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Cancer Epigenetics:
Modications, Screening,
and Therapy
Einav Nili Gal-Yam, Yoshimasa Saito,
Gerda Egger, and Peter A. Jones
Department of Urology, Biochemistry and Molecular Biology, USC/Norris
Comprehensive Cancer Center, Keck School of Medicine, University of Southern
California, Los Angeles, California 90089; email: jones p@ccnt.hsc.usc.edu

Annu. Rev. Med. 2008. 59:26780

Key Words

First published online as a Review in Advance on

October 15, 2007

DNA methylation, histone modication, CpG islands

The Annual Review of Medicine is online at

http://med.annualreviews.org
This articles doi:
10.1146/annurev.med.59.061606.095816
c 2008 by Annual Reviews.
Copyright 
All rights reserved
0066-4219/08/0218-0267$20.00

Abstract
Deregulation of gene expression is a hallmark of cancer. Although
genetic lesions have been the focus of cancer research for many years,
it has become increasingly recognized that aberrant epigenetic modications also play major roles in the tumorigenic process. These
modications are imposed on chromatin, do not change the nucleotide sequence of DNA, and are manifested by specic patterns
of gene expression that are heritable through many cell divisions.
We review these modications in normal and cancer cells and the
evolving approaches used to study them. Additionally, we outline
advances in their potential use for cancer diagnostics and targeted
epigenetic therapy.

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INTRODUCTION

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DNMT: DNA
methyltransferase
Nucleosome: the
core unit of
chromatin,
composed of 147
base pairs of DNA
wrapped around a
histone octamer. It
was initially viewed
as a structural
component of
chromatin, but now
its composition and
position are
considered
important in the
control of gene
expression
HDAC: histone
deacetylase

During normal development, somatic cells

that are descended from a single progenitor, and contain a similar genotype, differentiate to acquire diverse functions and
features by expressing and repressing different sets of genes. This process is brought
about by modications that affect how the genetic material is packaged and utilized without changing its nucleotide sequence. Importantly, these epigenetic modications are
maintained through cell division. The involvement of these modications in cancer
states has been increasingly recognized and
is the subject of this review.

THE INTERPLAY OF
EPIGENETIC MODIFICATIONS
Epigenetic modications can be generally divided into three interacting processes: DNA
methylation, histone modication, and chromatin remodeling.
DNA methylation is catalyzed by at least
three DNA methyltransferases (DNMTs) that
add methyl groups to the 5 portion of the cytosine ring to form 5 methyl-cytosine. During S-phase, DNMTs, found at the replication fork, copy the methylation pattern of
the parent strand onto the daughter strand,
making methylation patterns heritable over
many generations of cell divisions. In mammalian genomes, this modication occurs almost exclusively on cytosine residues that precede guaninei.e., CpG dinucleotides. The
term CpG applies to both methylated and
unmethylated dinucleotides; the p refers to
the phosphate moiety that connects deoxycytidine and deoxyguanosine. CpGs occur in the
genome at a lower frequency than would be
statistically predicted because methylated cytosines can spontaneously deaminate to form
thymine. This is not efciently recognized by
the DNA repair machinery, so C-T mutations accumulate during evolution. As a result,
99% of the genome is CpG depleted. The
other 1% is composed of discrete regions

268

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that have a high (G+C) and CpG content.

These regions are called CpG islands (1, 2).
CpG islands are mostly found at the 5 regulatory regions of genes, and 60% of human
gene promoters are embedded in CpG islands.
Although most of the CpG dinucleotides are
methylated, the persistence of CpG islands
suggests that they are not methylated in the
germ line and so did not undergo CpG depletion during evolution (3). Around 90% of
CpG islands are estimated to be unmethylated
in somatic tissues (4), and the expression of
genes that contain CpG islands is not generally regulated by their methylation. However,
under some circumstances CpG islands do
get methylated, resulting in long-term gene
silencing.
DNA methylation is essential for normal
development, as mice lacking any one of the
enzymes responsible for placing the mark
die in the embryonic stages or shortly after birth (5, 6). As a silencing mechanism,
it plays a role in the normal transcriptional
repression of repetitive and centromeric regions, X chromosome inactivation in females,
and genomic imprinting (7). The silencing mediated by DNA methylation occurs
in conjunction with histone modications
and nucleosome remodeling, which together
establish a repressive chromatin structure
(Figure 1A).
The functional link between DNA methylation and histone modications was initially established by studies showing that histone deacetylases (HDACs) are recruited
to methylated DNA by methyl-CpG binding proteins (8, 9). Histones, which are the
building blocks of nucleosomes, undergo numerous post-translational modications that
regulate chromatin structure, gene expression, and DNA repair (10). The most studied histone modications are methylation and
acetylation of lysine residues. Until recently,
histone methylation was considered a permanent mark placed on chromatin. However, several histone demethylating enzymes
have been discovered in recent years (10),
and both acetylation and methylation are now

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A
Normal

Exon

Exon

Repeat

nonmethylated Cytosine

Cancer

methylated Cytosine

x
Exon

Exon

Repeat

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B
nucleosome

Normal

repressive complex (e.g. PcG)

x
Cancer

methylated DNA binding protein

H3K4 methyl mark
H3K9/K27 methyl mark
acetylation mark

Figure 1
Epigenetic patterns in normal and cancer cells. (A) DNA methylation. In normal cells, nearly all of the
CpG dinucleotides are methylated whereas CpG islands, mostly residing in 5 regulatory regions of
genes, are unmethylated. In cancer cells, many CpG islands become hypermethylated, in conjunction
with silencing of their cognate genes, while global hypomethylation, mostly at repetitive elements,
occurs. (B) Chromatin and histone modication. Active genes are associated with acetylation of histone
tails, methylation of lysine 4 on histone H3 (H3K4), and nucleosome depletion at their promoters. The
promoters of silenced genes (drawn here in conjunction with DNA hypermethylation) become associated
with nucleosomes, lose acetylation and H3K4 methylation marks, and gain repressive methylation marks
such as lysine 9 or 27 on histone H3, which recruit repressive complexes. Methylated DNA binding
proteins link methylated DNA with the histone modication and nucleosome remodeling machineries
(not shown).

considered reversible modications catalyzed

by enzymes having opposing activities. In general, regions silenced by DNA methylation
also show hypoacetylation and hypermethylation of specic histone lysine residues, such as
lysine 9 or 27 in histone H3 (10). In contrast,
promoters of actively transcribed genes show
hyperacetylation of histones H3 and H4, and
methylation of lysine 4 of histone H3 (H3K4)
(11, 12).
DNA methylation and histone modications function in close interplay with nucleosome remodeling and positioning complexes
that bind specic histone modications, such
as trimethylated H3K4 (13, 14) and methyl
CpG binding proteins (15), and move nucle-

osomes on DNA by ATP-dependent mechanisms. Nonmethylated CpG island promoters

are usually hypersensitive to nucleases and are
relatively depleted of nucleosomes, whereas
methylated promoters have nucleosomes on
them and are nuclease resistant (16, 17, 17a)
(Figure 1B).

CANCER: A MODIFIED
EPIGENOME
When a general role for DNA methylation
in gene silencing was established more than
25 years ago (18), it was proposed that aberrant patterns of DNA methylation might play
a role in tumorigenesis (19). Initial studies
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MicroRNAs: small,
noncoding RNA
molecules,
approximately 22
nucleotides long that
bind to the mRNA
of target genes to
negatively control
their expression.
MicroRNAs have
essential roles in
normal development
and their expression
patterns are linked to
cancer development
Methylomes:
Distinct DNA
methylation proles
in tumors, tissues, or
different cell types
CpG island
methylator
phenotype (CIMP):
a trait exhibited by a
subset of tumors that
show an
exceptionally high
frequency of
methylation of
distinct CpG islands

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found evidence for a decrease in the total

5-methylcytosine content in tumor cells (20),
and the occurrence of global hypomethylation in cancer was rmly established in
subsequent studies. Hypomethylation occurs
mainly at DNA repetitive elements and
might contribute to the genomic instability
frequently seen in cancer (20). Hypomethylation might also contribute to overexpression
of oncogenic proteins and was shown to be
associated with loss of imprinting of IGF2
(insulin growth factor 2), leading to aberrant
activation of the normally silent maternally
inherited allele. This was found to be associated with an increased risk for colon cancer
(21). The mechanisms underlying global
hypomethylation patterns are currently
unknown.
Aberrant hypermethylation at normally
unmethylated CpG islands occurs parallel to
global hypomethylation (Figure 1A). The
CpG island promoter of the Rb (Retinoblastoma) gene, found to be hypermethylated in
retinoblastoma, was the rst tumor suppressor shown to harbor such a modication (22).
This discovery was soon followed by studies
showing promoter hypermethylation and silencing of other tumor suppressor genes such
as VHL (von HippelLindau) in renal cancer
(23), the cell cycle regulator CDKN2 A/p16 in
bladder cancer (24), the mismatch repair gene
hMLH1 in colon cancer (25), and many others. On the basis of these ndings, it was proposed that epigenetic silencing of tumor suppressor genes by DNA methylation can serve
as an alternative hit to mutation and/or
deletion in Knudsons two-hit carcinogenesis model (26). This led to the notion that
nding hypermethylated genes would result
in the discovery of new tumor suppressors.
An example is ID4, a proposed tumor suppressor, which was found to be hypermethylated in hematological malignancies but for
which no mutations were detected in tumors
(27). The development of large-scale unbiased
methods for detecting methylation, such as restriction landmark genomic scanning (RLGS)
and array-based techniques (see below), led
Gal-Yam et al.

to a urry of studies reporting numerous hypermethylated genes in cancer (see Reference

28 for a partial list). It is now established
that aberrant hypermethylation at CpG island
promoters is a hallmark of cancer. Notably,
not only protein-coding genes undergo these
modications; CpG island promoters of noncoding microRNAs were shown to be hypermethylated in tumors, possibly contributing
to their proposed roles in carcinogenesis (29,
30).
What is the origin for the deregulated
methylation patterns in cancer? Initially it
was suggested that like genetic mutations, de
novo hypermethylation events are stochastically generated, and that the nal patterns observed are a result of growth advantage and
selection (30a). However, several observations
made in recent years should be noted: First,
hypermethylation events are already apparent at precancerous stages, such as in benign
tumors and in tumor-predisposing inammatory lesions (31, 32). Second, there seem to be
dened sets of hypermethylated genes in certain tumors. These differential methylation
signatures, or methylomes, may even differentiate between tumors of the same type, as
was recently shown for the CpG island methylator phenotype (CIMP) in colon cancer (33).
Third, although many hypermethylated genes
have tumor-suppressing functions, not all are
involved in cell growth or tumorigenesis. Furthermore, some of these genes are not expressed in the corresponding normal tissue,
so their methylation does not result in their
de novo silencing in the cancer cells (34; E.
Nili Gal-Yam, G. Egger, A. Tanay, P. A. Jones,
unpublished data).
Thus, although the hypothesis of stochastic methylation and selection is probably
true for some cases, the observations detailed
above suggest that these patterns may be generated by upstream-acting programs that
have gone wrong. Evidence for such a program involving the Polycomb group complexes (PcGs) is emerging. PcGs are protein complexes responsible for maintenance
of long-term silencing of genes, which is

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mediated by methylation of lysine 27 of histone H3 at the repressed regions. The enzyme

that catalyzes this modication is EZH2,
which is known to be upregulated in tumors
and involved in tumor progression (35). In
embryonic stem cells, repression of a large set
of developmental genes mediated by PcGs is
thought to maintain these cells in a pluripotent state (36, 37). Several studies have recently shown that these genes are prone to be
hypermethylated in cancer, suggesting a functional link between the two repressing systems
and lending support to the idea of an epigenetic stem cell signature in cancer (3840).
Future studies that analyze global methylation patterns after manipulation of PcG components are needed to provide further insights
into the role of this system in aberrant DNA
methylation.
As discussed above, silenced hypermethylated promoters are generally associated with
hypoacetylation of lysine residues on histones
H3 and H4 and hypermethylation of lysine 9
or lysine 27 on histone H3, which mediate the
formation of a repressive chromatin structure
(Figure 1B). Global histone modications are
also altered in cancer: Leukemias, colon cancers, and cell lines derived from them exhibit

loss of acetylation at lysine 16 and trimethylation at lysine 20 of histone H4. These changes
seem to occur at hypomethylated repetitive
elements (41). The mechanisms responsible
for alterations of these global patterns are
mostly unknown but may involve the disruption of the enzymes responsible for these
modications (28).

DETECTION OF EPIGENETIC
MODIFICATIONS
DNA Methylation
Various approaches exist to study DNA
methylation at specic loci (Figure 2). The
oldest approach relies on the use of
methylation-sensitive restriction enzymes
(MSREs), which distinguish between methylated and nonmethylated sites. These were
initially used in conjunction with Southern blotting to analyze methylation status at candidate genes. This technique is
labor-intensive, requires large quantities of
high-quality DNA not readily obtained from
tumors, and depends on the existence of the
enzymes specic recognition sites. Nevertheless, MSRE-based techniques are also being

DNA methylation

Bisulfite Conversion

Restriction

Histone modifications

Immunoprecipitation

Bisulfite Treatment

PCR
T G T T A C G G

Sonication

IP

U G T U A C G G

Expression
untreated

C G T C A C G G

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Chromatin IP (ChIP)

5-aza-CdR

RNA

Crosslink, Sonication

Reverse Transcription
PCR

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IP

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methyl group

Figure 2
Approaches for detection of epigenetic marks. DNA methylation can be detected by three main
approaches: one based on bisulte conversion, which changes the nucleotide sequence depending on the
methylation state of cytosines; another based on methylation-sensitive restriction enzymes, which
differentially digest methylated and unmethylated DNA; and a third based on pulldown of methylated
DNA by 5 -methylcytosine binding proteins. Alternatively, specic activation of genes after treatment
with the demethylating agent 5 -aza-2 deoxycytidine identies potentially methylated genes that need to
be conrmed by direct analyses. Histone modications are usually detected by chromatin
immunoprecipitation. These approaches, initially used to detect modications at candidate regions, have
also been adopted for genome-wide studies (see text for details).
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Oligonucleotide
tiling arrays:
microarrays on
which overlapping
oligonucleotides,
usually 2550 base
pairs long, are
printed, covering
contiguous regions
of the genome. Used
to interrogate
enrichment of
genomic regions that
are bound by specic
factors or
modications

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adopted for large-scale analyses, as detailed

below.
Methods based on bisulte conversion
provide the most accurate methylation detection at the genomic-sequence level. Bisulte treatment of DNA results in deamination
of nonmethylated cytosines to uracils while
methylated cytosines are not altered (42). This
change in the nucleotide sequence, reecting
the initial methylation pattern, can be interrogated by various methods. Genomic bisulte sequencing, performed after PCR amplication and cloning of the region of interest,
is considered the gold standard for methylated cytosine detection; this method gives
the exact methylation status for each CpG
site. However, because of the large amount
of locus-specic amplication and sequencing involved, this is currently not the preferred method for high-throughput methylation analyses. Methylation-specic PCR
(MSP) or its quantitative derivatives, such as
Methyl-light (42a), amplify converted DNA
using primer sets that are specic either for
the methylated or unmethylated DNA (43).
These sensitive techniques have become the
most common methylation detection tools using a candidate gene approach, and they allow for the analysis of small quantities of
DNA derived from archived tissue. However,
as only totally methylated or totally unmethylated molecules are amplied in these techniques, the exact pattern of methylation is
not reected in the result. Additionally, owing to their high sensitivity, rigorous negative
(unmethylated) and positive (totally methylated) controls should be used. Other methods based on bisulte-converted DNA, such
as MS-SNuPE or pyrosequencing, have been
adapted from the eld of single nucleotide
polymorphism (SNP) detection; these enable
the accurate quantication of methylation
at discrete CpG sites within a given region
(44, 45).
With the realization that aberrant methylation patterns are common in cancer and
the advent of genomic technologies to detect them, the eld has moved from candiGal-Yam et al.

date gene approaches to methods that detect methylation on a large scale in an unbiased manner. In restriction landmark genomic
scanning (RLGS), the DNA from tumor
and healthy tissue is cleaved by methylationsensitive enzymes, radiolabeled, separated by
two-dimensional gel electrophoresis with further enzyme digestion, and autoradiographed.
Comparison between the normal and tumor
gels reveals spots with differential intensity,
representing differential methylation and/or
copy number at specic loci. Although only
1000 CpG islands can be interrogated in this
manner, this was one of the rst techniques
that compared global methylation proles in
a large number of tumor samples, and a nonrandom and type-specic pattern of promoter
hypermethylation was found in tumors (46).
Methods relying on microarray technologies have further advanced the study of
genomic methylation. An early example was
the differential methylation hybridization
method (DMH), in which DNA is cleaved by
MSREs, labeled, and hybridized to a CpG island array. A differential hybridization signal
between normal and tumor DNA reects differential methylation at a specic CpG island
(47). More recently developed techniques
rely on the ability of proteins or antibodies
to bind specically to methylated DNA (48,
49). The methylated DNA immunoprecipitaion (MeDIP) technique, for example,
utilizes antibodies that specically recognize
5-methylcytosine to immunoprecipitate
methylated DNA, resulting in its enrichment
in the sample. Coupling this method with
oligonucleotide tiling arrays covering the
majority of human promoters (50) or the
complete Arabidopsis thaliana genome (51)
resulted in the rst high-resolution methylomes to date and promises to be a powerful
tool for genome-wide methylation detection
in various applications.
An alternative approach to detect aberrantly methylated regions relies on the treatment of cells with demethylating compounds
such as 5-aza-2 deoxycytidine, which results in the demethylation and transcriptional

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upregulation of specic genes (52). The use of

these compounds in conjunction with expression microarrays enables large-scale screening
for differentially expressed genes in treated
compared to nontreated cells. An advantage
of this approach is that it detects functionally relevant changes in methylation, which
are assumed to affect the tumorigenic process, rather than simply the hypermethylation itself. However, as elevated expression of
a gene after drug treatment could be due to
indirect effects of the drug, the actual methylation status of the identied genes needs to
be conrmed by other methods such as those
described above. Another drawback is that the
actual experiments can only be performed on
cultured cell lines, which do not necessarily
reect the situation in the tumors themselves.

HISTONE MODIFICATIONS
The detection of histone modications largely
relies on the existence of high-quality antibodies that recognize specic modication
on various amino acid residues of histones.
Western blots and immunostaining can be
used to detect global levels or localization
patterns for these modications in the nucleus. The now commonly used chromatin
immunoprecipitation (ChIP) technique enables researchers to measure the enrichment
of specic histone modications at dened genomic regions. This technique can be scaled
to global studies, mainly by combining it with
microarray technology (ChIP-chip). ChIPchip can be used to study modications at
dened genomic entities such as promoters
or CpG islands, or in contiguous genomic
regions or even whole chromosomes using
recently developed oligonucleotide tiling arrays. A drawback to ChIP-chip is the inability
to study repetitive elements, as their inclusion
in the arrays will interfere with hybridizations
and skew the results. Additionally, a bias may
be introduced by the amplication performed
to obtain the large amounts of DNA needed
for hybridizations. ChIP-derived DNA can
also be sequenced, with the number of se-

quence reads aligning to a specic genomic

locus dened as enrichment at this locus (53).
Advantages of this approach are relative ease
of analysis, unbiased results, and the fact that
the nucleotide sequence of the pulled down
fragments is precisely known. Furthermore,
rapid developments in sequencing techniques
may eventually render ChIP sequencing
cheaper and more timely than conventional
ChIP-chip (54).

EPIGENETIC DIAGNOSTICS
Early detection and risk assessment remain
high priorities in oncology. Ideal tumor markers would have high sensitivity and specicity
and be present in sufcient amounts to reveal minimal disease in peripheral samples.
Detection of hypermethylated DNA is considered a promising diagnostic tool in cancer because aberrant methylation events are
abundant in tumors, occur early in the tumorigenic process, and different cancers exhibit
specic hypermethylation patterns. Because
they are DNA markers, they are more stable
than RNA or proteins. Furthermore, whereas
detection of other DNA aberrations such as
point mutations often requires examination
of different sites within a gene in various
patients, promoter hypermethylation usually
occurs over the same region of a given gene,
simplifying the design of a detection assay.
During the past decade, many studies have
detected tumor-derived free circulating hypermethylated DNA in plasma or serum of patients with cancer. Additionally, hypermethylated DNA was obtained from various body
uids of cancer patients, such as urine, stool,
saliva, bronchoalveolar lavage (BAL), sputum,
mammary aspiration uid, pancreatic juice,
peritoneal uid, and vaginal secretions (55).
Many of these samples can be obtained with
minimal invasiveness and thus are suitable
for large population screening. Most of these
studies were performed using the highly sensitive bisulte-based MSP methods and provide a basis for future clinical trials using DNA
methylation markers in cancer detection and
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Chromatin immunoprecipitation
(ChIP): A
commonly used
method to detect
binding of histones,
modied histones, or
other factors to
specic genomic
regions. Chromatin
is cross-linked and
sheared followed by
pull down with
specic antibodies to
the histones and
their bound DNA.
This is further
interrogated by PCR
amplication of
specic regions or
microarray analysis
(ChIP-chip)

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surveillance. However, various confounding

issues, such as the specicities of the markers for the different tumors, need to be claried. For example, many of the markers, such
as RASSF1 and CDKN2A/p16, appear to be
methylated in various tumors or preneoplastic conditions and are therefore not tumorspecic. Additionally, methods used for sample collection and methylation detection need
to be standardized to achieve sufcient reproducibility of the results. Ideally, one marker
could be used for the diagnosis of each tumor
type. In prostate cancer, hypermethylation of
GSTp1 may be promising in that respect (56).
In other cases, highly dened panels of genes
will probably be used for screening. One example of the latter is a prospective study in
which sputum was collected from individuals
who were at high risk for lung cancer but were
cancer-free upon entering the trial. Methylation status of six genes predicted the occurrence of lung cancer within two years of
trial initiation with a specicity and sensitivity of 65% (57). Although further optimization of this panel is needed to reach sufcient
sensitivity and specicity, this study provides
a proof of concept for the prospective use
of methylation markers in early detection of
cancer.
DNA methylation markers can also be
used for disease classication, and to predict
prognosis and response to therapy. For instance, methylation of RASSF1A in many tumors, including lung, breast, and prostate cancers, has been shown to be associated with
poor prognosis (58). In another example, neuroblastomas harboring the CIMP phenotype
were highly correlated with poor prognosis
(59). Metastatic potential can be predicted on
the basis of the E-cadherin promoter methylation in breast and oral cancers. In terms
of response to therapy, the most compelling
example to date is the hypermethylation of
the MGMT (O6 -methylguanine methyltransferase) promoter, which increases the sensitivity of glioblastomas to alkylating agents (60).
In addition to the study of single genes,
large-scale techniques are now generating

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DNMTi: DNA
methylation
inhibitor(s)

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tumor methylation proles, or methylomes,

which can be used for molecular classication. Furthermore, high-throughput platforms that can analyze the methylation state
of a large number of loci in a large number
of samples have been developed. One such
recently described technology adapts a highthroughput single nucleotide polymorphism
(SNP) genotyping system to detect methylation based on genotyping bisulte-converted
DNA (60a). By using this technology, 1500
CpG sites in 400 genes from 96 samples
can be analyzed simultaneously. Studies using
this technology identied panels of methylation markers that distinguished lung or bladder cancers from their normal counterparts
at high specicity (61; G. Liang, E. Wolf,
P. A. Jones, unpublished results). These panels are promising in terms of their implementation in DNA methylation analyses in large
populations.

EPIGENETIC THERAPY
Because of their dynamic nature and potential
reversibility, epigenetic modications are appealing therapeutic targets in cancer. Various
compounds that alter DNA methylation and
histone modication patterns are currently
being examined as single agents or in combination with other drugs in clinical settings.
Most DNA methylation inhibitors
(DNMTi) that have been clinically tested
belong to the nucleoside analog family. These
drugs are converted into deoxynucleotide
triphosphates intracellularly and are incorporated into replicating DNA in place of
cytosine. Their main mechanism of action
is probably through trapping of the methyl
transferases at sites of nucleoside incorporation (3), which depletes the cells of enzymatic
activity, resulting in heritable demethylated
DNA. Because incorporation occurs during
DNA synthesis, only replicating cells are
demethylated by DNMTi (62), which may
confer the preference for highly proliferating cancer cells. The hypomethylation
that ensues over the following cell division

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reactivates various silenced tumor suppressor

genes, which is proposed to undermine the
antineoplastic properties of the drugs.
The prototypes of DNMTi are 5-aza cytidine and 5-aza-2 deoxycytidine. Initially described as cytotoxic agents (63), they were
later found to cause DNA demethylation
and differentiation and to reactivate silenced
genes at much lower doses than those initially used (62). These low doses are now
used, mainly for hematological malignancies,
leading to better responses and lower toxicity. Both drugs were recently approved by the
U. S. Food and Drug Administration (FDA)
for the treatment of myelodysplastic syndrome, a preleukemic disease (64).
Zebularine is a new addition to the family
of nucleoside analogs that has demethylating
properties. The drug can be delivered orally,
is less toxic than the 5-aza analogs, acts preferentially on cancer cells, and inhibits polyp formation in female APC/MIN-decient mice
(65; C. Yoo, P. A. Jones, unpublished results).
However, the need for high concentrations
of zebularine and its limited bioavailability
in primates have slowed its advancement into
clinical trials (66).
As discussed above, epigenetic silencing
is tightly coupled with histone deacetylation.
Various compounds that inhibit HDACs have
demonstrated antitumor, growth inhibitory,
proapoptotic, and prodifferentiation properties (67). One of the universal targets of
HDAC inhibitors (HDACi) is the cell cycle
inhibitor p21, which is consistently upregulated by treatment with these drugs in conjunction with histone hyperacetylation at its
promoter (68). Several silenced proapoptotic
genes, which are members of the death receptor pathway, are also targets of HDACi
treatment in leukemic cells, resulting in their
promoter hyperacetylation and upregulation
(69). Notably, tumor cells are almost always more sensitive to HDACi activity than
healthy cells (70). It should be emphasized
that in addition to their effects on transcription, the antitumoral activity of HDACi
is probably mediated by other mechanisms,

such as disruption of higher-order chromatin

structure and DNA repair pathways (67). In
the clinic, many phase I trials show that these
drugs are well-tolerated, and one of the initial HDACi, suberoylanilide hydroxamic acid
(SAHA), has recently been approved by the
FDA for the treatment of T cell cutaneous
lymphoma. More are being developed and
tested in clinical trials for both hematological and solid tumors (71).
As histone methylation is also a major
player in establishing long-term silencing,
drugs targeting the enzymes involved in this
modication are being developed. For example, 3-Deazaneplanocin A (DZNep) was
recently shown to deplete Polycomb group
components, inhibit histone H3K27 methylation, and induce selective apoptotic cell death
in breast cancer cells (72). In another study,
the use of polyamine analogs inhibited the enzyme that removes the active H3K4 methylation mark, resulting in upregulation of aberrantly silenced genes in a cancer cell line (73).
The specicities of these drugs and their potential clinical effectiveness need to be carefully established in further studies.
As the interplay between epigenetic pathways is unraveled, the combination of epigenetic drugs with each other or with standard chemotherapies has become a focus of
interest. HDACi and DNMTi show synergistic effects on transcriptional activation (74),
and initial clinical trials using combinations
of both have been promising (75). Further
randomized trials are needed to prove their
synergy in patients. Both classes of epigenetic drugs might sensitize cells to the action of biological agents such as all-trans
retinoic acid, standard chemotherapeutics,
or potential immunotherapies. Clinical trials using these combinations are ongoing
(75).
Despite the promise of epigenetic therapy, several concerns remain, mainly stemming from the nonspecicity of the drugs. Induction of genomic hypomethylation in mice
caused chromosome instability and promoted
tumor formation (76, 77), and the question
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deacetylase
inhibitor(s)

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arises whether the use of hypomethylating

drugs will also have carcinogenic effects. One
study examining this has not found such effects, although the number of patients was
small and the time period short (75). Furthermore, in other mouse models, inhibition
of DNMTs prevented tumor development
(78). As clinical use of these drugs increases,
these concerns will be answered in the coming years. However, the search for more specic drugs targeting epigenetic modications
is warranted.

CONCLUDING REMARKS
With the recognition of the role of aberrant
epigenetic processes in cancer and the rapid
advent of new technologies to study them,
this is an exciting time for the cancer epigenetics eld. National and international collaborations are forming to launch a human
epigenome project (79). The ultimate aim of
this project would be to map all epigenetic
modications, resulting in a comprehensive

description of these in both normal and diseased cells. Additionally, a pilot project to the
Cancer Genome Atlas Project was recently
launched, which aims to systematically explore the entire spectrum of genomic changes
involved in human cancer, including epigenetic changes such as DNA methylation (80).
The data derived from these projects will be
able to answer questions such as how many
genes are actually affected by epigenetic aberrations in a given tumor. They will also shed
further light on the underlying mechanisms.
Although screening using epigenetic markers
is a promising prospect, specic and sensitive
screening panels are yet to be developed and
tested in large prospective clinical studies. It
is important to directly compare the efcacy
of these panels with classic screening procedures and other evolving screening techniques
based on proteomics, mRNA expression, or
microRNA arrays. Knowledge of the prevalence and mechanisms of epigenetic modications will allow the design of rational intervention strategies to target them.

DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.

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Annual Review of
Medicine

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Contents

Volume 59, 2008

The FDA Critical Path Initiative and Its Inuence on New Drug
Development
Janet Woodcock and Raymond Woosley p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p1
Reversing Advanced Heart Failure by Targeting Ca2+ Cycling
David M. Kaye, Masahiko Hoshijima, and Kenneth R. Chien p p p p p p p p p p p p p p p p p p p p p p p p 13
Tissue Factor and Factor VIIa as Therapeutic Targets in
Disorders of Hemostasis
Ulla Hedner and Mirella Ezban p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 29
Therapy of Marfan Syndrome
Daniel P. Judge and Harry C. Dietz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 43
Preeclampsia and Angiogenic Imbalance
Sharon Maynard, Franklin H. Epstein, and S. Ananth Karumanchi p p p p p p p p p p p p p p p p p 61
Management of Lipids in the Prevention of Cardiovascular Events
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Genetic Susceptibility to Type 2 Diabetes and Implications for
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Allan F. Moore and Jose C. Florez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 95
Array-Based DNA Diagnostics: Let the Revolution Begin
Arthur L. Beaudet and John W. Belmont p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p113
Inherited Mitochondrial Diseases of DNA Replication
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Childhood Obesity: Adrift in the Limbic Triangle
Michele L. Mietus-Snyder and Robert H. Lustig p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p147
Expanded Newborn Screening: Implications for Genomic Medicine
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Is Human Hibernation Possible?
Cheng Chi Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p177
Advance Directives
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Genetic Determinants of Aggressive Breast Cancer

Alejandra C. Ventura and Soa D. Merajver p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p199
A Role for JAK2 Mutations in Myeloproliferative Diseases
Kelly J. Morgan and D. Gary Gilliland p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p213
Appropriate Use of Cervical Cancer Vaccine
Gregory D. Zimet, Marcia L. Shew, and Jessica A. Kahn p p p p p p p p p p p p p p p p p p p p p p p p p p p p p223
A Decade of Rituximab: Improving Survival Outcomes in
Non-Hodgkins Lymphoma
Arturo Molina p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p237
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Nanotechnology and Cancer

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Cancer Epigenetics: Modications, Screening, and Therapy
Einav Nili Gal-Yam, Yoshimasa Saito, Gerda Egger, and Peter A. Jones p p p p p p p p p p p p267
T Cells and NKT Cells in the Pathogenesis of Asthma
Everett H. Meyer, Rosemarie H. DeKruyff, and Dale T. Umetsu p p p p p p p p p p p p p p p p p p p p281
Complement Regulatory Genes and Hemolytic Uremic Syndromes
David Kavanagh, Anna Richards, and John Atkinson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p293
Mesenchymal Stem Cells in Acute Kidney Injury
Benjamin D. Humphreys and Joseph V. Bonventre p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p311
Asthma Genetics: From Linear to Multifactorial Approaches
Stefano Guerra and Fernando D. Martinez p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p327
The Effect of Toll-Like Receptors and Toll-Like Receptor Genetics in
Human Disease
Stavros Garantziotis, John W. Hollingsworth, Aimee K. Zaas,
and David A. Schwartz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p343
Advances in Antifungal Therapy
Carole A. Sable, Kim M. Strohmaier, and Jeffrey A. Chodakewitz p p p p p p p p p p p p p p p p p p361
Herpes Simplex: Insights on Pathogenesis and Possible Vaccines
David M. Koelle and Lawrence Corey p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p381
Medical Management of Inuenza Infection
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Bacterial and Fungal Biolm Infections
A. Simon Lynch and Gregory T. Robertson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p415
EGFR Tyrosine Kinase Inhibitors in Lung Cancer: An Evolving Story
Lecia V. Sequist and Thomas J. Lynch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p429
Adaptive Treatment Strategies in Chronic Disease
Philip W. Lavori and Ree Dawson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p443
vi

Contents

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Antiretroviral DrugBased Microbicides to Prevent HIV-1 Sexual

Transmission
Per Johan Klasse, Robin Shattock, and John P. Moore p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p455
The Challenge of Hepatitis C in the HIV-Infected Person
David L. Thomas p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p473
Hide-and-Seek: The Challenge of Viral Persistence in HIV-1 Infection
Luc Geeraert, Gnter Kraus, and Roger J. Pomerantz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p487

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Advancements in the Treatment of Epilepsy

B.A. Leeman and A.J. Cole p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p503
Indexes
Cumulative Index of Contributing Authors, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p525
Cumulative Index of Chapter Titles, Volumes 5559 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p529
Errata
An online log of corrections to Annual Review of Medicine articles may be found at
http://med.annualreviews.org/errata.shtml

Contents

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