice and washed with PBS. The intestinal tract was cut
longitudinally and rinsed with PBS. Hank’s balanced salt solution (HBSS) (Ca
++
,
Mg
++
-free, 30mM EDTA, pH 8.0) was used to wash 1cm cubes lacking tumor from
small and large intestines. The mucosa was incubated in HBSS with EDTA for 20
min at 37ºC and placed on a shaker at maximum speed at room temperature for 15
min. The solution was centrifuged at 1000 rpm for 2 min, supernatant was removed
and the epithelial cell pellet was stored at -80ºC for extraction of nucleic acids at a
later time point. The remaining intestine was fixed with 70% ethanol and was
187
subjected to polyp analysis under dissecting microscope. All adenomas present along
the entire length of the intestine were counted.
Nucleic acid isolation. DNA was prepared form frozen tissue samples and intestinal
epithelial cells using methods described previously (Laird et al., 1991). Total RNA
was collected from intestinal epithelial cells using the mirVana miRNA Isolation Kit
(Ambion, Austin, TX) according to the manufacturer’s protocol.
Microarray analysis. The microarray analysis was performed at the Microarray
Core Facility at Children’s Hospital Los Angeles (Los Angeles, CA). Purified RNA
from two mice per sample were pooled together and submitted for cRNA preparation
and array hybridization. An Affymetrix GeneChip Mouse Gene 430 2.0 Array was
used for hybridization on GeneChip Scanner 3000 (Affymetrix Inc., Santa Clara,
CA). Data analysis was done by Dennis Mock at the Microarray Core Facility using
S-score and dChip algorithms (Kennedy et al., 2006; Li and Wong, 2001).
Quantitation of DNA methylation by pyrosequencing. This assay was done by Dr.
Hyangmin Byun from Dr. Allen Yang’s laboratory (USC, LA, CA). Bisulfite-
converted DNA was used for pyrosequencing analysis as previously described (Yang
et al., 2004). Briefly, PCR product of each gene was used for individual sequencing
reaction. Streptavidin–Sepharose beads (Amersham Biosciences, Uppsala, Sweden)
and Vacuum Prep Tool (Biotage AB, Uppsala, Sweden) was used to purify the
188
single-stranded biotinylated PCR product according to the manufacturer’s
recommendation. The appropriate sequencing primer was annealed to the purified
PCR product and used for a Pyrosequencing reaction using the PSQ 96HS system
(Biotage AB, Uppsala, Sweden). Raw data were analyzed with the allele quantitation
algorithm using the provided software.
Hematoxylin and eosin staining. H&E stained specimen were prepared at the
Pathology Core Facility at Norris Comprehensive Cancer Center according to the
accepted procedures and analyzed with the help of Dr. Louis Dubeau.
189
RESULTS
Gender-specific abrogation of intestinal polyp formation in Apc
min/+
(min) mice
by chronic zebularine treatment.
To determine whether chronic administration of zebularine would have a
substantial effect on the growth and development of normal tissues and prevent the
formation of intestinal polyps, we treated Apc
min/+
(min) mice with zebularine
(0.2mg/ml) in the drinking water starting at the seventh day after birth until the mice
were 120 days old. Min mice have a mutation in the Adenomatous polyposis coli
(Apc) gene which results in the formation of intestinal polyps (Su et al., 1992). These
polyps are concentrated in the small intestine and are rarely sighted in the colon.
After zebularine treatment, the number of intestinal polyps in female mice decreased
from 58 to 1 while the polyp count remained unaffected in the males (Fig 5.1). All of
the males had developed polyps, while only 3 out of 11 females had developed
polyps detectable with a dissecting microscope (Fig 5.1). Examination of the
intestinal polyps in the small intestine of male and female mice showed that the
polyps found in the untreated male and female and the treated male were
macroscopic in size and fully developed histopathologically. On the other hand,
those found in the treated females were microscopic and immaturely developed (Fig
5.2). Therefore, zebularine had effectively delayed the formation of polyps in a
gender-specific manner.
190
Figure 5.1. Gender-specific effects of chronic zebularine administration on the
formation of intestinal polyps in min mice. Mice were given 0.2 mg/ml zebularine
dissolved in 3% sucrose water daily for 120 ± 3 days. Mice were sacrificed at day
120. The gastrointestinal tract was removed, cut longitudinally, and washed with
PBS. Polyps were counted under dissecting microscope.
0
10
20
30
40
50
60
70
80
90
12 3 4 56 7 89 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Number of Polyps
Male
Female
Untreated Treated Treated
191
Fig 5.2. H&E staining of intestinal polyps after chronic zebularine treatment of
min mice. Untreated male (A), untreated female (B), treated male (C, E), and treated
females (D, F).
A. B.
C. D.
E. F.
192
Gender- and tissue-specific demethylation by chronic zebularine treatment of
min mice.
In order to assess the effect of zebularine treatment in normal tissues, we
measured the changes in DNA methylation after treatment in various of these mice.
Methylation analysis of brain, heart, lungs, liver, kidneys, epidermal skin cells,
spleen, stomach, small and large intestines was conducted by pyrosequencing at the
B1 repetitive element. No change in methylation in the male mice was detected in all
tissues examined after zebularine treatment (Fig 5.3A). However, a dramatic
decrease in the methylation of B1 was observed in the large intestine and a small
decrease in methylation was seen in the small intestine (Fig 5.3B). Methylation
analysis of DNA from individual female mouse showed that demethylation was seen
only in mice that did not develop intestinal polyps and the methylation level in
intestines of females that developed few polyps was comparable to that of the
untreated mice. These results imply that DNA methylation plays a causal role in the
formation of polyps in these mice. In addition, demethylation was found in colon but
not in other organs, suggesting that zebularine is much more specific than was
presumed. Furthermore, female mice were consistently found to respond favorably to
zebularine treatment while the males remained insensitive to the drug.
193
Figure 5.3. Gender- and tissue-specific demethylation of methylation by
zebularine in min mice. Mice were given 0.2 mg/ml zebularine dissolved in 3%
sucrose water daily for 120 ± 3 days. Mice were sacrificed after treatments were
completed. DNA was purified from various tissues and subjected to methylation
analysis by pyrosequencing at the B1 repetitive element. The methylation of treated
males (A) was not affected by prolonged zebularine treatment while the DNA
methylation in gastrointestinal tracts of treated females (B) was affected. White bars
indicated methylation level of untreated mice and black bars indicate zebularine-
treated group.
A.
0
10
20
30
40
Brain Heart Lung Liver Kidney Epidermal
Cells
Spleen Stomach Small
Intestine
Large
Intestine
% DNA Methylation
Untreated
Zebularine
B.
0
10
20
30
40
Brain Heart Lung Liver Kidney Epidermal
Cells
Spleen Stomach Small
Intestine
Large
Intestine
% DNA Methylation
Untreated
Zebularine
194
Effects of chronic zebularine administration on min mice.
The majority of the mice survived the entire zebularine treatment period and
displayed normal behavior and remained healthy. However, 3 out of 21 (14%)
became moribund between week 5 to 7 of the treatment. No gender difference to
zebularine toxicity was observed. Two male and one female mice were found to be
unhealthy. During the course of the treatment, the weights of these mice were
measured weekly and the zebularine-treated mice did not show retardation in growth
compared to the control mice (Fig 5.4). At the time of euthanasia, splenomegaly was
observed in both male and female untreated mice and treated males. However, a
significant decrease in the weight and size of spleen was noted in the treated female
group (p=0.0004). This indicates that these mice were under much physical stress
due to the genetic mutation, despite their healthy outward appearance and behavior,
and that splenomegaly was relieved in the females upon zebularine treatment. It has
been documented that min mice suffer from anemia as a result of intestinal bleeding
(Moser et al., 1990). A significant increase in the red blood cell (RBC) count was
observed in the females after zebularine treatment (p=0.017), suggesting that the
females responded to zebularine more readily and lost phenotypes characteristic of
the min mice. In summary, the growth of the majority of zebularine-treated mice was
not stunted and females responded to zebularine more favorably, resulting in
regression of splenomegaly and increased RBC count.
195
Figure 5.4. Effects of chronic administration of zebularine on the weights of
male and female min mice. Mice were given 0.2 mg/ml zebularine dissolved in 3%
sucrose water daily for 120 ± 3 days. The mice were sacrificed immediately after
treatments ended.
0
5
10
15
20
25
30
35
36 9 12 15
Age (weeks)
Weight (g)
Untreated Male n=10
Treated Male n=7
Untreated Female n=12
Treated Female n=11
p 0.3246
p 0.2244
196
Microarray analysis of colonic epithelial cells in female min mice after chronic
zebularine treatment.
To assess the effect of chronic zebularine treatment on the global gene
expression profile, we performed a microarray analysis using normal colonic
epithelial cells from female mice. Since most of the DNA demethylation was
observed in the colon, we expected to see the most dramatic change in gene
expression changes in this tissue. Microarray results were analyzed using the
following algorithms to ensure that any bias concerning analysis method was
eliminated: dChip, which is a model-based analysis that estimates gene expression
indexes (Li and Wong, 2001), and S-score, which calculates the relative change in
probe pair intensities directly (Kennedy et al., 2006) were employed. Using the
dChip method, we found that 1,713 (3.8%) probe sets out of a total of 45,101 probe
sets were upregulated 2-fold or more and 1,066 (2.4%) probe sets were
downregulated 2-fold or more. According to the S-score algorithm, the number of
probe sets that yielded a significant signal was 1,392 (p=0.01). To summarize, dChip
and S-score revealed that the expression profile of 6% and 3% of the genome were
affected by zebularine treatment, respectively. This is quite surprising that less than
10% of the genes showed differential expression in the colonic epithelial cells, while
methylation level dropped by nearly 50% in the same tissue. We further analyzed our
data using the S-score to select the significant probe sets (p=10
-5
) and analyzed it
using the L2L, a database that allows the comparison of published mammalian
microarray studies to our data and provides insights into the biological processes at
197
Table 5.1. Genes upregulated in colonic epithelial cells after chronic zebularine treatment of min mice. Mice
were given 0.2 mg/ml zebularine dissolved in 3% sucrose water daily for 120 ± 3 days. Mice were sacrificed on day
120. RNA from colonic epithelial cells was subjected to microarray analysis and analyzed using S-score algorithm
(p<10-5). An L2L array was used to obtain biological processes that were significantly affected (Newman and Weiner,
2005).
Biological Process
Gene
Symbol Probe ID Description
peptide cross-linking Tgm2 1455900_X_AT transglutaminase 2
1433428_X_AT
1437277_X_AT
digestion Ppy 1420440_AT pancreatic polypeptide
Akr1b10 1448894_AT
aldo-keto reductase family 1, member B10
(aldose reductase)
Fabp2 1418438_AT fatty acid binding protein 2, intestinal
acute-phase response Reg3a 1448290_AT regenerating islet-derived 3 alpha
1416297_S_AT
Reg3g 1448872_AT regenerating islet-derived 3 gamma
regulation of cell adhesion Tgm2 1455900_X_AT transglutaminase 2
1433428_X_AT
1437277_X_AT
arginine metabolism Ddah1 1429298_AT dimethylarginine dimethylaminohydrolase 1
Otc 1420525_A_AT ornithine carbamoyltransferase
urea cycle intermediate metabolism Ddah1 1429298_AT dimethylarginine dimethylaminohydrolase 1
Otc 1420525_A_AT ornithine carbamoyltransferase
aldehyde metabolism Aldh1a1 1416468_AT aldehyde dehydrogenase 1 family, member A1
Akr1b10 1448894_AT
aldo-keto reductase family 1, member B10
(aldose reductase)
198
Table 5.2. Genes downregulated in colonic epithelial cells after chronic zebularine treatment of min mice. Mice
were given 0.2 mg/ml zebularine dissolved in 3% sucrose water daily for 120 ± 3 days. Mice were sacrificed on day
120. RNA from colonic epithelial cells was subjected to microarray analysis and analyzed using S-score algorithm
(p<10-5). An L2L array was used to obtain biological processes that were significantly affected (Newman and Weiner,
2005).
Biological Process
Gene
Symbol Probe ID Description
peptide cross-linking Tgm3 1440150_AT transglutaminase 3
digestion Slc15a1 1419343_AT
solute carrier family 15 (oligopeptide
transporter), member 1
Ctse 1418989_AT cathepsin E
regulation of cell adhesion Lama3 1427512_A_AT laminin, alpha 3
iron ion homeostatsis Slc40a1 1417061_AT
solute carrier family 40 (iron-regulated
transporter), member 1
1447227_AT
199
hand (Newman and Weiner, 2005). From this analysis, we found 10 upregulated and
5 downregulated genes that were involved in various cellular and biological
processes (Table 5.1 and 5.2). However, the number of genes that were identified in
this analysis was too small to draw any meaningful conclusion. Taken together, the
gene expression profile of colonic epithelial cells of min mice was minimally
affected. This suggests that zebularine, when chronically administered, does not
disturb the normal gene expression patterns and may be less detrimental to the
normal tissues than anticipated despite the degree of demethylation observed in the
same tissue.
Histopathology of min mice after chronic zebularine treatment.
To demonstrate that zebularine does not disrupt the normal development and
structure of tissues, we examined the hematoxylin and eosin (H&E) stained liver and
intestines. The architecture of liver tissue was intact, and no histological changes
have been noted in the liver of males and females after chronic zebularine treatment
(Fig 5.4). Similarly, no gross abnormalities were observed in the small intestines of
these animals (Fig 5.5). In addition, the absence of acute or chronic inflammation
and the lack of disruption of the integrity of the normal architecture provide evidence
that zebularine caused minimal toxicity in these organs during chronic administration
of the drug. A few localized fibroses were found in the large intestine in 2 out of 11
treated females, but since these fibroses are not a general feature of the entire
intestine it is unlikely that they are zebularine-related. This suggests that zebularine
200
Figure 5.5. H&E staining of liver after chronic zebularine treatment of min mice.
Untreated male (A), treated male (B), untreated female (C), and treated female (D).
A. B.
C. D.
201
Fig 5.6. H&E staining of small intestine after chronic zebularine treatment of
min mice. Untreated male (A), treated male (B), untreated female (C), and treated
female (D).
A. B.
C. D.
202
had delayed the progression of carcinogenesis and reduced the size and number of
the intestinal polyps while causing very little damage to the formation and
maintenance of these organs.
203
DISCUSSION
The potential use of zebularine as a chemopreventive agent has been explored
in this chapter. When administered chronically beginning shortly after birth,
zebularine inhibited the formation of adenomas in the small intestine of min mice in
a gender-specific manner. Other phenotypes characteristic of Apc
min/+
such as anemia
and splenomegaly was absent in the female mice after treatment. Demethylation was
observed in large and small intestines of female mice, but no methylation change
was noted in other tissues demonstrating the tissue specificity of the drug. The
majority of mice undergoing zebularine treatment remained healthy and did not show
growth inhibition compared to the untreated group. Microarray analysis showed that
the global gene expression profile was minimally affected. Additionally, tissue
analysis showed absence of acute or chronic inflammation and normal histological
architecture, indicating that zebularine did not have adverse effects in the min mice.
Normal histology was observed in both male and female mice, regardless of
zebularine’s ability to prevent polyp formation, and attested to the low or lack of
toxicity caused by the compound when administered in vivo.
The most intriguing aspect of our study was the gender specific zebularine
response of the min mice. In plasma, zebularine is rapidly converted into uridine,
uracil, and dihydrouracil by a liver enzyme called aldehyde oxidase (AO) (Beumer et
al., 2006a; Beumer et al., 2006b; Newman and Weiner, 2005). AO is an enzyme
responsible for the metabolism of many antiviral and anticancer agents, including
204
methotrexate (Garattini et al., 2003; Kitamura et al., 1999). Interestingly, AO activity
is influenced by the levels of testosterone and growth hormones, leading to gender,
species, and strain differences in the enzyme activity (Al-Salmy, 2002; Kurosaki et
al., 1999; Yoshihara and Tatsumi, 1997a; Yoshihara and Tatsumi, 1997b). Recent
articles has shown that zebularine metabolism is also influenced by the differential
AO activity, and the enzyme activity of male mice ranked the highest among the
animals tested while the female mice had virtually no AO activity (Klecker et al.,
2006). This may explain the gender-specific effect of zebularine seen in the min
mice. To show that the differential activity of AO is responsible for the gender
specificity, an inhibitor of AO may be administered with zebularine in male mice to
extend the efficacy of the drug. AO has numerous inhibitors, many of which are
commonly used clinical drugs such as raloxifene (Obach, 2004; Obach et al., 2004).
The combined use of raloxifene and zebularine may make it possible to use
zebularine as a chemopreventive agent in individuals with high levels of aldehyde
oxidase activity. Fortunately, the AO activity in humans did not show high gender
differences, implying that the degree of zebularine response in human patients may
be comparable in both gender groups and validates the need for further studies using
human subjects (Klecker et al., 2006).
Another fascinating component of our results was the tissue-specific
demethylation of the female mice. It is possible that demethylation occurred in the
most actively dividing cells and/or tissues, which in this case was the colon and the
small intestine. The life spans of colonic and small intestine epithelial cells are 4-8
205
days and 3-4 days (Croft and Levitan, 1970; Gavrieli et al., 1992). This is
considerably shorter than the turn over of skin epidermal cells (45 days)
(Bergstresser and Taylor, 1977). These three types of tissues were analyzed for
methylation and a considerable amount of demethylation was detected in large and
small intestines whereas no decrease in methylation was seen in skin epidermal cells
of the treated mice. However, when we looked at the methylation levels of colonic
mucosa and the colonic mesodermal layer, there was no difference in the methylation
levels of these two histologically distinct populations of cells and the entire gut had
been affected which was contrary to our original hypothesis. Another possible
explanation for this is that zebularine was active only in those tissues with which it
had contact initially. Since the drug was administered orally, it may have
demethylated the DNA in the small and large intestines as it came in contact with
these organs. Surprisingly, we did observe a marginal decrease in DNA methylation
in stomach in the treated females, which suggests that the “first-contact” may indeed
be the reason for the tissue specificity. However, a similar experiment using a rat
breast cancer model showed zebularine efficacy, suggesting that there may be more
mechanisms involved than the first-contact hypothesis (personal communication, Dr.
Victor E. Marquez). The mechanism of tissue-specific demethylation by zebularine
is not yet elucidated. It would be of interest to determine whether zebularine activity
is dependent on the rate of cell division, however, the tissue specificity of zebularine
make it an attractive property for a potential chemopreventive agent.
206
The third facet of our study dealt with the issue of toxicity resulting from the
chronic administration of a pharmacological agent and the feasibility of prolonged
zebularine use in patients and high-risk individuals. Our study demonstrated the in
vivo effects of the DNA demethylating agent in normal tissues for the first time.
Zebularine caused an appreciable level of demethylation while causing little toxicity.
The majority of the mice did not show weight loss with the exception of 3 mice that
had become moribund during zebularine treatment; hence we concluded that the rest
of the mice did not suffer from toxicity.
However, weight loss alone cannot completely establish the well-being of
these animals. Therefore, we examined the gene expression changes incurred as a
result of demethylation. The greatest decrease in methylation was observed in
colonic epithelia, however, the expression of 95% of genes in that tissue was not
disrupted. This is quite remarkable, given that 50% of genes are associated with CpG
islands in their promoter (Takai and Jones, 2002) and that gene expression is easily
disturbed as a result of drug treatment. Perhaps other mechanisms such as
nucleosome modification and transcription factors that are essential for gene
upregulation were absent in the colonic epithelial cells, leading to a relatively
marginal change in gene expression profile. This is a significant finding which may
relieve the suspicion that the use of demethylating agents is oncogenic (Eden et al.,
2003; Gaudet et al., 2003). We were not content with the microarray results, since a
small change in a single gene expression may lead to tumorigenesis. It is then
perhaps safe to say that the expression level of the vast majority of the genes
207
remained unchanged after chronic zebularine treatment. However, it is presumptuous
to state that it caused minimal toxicity.
Our next concern was to demonstrate that the chronic administration of a
pharmacologic inhibitor would not generate inflammation or tissue damage. Since
zebularine is mainly metabolized by AO in liver and causes the greatest change in
methylation in the intestines, we examined these two organs. Chronic use of any
chemical compound may be of concern since it may cause extensive damage in liver
and kidneys. Our results showed that after almost four months of zebularine
treatment, no histological damages were found in the liver and the intestines of the
animals. Taken together, zebularine is an extremely safe and sound therapeutic with
high tissue specificity for hypomethylating activity. It has advantages over
chemically labile 5-azacytidine and 5-aza-2’-deoxycytidine and may serve as a
surrogate agent with chemical stability and low toxicity. Further studies, especially
using animal tumor models, would warrant its potential development as either a
chemotherapeutic or chemopreventive drug.
208
CHAPTER 6
SUMMARY AND CONCLUSIONS
Epigenetics is an undeniably important aspect of development and
tumorigenesis. While methylation of the cytosine base in DNA serves to maintain the
epigenetics of a particular cell, aberrant methylation patterns may play a pivotal role
in the formation and progression of cancer. Unlike genetic alterations which cannot
be reversed, the plasticity of DNA methylation makes it an attractive therapeutic
target. Both hypomethylation and hypermethylation of DNA is observed in cancer,
however, the reactivation of tumor suppressor genes with chemotherapeutic
intervention remains the main aim of epigenetic therapy. This thesis is divided
largely into three major sections, all of which describe the use of DNA methylation
inhibitors for chemotherapy and chemoprevention of cancer. The first chapter dealt
with the characterization of zebularine as a DNA methylation inhibitor and a
potential chemotherapeutic agent. In the second part of the thesis, we sought to
identify novel inhibitors of DNA methylation. The two best known inhibitors of
methylation, 5-aza-CR and 5-aza-CdR suffer from instability in aqueous solution and
short half-life in vivo. In addition, the novel demethylating agent, zebularine, has its
shortcomings in terms of potency. Therefore, it was logical for us to identify other
DNA methylation inhibitors that are more potent and more stable than the existing
ones. Lastly, aberrant DNA methylation is thought to be an early event in
tumorigenesis (Nephew and Huang, 2003). Therefore, we explored the
209
chemoprevention aspect of epigenetic therapy using zebularine in a mouse model of
cancer in the final chapter.
In Chapter 2, we performed an in-depth characterization of zebularine as a
novel inhibitor of DNA methylation. Although much was already known about the
anti-tumor, anti-proliferative, and demethylating properties of zebularine, better
understanding of the drug was needed. We showed that the serum content in growth
medium of cultured cells is inversely related to the rate of incorporation of
zebularine into DNA. Zebularine incorporation was completely obliterated with
competition by 0.1% cytidine, a naturally occurring ribonucleoside. Since zebularine
is activated upon incorporation into a newly synthesized DNA, folic acid played an
important role in driving the cell cycle forward and achieving the desired effect of
DNA methylation inhibition. The comparison of effects of zebularine in normal and
cancer cells demonstrated the highly selective nature of the drug. Zebularine was
incorporated into the DNA of cancer cells more readily than the DNA of normal
fibroblasts, partly due to the differential activity of uridine/cytidine kinase. As a
result, cancer cells exhibited greater depletion of DNMT enzymes, showed greater
demethylation at methylated loci, displayed greater growth inhibition, and
demonstrated induction of cell cycle regulators such as p16 and p21. Interestingly,
despite these preferential activities of zebularine in cancer cells, expression profile of
a select group of genes were shown to be disturbed according to microarray analysis,
demonstrating that zebularine does not affect the global gene expression profile
enormously and is very specific in its action. Among the genes that were upregulated
210
were cancer/testis antigens, whose expression on the cell surface may be useful as
targets of immunotherapy in a combinatorial approach. Further experiments
confirmed that indeed the expression of these antigens is controlled by DNA
methylation and suggested that their expression status may give an indication as to
the degree of resistance to zebularine treatment in cancer cells.
We attempted in Chapter 3 to identify a zebularine prodrug which can
overcome the metabolic hurdles that the drug faced. There was a need to find a
prodrug because, despite its stability and low toxicity, zebularine was considerably
less potent than 5-aza-CR and 5-aza-CdR in terms of inhibiting DNA methylation.
The identification of novel DNA methylation inhibitors is an active area of research;
while most investigators concentrate on small molecule inhibitors, we felt that the
mechanism-based nucleoside analog inhibitors are best suited since the mechanism
of action of these compounds have been elucidated. The first group of compounds
that we screened is nucleoside analogs with modifications in the pyrimidine ring.
These compounds did not induce the expression of the methylation-silenced p16
gene in several cancer cell lines. We then tested the deoxy-zebularine
monophosphate moieties with different protecting groups, which allow facile cellular
uptake of the monophosphate and are either spontaneously or enzymatically cleaved
once in the cells. The deoxy-zebularine phosphoramidites were active in Cf-Pac-1
pancreatic cells in the presence of thymidine, which led us to test a cyclic dimer
containing both deoxy-zebularine and thymidylate monophosphate. Although we
were not able to identify a zebularine pronucleotide which can be activated without
211
the aide of another supplement and is more potent than the parent compound, we
learned a great deal about the zebularine metabolic pathways as well as cellular
responses elicited by a demethylating agent.
Our next approach to improve the existing DNA methylation inhibitors was
to give stability to 5-aza-CdR while retaining its potency. In Chapter 4, we
synthesized short oligonucleotides that contain one or more 5-aza-CdR in its
sequence. Unlike 5-aza-CdR which is subject to hydrolysis by water and
deamination by cytidine deaminase (CDA), these oligonucleotides are hydrolyzed
but not degraded enzymatically, potentially prolonging the half-life of 5-aza-CdR.
We demonstrated using recombinant CDA and human plasma that indeed these short
oligonucleotides are longer lived than 5-aza-CdR at physiological conditions. Further
experiments showed that these oligonucleotides are comparable to 5-aza-CdR in
terms of stability in aqueous solution, toxicity, ability to inhibit DNA methylation
and induce gene expression. The short oligonucleotides provided an attractive
method of delivering nucleoside analogs in which the metabolic degradation of the
drug is of concern. This approach is not limited to delivering DNA methylation
inhibitors but may be particularly useful in clinical setting where many existing
therapies utilize nucleoside analogs.
Lastly, we demonstrated the chemopreventive property and reiterated the
selective and non-toxic nature of zebularine using a murine colon cancer model.
APC
min/+
mice were allowed to drink zebularine-containing water ad libitum for 113
days and examined for the presence of intestinal polyps. These mice displayed a
212
gender-specific response to zebularine; the number of polyps had dramatically
decreased in females only. Furthermore, analysis of DNA methylation at repetitive
loci showed that demethylation was seen in intestines of female mice. Majority of
mice (80%) undergoing chronic zebularine treatment did not suffer from toxicity and
histopathology of liver and intestines of these mice attested to the normal
development and growth during the course of the treatment. Additionally, the
assessment of global gene expression profile in the colonic epithelial cells from
females showed that less than 5% of the genes were affected due to zebularine
treatment. The tissue- and gender-specificity of zebularine as well as the absence of
severe toxicity in these mice stress the fact that zebularine is highly specific and
selective agent, contrary to previous findings regarding 5-aza-CR and 5-aza-CdR
which suggested that zebularine may also be a non-specific inhibitor.
This thesis described the role of DNA methylation inhibitors in epigenetic
therapy of cancer, which encompasses both treatment and prevention of the disease.
An in-depth characterization of a novel demethylating agent, zebularine was given.
We explored the possibility of improving the existing drugs for use in the clinic. The
deoxy-zebularine monophosphate moiety provided an alternative to zebularine with
greater potency than its parent molecule. Short oligonucleotides containing 5-aza-
CdR introduced a new concept of drug delivery system and greatly improved the
stability of the drug. Finally, chronic zebularine treatment in mice demonstrated that
the formation of polyps in these mice can be prevented, suggesting that the
progression of tumor may be delayed with the intervention of an epigenetic drug. In
213
summary, this thesis provided evidence that DNA methylation inhibitors can be used
effectively against cancer as well as offered means for developing an ideal cancer
therapeutic agent.
214
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Abstract (if available)
Abstract
Aberrant DNA methylation is a common feature of cancer, which has been targeted for pharmacologic intervention because of its reversible nature. Nucleoside analogs such as 5-azacytidine, 5-aza-2'-deoxycytidine, and zebularine are mechanism-based inhibitors of DNA methylation that are incorporated into the DNA during replication and deplete the DNA methyltransferase enzymes. 5-Azacytidine and 5-aza-2'-deoxycytidine are extremely potent inhibitors and may be ideal for use in chemotherapy of cancer. Delivery of these inhibitors has been facilitated with the use of short oligonucleotides that prevent enzymatic degradation. On the other hand, zebularine, a more stable and less toxic surrogate of the other two compounds may be best utilized as a chemopreventive agent. Chronic oral administration of zebularine in a murine colon cancer model highlighted its low toxicity and gender- and tissue-specific action against DNA methylation.
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Yoo, Christine Bora
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Core Title
Use of DNA methylation inhibitors for chemotherapy and chemoprevention of cancer
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Keck School of Medicine
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Doctor of Philosophy
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Biochemistry and Molecular Biology
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2007-08
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06/07/2007
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04/27/2007
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Jones, Peter A. (
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