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Abnormalities of chromatin in tumor cells
Bojan Drobic, Katherine L. Dunn, Paula S. Espino and James R. Davie
Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, R3E 0V9 Canada
Summary. Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin
organization are used by pathologists as diagnostic markers for cancer [1]. Tumorigenesis involves a
series of poorly understood morphological changes that lead to the development of hyperplasia, dys-
plasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma.
Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of
chromatin, which appears to correlate with malignancy [2]. Multistep tumorigenesis is a process that
results from alterations in the function of DNA. These alterations result from stable genetic changes,
including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially
reversible epigenetic changes, which are modifications in gene function without a change in the DNA
sequence [3–5]. DNA methylation and histone modifications are two epigenetic mechanisms that are
altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic
alterations with a focus on histone modifications on chromatin structure and function in cancer cells
are reviewed here.
Key words: Chromatin, histones, MAPK, nucleus, ras gene, tumors.
Introduction
Histones are basic proteins that have a vital role in the organization of DNA in
the human cell nucleus. In addition to establishing a hierarchy of chromatin
structures, resulting in compaction of the nuclear DNA about 10,000-fold, the
histones have critical roles in differential packaging of decondensed euchro-
matin and condensed heterochromatin regions of the genome. Both euchro-
matin and heterochromatin are organized by the basic repeating structural unit
in chromatin, the nucleosome [6]. The nucleosome core particle consists of a
histone octamer core around which 146 base pairs of DNA are wrapped. The
core histones are arranged as a (H3–H4)2tetramer and two H2A-H2B dimers
positioned on both sides of the tetramer. The core histones have a similar struc-
ture with a basic N-terminal domain, a globular domain organized by the his-
tone fold, and a C-terminal tail (Fig. 1). The histone-fold domains of the four
core histones mediate histone-histone and histone-DNA interactions [6].
The nucleosomes are joined by linker DNA, which is of varying length. A
fifth class of histone, the H1 histones or linker histones, binds to the linker
DNA and to core histones. H1 has a tripartite structure consisting of a central
globular core and lysine-rich N- and C-terminal domains (see Fig. 2). The
globular domain binds to one linker DNA strand as it exits or enters the
nucleosome and to nucleosomal DNA near the dyad axis of symmetry of the
Cancer: Cell Structures, Carcinogens and Genomic Instability
Edited by Leon P. Bignold
© 2006 Birkhäuser Verlag/Switzerland
25
nucleosome [7]. H1 and the histone tails stabilize the higher order compaction
of chromatin.
The core histones undergo a wide range of post-synthetic modifications,
most of which are reversible. These modifications include acetylation, phos-
phorylation, methylation, ubiquitination, poly ADP ribosylation, and biotiny-
lation [8, 9] (Fig. 1). The majority of modified amino acids reside in the tail
domains, but there is an increasing awareness of modified residues occurring
in the histone-fold domains [10]. The amino-terminal tails of the four core his-
tones play an important role in chromatin compaction. These tails protrude
from the nucleosome, with that of H3 protruding the farthest. The tails of H3,
being the longest and positioned in such a way as to allow several contacts
26 B. Drobic et al.
SGRGKQGGKARAKAKTRSSRAGLQ
P
510
15 20
Ac Ac
AVLLPKKTESHHKAKGK
119
U
H2A
Ac Ac
mm
125 127
K99 M
K74
m
K75
m
R77
m
K85 Ac
AVSEGTKAVTKYTSSK
120
U
PEPAKSAPAPKKGSKKAVTKAQKKDGKK
510 15 20 25
Ac Ac Ac Ac
H2B K
108
Ac
116
Ac
R99 M
K43 M
IRGERA
Ac Ac
H3
K79 M
ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPH
P P
M M M
Ac Ac Ac
M
M M
M
510152025
30 35
P
K115
K122 Ac
Ac
SGRGKGGKGLGKGGAKRHRKVLRDNI
510152025
M
P
Ac Ac Ac Ac M
H4 K59 M
R92 M
K77
Ac
K79
Ac
K91
Ac
B B
Figure 1. Core histone modifications. The N-terminal and in some cases C-terminal amino acid
sequences of human histone are shown. The modifications include methylation (M), acetylation (Ac),
phosphorylation (P), ubiquitination (U), and biotinylation (B). Methylation sites that are uncertain are
denoted as (m).
with linker DNA, are crucial for the formation of higher order chromatin fibers
[11]. Acetylation and phosphorylation of the N-terminal tail of H3 promotes
Abnormalities of chromatin in tumor cells 27
Y-P
RTKs
Grb2 Sos
Ras Raf
GDP
GTP
Mos
MEK
EGF
RasGRP
DAG
TPA
ERK
NUCLEUS
cyclin E- Cdk2
Histone H1
Msk1
HDAC
Complex
PIC
SWI/SNF
Complex
CBP
ERK
Transcription
Factor
PKC
UV-B
MEKK3
MEKK6
p38/SAPK2
p38/SAPK2
KKKKK
Ac Ac
Ac Ac Ac
SS
N
H3
91014 18 23 2728
H1-3
NEET
ESETTSSK
2715 17 35 114 134 151 169 185 222
PPPPP
PP
Figure 2. MAPK signal transduction pathways and the modification of chromatin. The Ras-MAPK
pathway is activated by EGF and TPA. TPA acts through PKC and/or RasGRP. UV-B activates both
the Ras-MAPK and the p38 kinase pathways. Inserts: Left panel, The sites of phosphorylation of H1
subtype H1.3 are located on the N- and C-terminal tails. Right panel, The H3 phosphorylation sites
are nestled in a region of acetylation (P, phosphorylation, Ac acetylation). CBP, CREB binding pro-
tein (histone acetyltransferase); Cdk2, cyclin-dependent kinase 2; DAG, diacylglycerol; GDP, guano-
sine diphosphate; GTP, guanosine 5'-triphosphate; HDAC complex, histone deacetylase complex;
PIC, preinitiation complex; RasGRP, Ras guanyl nucleotide-releasing protein; RTKs, receptor tyro-
sine kinases; SOS, Son of Sevenless; TPA, 12-O tetradecanoylphorbol-13-acetate.
transcriptional activation of local genes by disrupting chromatin compaction.
An understanding of the significance of the histone modification type and
position in the nucleosome is starting to emerge. Transcribed regions of the
genome are associated with hyperacetylated H3 (including acetylation of K9
and K14), hyperacetylated H4 and H3 hypermethylated at K4 and K79, while
inactive genes have poorly acetylated histones and H3 that is hypomethylated
at K4 and K79 [12, 13]. Methylation of K9 of H3 is involved in maintaining
the condensed structure of heterochromatic regions. The targeting of histone-
modifying enzymes to specific regions of chromatin results in the distinct dis-
tribution of the modified histone isoforms.
A universal distinguishing feature of transcribed and potentially active
(referred to as competent) chromatin is that these regions of the genome have
an increased sensitivity to nuclease digestion (for example to DNase I and
micrococcal nuclease). The binary distribution of modified histones among the
transcribed and silent genes is thought to be one of the factors in conferring
nuclease sensitivity onto transcribed chromatin [12, 13].
Activation of an oncogene or deactivation of a tumor suppressor gene
results in the decondensation of chromatin [14–16]. Alterations in modifica-
tion of core and linker histones are thought to be responsible for the more
relaxed chromatin structure of these cells. In the following sections, we
explore the diverse mechanisms by which cancer cells divert the targeting or
alter the activity of histone-modifying enzymes to remodel chromatin structure
and adjust gene expression.
Histone acetyltransferases in malignancies
Acetylation of core histones destabilizes histone-DNA interactions as well as
histone-histone contacts between adjacent nucleosomes and interactions
occurring between histones and specific regulatory proteins. Histone acetyla-
tion is catalyzed by a group of enzymes termed histone acetyltransferases
(HATs). The highly organized and repressive nature of chromatin stresses the
integral role that HATs play in the dynamic changes necessary for gene expres-
sion that control cellular processes. For most HATs, acetylation of substrates
extend beyond histones and addition of acetyl groups to transcription factors,
cell cycle regulators and structural proteins, demonstrating the extensive influ-
ence of HATs to normal cellular function and maintenance.
Two of the most extensively studied HATs in transcriptional activation are
p300 and its closely related homolog cyclic AMP response element-binding
(CREB)-binding protein (CBP). p300 was discovered through its interaction
with the adenovirus E1A protein, while CBP was identified through its associ-
ation with the phosphorylated form of CREB. p300 and CBP act as global tran-
scriptional coactivators, being involved in cellular processes such as cell cycle
control, differentiation and apoptosis [17]. p300 and CBP acetylate all four core
histones. p300/CBP stimulate transcriptional activation of specific genes
28 B. Drobic et al.
through direct or indirect interaction with various promoter-binding transcrip-
tion factors including CREB, nuclear hormone receptors and oncoprotein acti-
vators such as c-Fos, c-Jun and c-Myb. The broad spectrum of p300/CBP inter-
acting proteins provides a general mechanism for integration of several signal-
ing and transcription-response pathways that p300 and CBP modulate [18].
Defects in the expression and function of HATs have been reported in can-
cer cells. The mis-targeting, ill-timed activation or irregular increase in activi-
ty of HATs can lead to expression of genes that allow tumorigenesis [19].
Chromosomal translocations, deletions and mutations that affect genes encod-
ing potent HATs have been associated to the genesis of several malignant con-
ditions, particularly hematological disorders. The following section discusses
malignancies that result from aberrant HATs and subsequent consequences to
chromatin structure and function.
HAT fusion proteins in leukemias
Hematopoietic disorders such as leukemia frequently manifest chromosomal
translocations spanning genes that encode HATs, and result in protein fusions
with other transcription factors that lead to defective acetyltransferase activi-
ties (Fig. 3) [20]. Fusion proteins that result from these translocations form
hybrid transcriptional regulators and chimeric HATs that become dominant
over their wild-type counterpart, permitting either a gain or loss of function,
and enabling differential expression of target genes. The resulting
myelomonocytic neoplasms or other malignancies are attributed to changes in
proteins involved in cell cycle control, differentiation and apoptosis. Altered
acetylation of histones and protein substrates contribute to defects in chro-
matin remodeling and allow oncogenesis [21].
The hematological malignancy acute myeloid leukemia (AML) subtype
M4/M5 displays frequent somatic translocations that disrupt the 5' end of CBP
gene. Commonly, it has been detected that the MYST family putative acetyl-
transferase monocytic zinc finger (MOZ) gene is juxtaposed beside the CBP
gene after (8;16)(p11;p13) translocation, resulting in MOZ-CBP fusion pro-
teins. The chimera retains most of the interaction domains of both parent pro-
teins as well as the MOZ acetyltransferase domain establishing a likely gain of
function that is suspected to play a role in leukemogenesis. Although this is so,
further studies to directly address this claim and its effects on chromatin organ-
ization are lacking [21, 22]. Fusion proteins involving MOZ and p300 have
also been observed as rare variants [23]. Furthermore, chromosomal translo-
cations also affect the MOZ-related gene MORF, which has been observed to
form MORF-CBP and/or CBP-MORF chimeras, and are expressed in juvenile
AML subtype M5a and therapy-related myelodysplastic syndrome (MDS) [24,
25]. In some AML patients that present similar clinical conditions as those
expressing MOZ-CBP fusions, inversions of chromosome 8 between p11 and
q13 allows fusion of the MOZ gene with the nuclear coactivator TIF2 gene,
Abnormalities of chromatin in tumor cells 29
generating a chimeric protein. This MOZ-TIF2 protein can bind CBP and
mimic the MOZ-CBP fusion function that has been demonstrated to be neces-
sary for transformation and leukemogenesis in vitro and in vivo [26].
In many cases, the development of leukemias can arise during recession
after primary treatment. These disorders are not only limited to AML, they are
also found in chronic myeloid leukemia (CLL) and myelodysplastic syndrome
30 B. Drobic et al.
N-CoR (SMRT) SAP30
mSin3
RbAP48
HDAC1, 2
Hematological malignancies
PML-RARalpha
PLZF-RARalpha
AML1/ETO
LAZ3/ BCL6
non-Hodgkin
Lymphomas
CBP
MLL-CBP MORF-CBP
CBP-MORF
MOZ-CBP
CBP-MOZ ; MOZ-TIF2
Somatic
translocation
mutated
CBP
Rubenstein-Taybi
syndrome
Figure 3. Somatic translocations and mis-targeting of HATs and HDACs. Somatic translocations and
mutations involving the CBP gene can interfere with the normal function and targeting of CBP, a
potent histone acetyltransferase and coactivator, resulting in a variety of hematological malignancies.
Mis-targeting of HDAC complexes, which deacetylate histones and transcription factors and are core-
pressors, by fusion proteins arising from somatic translocations, can interfere with genetic programs,
resulting in a variety of hematological malignancies.
(MDS). Rearrangements and consequently formation of fusion partners of
mixed lineage leukemia (MLL) gene with CBP, p300 and MOZ have been
associated with development of therapy-related disease due to defects in chro-
matin structure modulation and the inability of HAT fusion proteins to regu-
late differentiation and cell cycle control [21, 27–29]. The ability of the MLL
gene to generate fusion partners with many HATs suggests that multiple
avenues of cellular homeostasis can be affected, disturbed and deregulated.
Aberrant HATs associated with other defects
Many studies have demonstrated the presence of somatic mutations in solid
tumors and carcinomas generating truncated, missense or nonfunctional HAT
proteins [23]. Biallelic inactivating somatic point mutations of p300, and more
rarely of CBP, have been observed in neoplasms such as gastric, colon, ovari-
an, breast and pancreatic cancers, which, depending on the mutation site and
the domains that are affected, can yield a nonfunctional HAT or a HAT with
decreased activities [20, 21, 23]. The HAT, amplified in breast cancer 1
(AIB1), is found commonly overexpressed in breast and ovarian cancer due to
an amplification of chromosomal region containing the gene [27]. Moreover,
loss of heterozygosity in 80% glioblastomas of p300 locus in chromosome 22
and of CBP locus in hepatocellular carcinomas has also been detected [21].
In patients affected by the autosomal dominant disorder Rubinstein-Taybi
syndrome, the crebbp gene, which encodes for CBP, has a germline allele
mutation that renders the HAT nonfunctional. Since CBP is a potent and mul-
tipurpose acetyltransferase involved in many cellular processes, affected indi-
viduals display a wide range of severity and clinical presentations from facial
and limb abnormalities to physiological anomalies, and are consequently pre-
disposed to developing other malignancies [30]. Surprisingly, although the
close homolog p300 remains intact in patients affected by this syndrome, it
does not replace CBP function, which further displays that each HAT has spe-
cific and nonredundant roles [21].
Since HATs have key roles in many cellular processes particularly in chro-
matin remodeling and transcriptional activation, the consequences of these
mutations and formation of fusion proteins in the development of malignan-
cies are manifested far beyond the molecular level. These fusion proteins,
depending on their specific orientation and on the domains they retain from
their parent proteins, can dictate the pattern of pathogenicity and phenotypic
presentation of disease.
Chromatin consequences of aberrant HATs
HATs are critical players in normal cellular function and work in concert with
deacetylases. They have roles in bridging the basal transcriptional machinery
Abnormalities of chromatin in tumor cells 31
to promoter coactivators and factors as well as stimulating chromatin remod-
eling. Furthermore, they are integral in the positive or negative coordination of
cell signaling pathways, growth, apoptosis, differentiation and embryogenesis.
Acetylation and deacetylation of histones and proteins not only affect local
areas of chromatin but also bulk conformations, dictating activity and three-
dimensional interactions of transcription factors involved [31]. The functional
availability of HATs alongside histone deacetylases (HDACs) have an effect
on which subsets of genes, a particular developmental stage or a cellular
process, are expressed at a given time, permitting the active “on” and inactive
“off” states. Formation of HAT fusion proteins from translocations and non-
functional HATs from mutations generate aberrant acetyltransferases that
influence the balance of gene activation and repression, and alter transcrip-
tional regulation, leading to genesis of cancers. Different HATs may function
differently according to their chromosomal contexts. Depending on the
domains retained or left intact after translocations and mutations (i.e., bro-
modomains, acetyltransferase domains), their DNA binding capabilities, fac-
tor binding domains and modifying potential are compromised allowing shifts
in the equilibrium of gene expression [32]. In the leukemic disorders men-
tioned above, the HAT fusion proteins involved potentially recruit anomalous
transcription factors that elevate HDAC complexes at transcription sites and
prompt the block in hematopoietic differentiation characteristic of the malig-
nancies [33]. In certain scenarios, some investigations point to the potential
role of HATs as tumor suppressors, and this claim has been verified in mice
models but continues to be studied in humans [23].
HATs as therapeutic targets
Nowdays, there are many compounds, both synthetic and naturally occurring,
that have been developed to inhibit HDAC activity. Human clinical trials of
these drugs have superceded that of studies investigating HAT inhibitors. Cole
and colleagues [34, 35] were first to demonstrate that selective inhibitors of
HATs can be synthesized. They designed peptide conjugates of acetyl-CoA
with compounds such as lysyl-CoA, specific for p300, and H3-CoA-20 that
targets PCAF [35]. However, further studies on the pharmacokinetic properties
of these synthetic analogues need to be analyzed to fully elucidate their appli-
cation in HAT-driven neoplasms. A recent study found that anacardic acid
from cashew nut shell liquid, which has been reported to exhibit antitumor
potential, is able to inhibit HAT-dependent transcription particularly for p300
[36]. This compound acts as a noncompetitive inhibitor with no effect on DNA
transcription, and investigators argue that it can be used as a pilot compound
to design novel anti-HAT drugs.
Further study of HAT inhibitors is needed to reveal the potential roles of
individual HATs in transcriptional regulation not only in different promoter
contexts but also in a cell-specific manner. Furthermore, targeting specific
32 B. Drobic et al.
aberrant HATs could be of great advantage as anticancer therapy and further
expand the repertoire of selective therapeutic agents currently in clinical trials.
HDACs in cancer
HDACs are chromatin-modifying enzymes that remove acetyl groups from the
N-terminal tails of histones. Deacetylation of histones is associated with
repression of transcriptional activity, thus HDACs are co-repressors of tran-
scription. In addition to the deacetylation of histones, HDACs are responsible
for the deacetylation of non-histone proteins including E2F, MyoD, p53,
Hsp90, GATA-1 and tubulin [37–40].
Mammalian HDACs belong to one of three families. The first class, consist-
ing of HDACs 1, 2, 3, and 8, is defined by its relationship to the yeast deacety-
lase Rpd3. Class II HDACs are larger proteins related to yeast Hda1 and include
HDACs 4–7, 9 and 10. HDAC 11 shares properties with both class I and II
HDACs, and thus tends not to be classified. The third class of HDACs is often
referred to as the Sir2 family, and encompasses those HDACs with homology
to yeast Sir2. These enzymes require nicotinamide adenine dinucleotide to
function [37]. HDACs do not bind DNA directly, and instead are tethered to tar-
get sites by mediating factors present in various protein complexes [41].
HDACs are members of large multi-protein complexes. Class I HDACs are
found in a variety of protein complexes including Sin3, NuRD, and Co-REST
and interact with factors such as Sp1, YY1, and retinoblastoma (Rb) binding
protein-1 [38]. Additionally, they are found in complexes with nuclear recep-
tor co-repressor (N-CoR) and silencing mediator for retinoic acid receptor and
thyroid hormone receptor (SMRT), which can contain other HDACs such as
HDACs 4, 5 and 7 [38].
Expression of HDACs does not appear to be altered in cancer [38].
However, HDACs are found in complexes with well-known tumor suppressors
and oncogenes, such as Rb and Mad; in a diseased state inclusion of HDACs
in these complexes could lead to abnormal recruitment of HDACs and aberrant
gene expression [42, 43].
Translocation and point mutation events in non-Hodgkin’s lymphoma often
result in overexpression of the BCL-6 oncogene (Fig. 3). The product of this
oncogene has been linked to the regulation of B cell proliferation and is able
to recruit HDAC activity through interactions with N-CoR and SMRT, thus
aberrant repression activities may be involved in this cancer [44, 45].
The mis-targeting of HDACs mediated by recruitment by fusion proteins is
evident in many hematological cancers (Fig. 3). Patients with acute promyelo-
cytic leukemia often have translocations in which RAR is fused to PML or
PLZF [38, 45, 46]. This results in an oncoprotein able to recruit HDAC activ-
ity through N-CoR and SMRT, and is thought to lead to selective transcrip-
tional repression [38, 45]. This prevents differentiation and results in the dis-
proportionate proliferation of cells seen in these patients [38]. Although acute
Abnormalities of chromatin in tumor cells 33
promyelocytic leukemia patients having the PML-RARαtranslocation can
achieve remission successfully through treatment with retinoic acid,
PLZF-RARαtranslocations do not respond well to this therapy [47]. HDAC-
containing co-repressor complexes are also implicated in other leukemias
including AML. The AML1 gene product acts to upregulate genes related to
hematopoiesis [41]. The fusion protein AML1-ETO, produced as the result of
a translocation event in a significant percentage of AMLs, is able to bind the
HDAC co-repressor complexes N-CoR and SMRT, thereby providing a mech-
anism by which HDAC can be aberrantly targeted to alter chromatin structure
and transcription status [48]. Chromosomal rearrangements of TEL, a tran-
scriptional repressor, result in common acute lymphoblastic leukemia [41].
The protein encoded by TEL represses transcription by working in complex
with mSin3A, SMRT and HDAC3 [49, 50]. The evidence indicates that abnor-
mal transcriptional repression of genes necessary for proper cellular differen-
tiation by HDACs is an important factor in the progression of hematological
malignancies [41].
HDAC inhibitors as therapeutic targets for cancer
Experiments performed on cultured cells and with animal models have shown
that treatment of transformed cells with HDAC inhibitors leads to growth
arrest, differentiation, and apoptosis [37, 51]. Profiling of cultured cells treat-
ed with HDAC inhibitors has determined that the treated cells exhibit an
altered expression profile for a small percentage of genes [52–55]. Alterations
in expression profiles include many increases such as p21WAF1 and metalloth-
ionein, while decreases were observed for ErbB2, vascular endothelial growth
factor and others [37].
Expression of p21WAF1 is induced by several HDAC inhibitors, such TSA,
sodium butyrate, phenyl butyrate, SAHA, FK-228, MS-275 and oxoflatins
[37]. Higher levels of p21WAF1 are also observed in embryonic cells deficient
in HDAC1 displaying a reduced rate of proliferation [56]. Studies have demon-
strated an increase in the acetylation of histones in the p21WAF1 promoter
region after HDAC inhibitor treatment [57, 58]. Thus events leading to the
change in expression of genes following HDAC inhibitor treatment may in
some cases be a direct result of an increase in acetylation levels of histones
associated with the affected genes [37].
Treatment with HDAC inhibitors also alters the acetylation status of sever-
al non-histone proteins including p53, MyoD, GATA-1, α-tubulin and Hsp90
[37]. An increase in acetylation of chaperone protein Hsp90 results in
decreased binding to other proteins and the subsequent degradation of those
proteins [37, 59]. Thus, HDAC inhibitors may modulate their effects by influ-
encing both gene expression and protein stability [37].
Studies have shown that both normal and tumor cells accumulate acetylat-
ed histones when treated with HDAC inhibitors [60–63]. Yet the growth arrest
34 B. Drobic et al.
of tumors occurred without excessive toxicity in animal models [37]. This
result may be explained by the finding that tumor cells have a tenfold higher
sensitivity to HDAC inhibitors than do normal cells [37]. Several HDAC
inhibitors are currently in clinical trials and provide a promising new strategy
to treat cancer [64].
Alterations to chromatin structure by activation of the Ras-MAPK
pathway
Signals from growth factors, stresses and cytokines are relayed by the mito-
gen-activated protein kinase (MAPK) pathway. Activation of either the Ras-
MAPK or p38 MAPK pathway leads to the activation of transcription at imme-
diate-early genes (Fig. 2). In 1999, Brown et al. [65] identified genes whose
transcription was activated within 15 min of stimulating human fibroblasts
with serum. A programmed transcriptional response occurred: genes such as
c-fos, Jun B and MAP kinase phosphatase-1 were activated within 15 min of
serum addition, followed later by the transcription of genes encoding proteins
associated with wound repair [65].
The Ras-MAPK pathway is activated by growth factors and phorbol esters
(12-O-tetradecanoylphorbol-13-acetate, TPA), while the p38 MAPK pathway
is activated by stressors such as UV irradiation (Fig. 2). TPA works through
PKC and/or RasGRP, which is expressed in a cell type-specific manner, to acti-
vate the Ras-MAPK [66–68]. Stimulation of these pathways activates a series
of protein kinases (see [69] for a review of this process), leading to the phos-
phorylation of histone H3 and HMGN1 and modification of chromatin struc-
ture. Stimulation of the MAPK induces phosphorylation of H3 on S10 and S28
[70–72], and phosphorylation of HMGN1 on S6 [73]. As evidenced by MSK1
and MSK2 knockout mice, impaired phosphorylation severely limits tran-
scription of immediate-early genes [70]. The location of H3 phosphorylated on
S28 after activation of the MAPK pathway has not been determined, but it is
known that phosphorylation on S10 occurs at immediate-early genes [74–77].
H3 phosphorylated at S10 in TPA-treated, serum-starved mouse 10 T1/2
fibroblasts appears in numerous small foci throughout the interphase nuclei
[74]. These sites may represent the nuclear locations of immediate-early genes
targeted for transcriptional activation. After stimulation of the MAPK path-
way, H3 phosphorylated on S10 is associated with the c-fos promoter and is
found at various regions of the c-jun gene [75, 77].
The MAPK pathway, histone phosphorylation and cancer
Several cancerous tissues and cell lines display constitutive activation of the
MAPK pathway. An estimated 30% of human malignancies contain a mutation
to one of the ras oncogenes that renders the protein constitutively active [78].
Abnormalities of chromatin in tumor cells 35
Kirsten-ras (Ki-ras) is the most commonly mutated, and defects in Harvey-ras
(Ha-ras) and Neuroblastoma-ras (N-ras) are also detected [78 –80]. Other
members of the MAPK pathway have also been implicated in malignant trans-
formation. An upregulation of ras-oncogene-related p21-rac1 and MAPK
p38αand a downregulation of genes associated with apoptosis have been
observed in low-grade dysplastic adenomas of the colon [81]. The study also
found upregulation of the rho GDP dissociation factor [81]. An upregulation in
members of the epidermal growth factor family has been observed in several
cancers, and various tumor cell lines exhibit high levels of activated Erk1/2
[82, 83]. Mutations to the BRAF gene encoding a MAPK kinase kinase are
frequently seen in cutaneous melanoma [84]. BRAF mutation can lead to con-
stitutive activation of the MEK/ERK pathway independent of Ras and increas-
es in B-RAF(V599E) activity, which could contribute to anchorage-independ-
ent growth [84].
Alterations in chromatin and nuclear structure of oncogene-transformed cells
Cancer can be diagnosed by changes to nuclear morphology. Fibroblasts trans-
formed with oncogenes such as v-Mos, v-Fes, v-Raf, v-Src, and H-Ras display
abnormal nuclear morphology such that the nucleus is more rigid and spheri-
cal [85]. The degree of changes in nuclear morphology correlate with metasta-
tic potential [85]. Oncogene-transformed cells also exhibit abnormalities in the
nuclear matrix [86]. Highly metastatic lines transformed with ras or kinase
oncogenes show similar nuclear matrix profiles [86], suggesting that compo-
nents of the nuclear matrix change as malignancy progresses [87].
Changes to chromatin structure have also been observed. Ras- and myc-
transformed cells display a relaxed chromatin conformation [15, 16].
Micrococcal nuclease digestion of chromatin from parental and ras-trans-
formed cells determined that the bulk chromatin, and chromatin at the imme-
diate-early genes ornithine decarboxylase and c-myc was less condensed in the
ras-transformed cells [16]. Experiments to determine the methylation status of
DNA at the ornithine decarboxylase gene showed similar methylation levels in
parental and transformed cells, thus it is not believed that hypomethylation
plays a role in the formation of a relaxed chromatin structure in this case [88].
Phosphorylation of H3 on S10 following stimulation of the Ras-MAPK is
known to occur at immediate-early genes [75–77]. Transcription requires that
chromatin structure become less condensed. Parental and ras-transformed
fibroblasts synchronized by serum starvation contain relatively condensed chro-
matin that becomes highly decondensed in late G1[16]. Both phosphorylation
of H3 and transcription at immediate-early genes is impaired if the H3 kinases,
MSK1 and MSK2, are knocked out [70]. This link between the MAPK path-
way, histone modifications and gene expression strengthens the hypothesis that
deregulation of the Ras-MAPK pathway leads to an abnormal chromatin con-
formation and aberrant gene expression in transformed cells [89].
36 B. Drobic et al.
Histone kinases and phosphatases and cancer
Phosphorylation and dephosphorylation of proteins play a major role in mech-
anisms controlling proper execution of cellular functions. The importance of
histone phosphorylation in intracellular processes has long been recognized.
Kinases and phosphatases responsible for the reversible process of histone
phosphorylation have been recognized as crucial mediators of various cellular
processes including mitosis, meiosis and transcriptional activation [90].
Further, abnormal expression of mitotic histone kinases have been implicated
in some human cancers, and deregulated activities of histone kinases and phos-
phatases could underline some of the mechanisms leading to oncogenic trans-
formation and malignancies. Histone kinases implicated in cell transformation
and tumorigenesis are reviewed in the following sections.
Histone kinases and activated Ras-MAPK pathway in cancer
Histone phosphorylation is linked to relaxation of chromatin structure [91].
Phosphorylation of histone H3 plays a crucial role during mitosis and meiosis.
Further, H3 phosphorylation at S10 and S28 has a role in transcriptional acti-
vation of immediate-early genes [92]. The kinases responsible for phosphory-
lating H3 during this process are mitogen- and stress-activated protein kinases
(MSKs). Both MSK1 and MSK2 phosphorylate H3 at S10 and S28 [70]. To
date, the expression and activities of MSKs in human cancers have not been
analyzed. However, H3 phosphorylation at S10 is elevated in oncogene-trans-
formed cells, and we recently reported that the increase in the steady-state
level of phosphorylated H3 is due to an increase in MSK1 activity and not its
expression [74, 93]. Further research needs to be conducted with respect to
MSKs to elucidate possible roles these H3 kinases have in oncogenesis and
tumorigenesis.
Histone H1 phosphorylation is also linked to chromatin relaxation, and
phosphorylation of H1 is elevated in oncogene-transformed mouse fibroblasts
[15]. The increased phosphorylation of H1 in the oncogene-transformed cells
was shown to be due to elevated activity of H1 kinase, cyclin-dependent kinase
2 (Cdk2). The activity of the H1 phosphatase, PP1, is not altered in the onco-
gene-transformed cells [94]. Cdk2 overexpression has been described in colon
carcinoma cell lines, and Cdk2 overexpression is postulated to be a prognostic
indicator of oral cancer progression [95, 96]. However, no direct studies relat-
ing Cdk2 activity and chromatin remodeling in cancers have been conducted.
Aurora kinases and cancer
Human mitotic serine/threonine kinases termed Aurora kinases belong to the
prototypic yeast Ipl1 and Drosophila Aurora kinase family. There are three
Abnormalities of chromatin in tumor cells 37
human Aurora kinases, Aurora A (Aurora-2, STK15, mouse STK6), Aurora B
(AIM-1, Aurora-1, STK12) and Aurora C (STK13). These kinases are respon-
sible for proper mitosis and meiosis in all eukaryotes [97]. Aurora kinases
have been shown to associate with interphase chromosomes, mitotic spindle
poles, mitotic microtubules and the spindle midbody, implicating these kinas-
es in the tight regulation of chromosomal ploidy in cells. Aurora kinases have
a highly conserved catalytic domain, a short C-terminal domain and an N-ter-
minal domain of varying size [98]. All three Auroras are able to phosphory-
late H3 in vitro [91], with only Aurora B being shown to phosphorylate H3 at
S10 and S28 in vivo. This phosphorylation event catalyzed by Aurora B has
been linked to proper mitotic chromosome condensation [99]. It has been
shown that H3 S10 phosphorylation is absolutely critical for both chromo-
some condensation and segregation in Tetrahymena [100]. However, in mam-
mals both S10 and S28 phosphorylation seem to be important for proper
mitotic processes. Further, H3 phosphorylation events during mitosis are
tightly governed not only by Aurora B but also by H3 phosphatase PP1 [99].
It is important to note that the H3 phosphorylation events are reversible, and
both kinase and phosphatase activities play critical roles in this regulation.
Moreover, overexpression of all three Aurora kinases has been observed in
various human cancers. Aurora A, which is implicated in centrosome matura-
tion and spindle assembly, is mapped to chromosomal 20q13 region often
amplified in human cancers [101]. Overexpression and amplification of
Aurora A has been detected in colon, bladder, ovarian, human breast and pan-
creatic cancers. Further, overexpression of Aurora A has been shown to cor-
relate with induced aneuploidy, centrosomal anomalies and prognosis of nat-
urally occurring tumors in animal model systems as well as with the induc-
tion of oncogenic transformation in cells [98]. The evidence implies the
involvement of Aurora A in the cellular processes that are most likely dereg-
ulated in many human cancers. In addition, Aurora B, which is required for
proper mitotic events and cytokinesis, has an abnormal expression profile
detected in various human tumor cell lines, including colorectal cancer cell
lines [98]. Elevated kinase activity and overexpression of Aurora B along with
the overexpression of Aurora B interacting proteins, such as INCENP and
Survivin [102], which target Aurora B to the centromere at metaphase for H3
phosphorylation, are likely to be responsible for anomalous effects observed
in cancer cells [98]. Increased H3 phosphorylation due to Aurora B has been
attributable to chromosome number instability, a feature often seen in many
human cancer cells. Aurora B overexpression in Chinese hamster cells led to
increased phosphorylation of H3 at S10, and Aurora B overexpressing cells
were able to form aggressive tumors in nude mice [103]. The involvement of
Aurora B in the generation of chromosomal instabilities in conjunction with
increased H3 phosphorylation reinforces the role this kinase could be under-
taking in carcinogenesis. Lastly, Aurora C overexpression has been detected
in various cancer cell lines. There is also some correlative evidence that
Aurora C could be involved in oncogenic signal transduction in somatic cells
38 B. Drobic et al.
[98]. Recently, a potent and selective inhibitor of all three human Aurora
kinases, VX-680 has been shown to decrease H3 phosphorylation at S10 in
the MCF-7 cell line as well as suppress tumor growth in vivo [104]. This find-
ing implicates Aurora kinases in the processes leading to malignant transfor-
mation and carcinogenesis, and shows promise for a new approach for anti-
cancer therapy since VX-680 was able to induce regression of a range of
human tumor types. The VX-680 inhibitor is progressing into clinical devel-
opment [104].
Histone methylation and chromatin
The four core histones are modified by methylation of lysines and arginines
located in the N-terminal tail and histone-fold domains (Fig. 1). Histone
methylation is catalyzed by histone methyltransferases, which are a large fam-
ily of enzymes that have specificity for a histone, the modification site (lysine
or arginine) and chromatin region (for review see [105]). In contrast to histone
acetylation and phosphorylation, histone methylation is a stable modification.
To date a histone demethylase has not been identified. However, histone
exchange occurring during transcription is one mechanism by which the core
histones are dislodged from the transcribed DNA and replaced by a histone
that is not methylated [106].
Analyses of the distribution of methylated histones in nuclei of normal and
tumor cell nuclei using an antibody recognizing methylated lysines independ-
ent of their lysine position in the histone revealed a differential distribution of
methylated histones in these nuclei. In contrast to the homogeneous distribu-
tion of chromatin with methylated histones in normal G0lymphocytes, the
leukemic T cell Jurkat cells had methylated histones located in numerous dis-
tinct clusters. Further, chromatin with lysine methylated histones was concen-
trated more peripherally in colon carcinoma compared to nuclei of normal
colon epithelial cells [107].
H3 methylated at K4 and K79 is located in transcribed regions of the
genome, while H3 methylated at K9 and H4 methylated at K20 are present in
heterochromatin regions, the histones of which are hypoacetylated [13, 108,
109]. H3 methyl K9 avidly binds to the chromodomain of heterochromatin
protein 1 (HP1), recruiting the protein to heterochromatic regions [105]. HP1
interacts with the H3 K9 methyltransferase SUVAR39H1. Thus, HP1 recruit-
ed by a nucleosome bearing an H3 methyl K9 will enable the HP1-bound H3
K9 methyltransferase to methylate neighboring nucleosomes, establishing a
self-propagating mechanism for the spreading of heterochromatin. In addition
to heterochromatic silencing, SUV39H1 H3 methyltransferase and HP1 are
involved in repression of euchromatic genes. Downregulation of HP1Hsαis
observed in metastatic breast cancer. The reduced expression of HP1 may
result in the reorganization of chromatin and activation of genes involved in
metastasis [110].
Abnormalities of chromatin in tumor cells 39
The transcription factor E2F has a pivotal role in regulating the expression
of S phase-specific genes. Repression of these genes is through the Rb protein,
which binds to E2F. Rb recruits histone methyltransferases and histone
deacetylases to repress gene expression [111]. Rb bound to E2F recruits
SUV39H1 to the S phase-specific gene promoter (e.g., cyclin E), which in turn
recruits HP1. Rb phosphorylation abolishes its association with histone
deacetylase and histone H3 K9 methyltransferase. Thus, inactivation of this
tumor suppressor gene will result in the deregulation of Rb-guided epigenetic
pathways.
EZH2 is an H3 K27 histone methyltransferase that is a component of the
EED (embryonic ectoderm development)-EZH2 complex. Sequence-specific
DNA binding proteins Pho and Pho1 bind to the Polycomb response element
and recruit the EED-EZH2 complex, resulting in the methylation of H3 K27.
Methylated H3 K27 recruits Polycomb group (PcG) proteins and the
Polycomb repressive complex 1 to silence specific genes. PcG proteins main-
tain the silenced state of homeotic genes. EZH2 is overexpressed in prostate
and breast cancer cells, and this deregulation of EZH2 may result in alteration
of chromatin structure and deregulation of the downstream targets of the EED-
EZH2 complex [112]. SMYD3 (SET- and MYND-domain containing protein
3) is an H3 K4 histone methyltransferase and sequence-specific DNA binding
protein that is overexpressed in colorectal carcinomas and hepatocellular car-
cinomas. Suppression of SMYD3 expression inhibited the growth of colorec-
tal carcinoma and hepatocellular carcinoma cells. SMYD3 is involved in the
activation of oncogenes and genes associated with cell-cycle regulation [113].
DNA methylation, histone methylation and cancer
DNA methylation is a key epigenetic process involved in gene silencing.
Methylation of DNA at CpG residues is catalyzed by DNA methyltransferase
(DNMT1). Deregulation of this epigenetic process is often observed in cancer
cells [114]. Hypermethylation of tumor suppressor genes silences their expres-
sion [115]. The Cdk inhibitor, p16, is silenced by hypermethylation in many
types of cancer. Without p16 expression, cell cycle regulation is lost, confer-
ring a growth advantage to affected cells. In colorectal cells, the promoter
DNA of the p16 gene is hypermethylated and associated with H3 methylated
at K9 [116]. Although mutational inactivation of the DNMT1 gene is a rare
event, loss of DNMT1 expression in human colorectal cells results in gross
nuclear changes, including a more relaxed nuclease sensitive chromatin, dis-
persed and diffuse localization of H3 trimethylated at K9, which is located in
discrete spots of heterochromatin in wild-type cells, and spatial disorganiza-
tion of HP1α[117].
Investigations into the order of events resulting from gene silencing have
shown that a low level of methylation of a promoter recruits the methylated
DNA-binding protein MBD2, which recruits HDACs and DNMT1. The
40 B. Drobic et al.
HDACs deacetylate residues such as acetylated K9 and K4 of H3. DNMT1
recruitment leads to further methylation of the promoter, resulting in the
recruitment of another methylated DNA-binding protein MeCP2. MeCP2 in
turn recruits an H3 K9 methyltransferase, leading to the methylation H3 K9
[118]. This program of events may be reversed when cancer cells are treated
with the DNA-demethylating drug 5-Aza-dC, with DNA demethylation of the
promoter resulting in the loss of methyl H3 K9 and acetylation of H3 and
methylation of H3 K4 after the resumption of transcription [119].
Histone ubiquitination and cancer
Histones H2A and H2B and their variant forms are reversibly ubiquitinated
[120]. The carboxyl end of ubiquitin is attached to the ε-amino group of lysine
(K119 in H2A; K120 in H2B) (Fig. 1). Ubiquitinated H2B and to a lesser
extent ubiquitinated H2A are associated with transcriptionally active DNA
[121]. Ubiquitination of H2B is the only core histone modification that is
dependent upon ongoing transcription. The level of ubiquitinated H2A in
SV40-transformed human fibroblasts (WI-38 SV40) and keratinocytes (K14)
is greater than their normal counterparts [122]. In AML, OCI/AML 1a cells
and cells from leukemia patients proteolysis of ubiquitinated H2A was evident
[123]. The impact of an increased level of ubiquitinated H2A or its proteoly-
sis on chromatin structure and function is not known.
Histone H1 subtypes and cancer
The H1 histones are a heterogeneous group of several subtypes that differ in
amino acid sequence [124]. Mammalian nuclei typically have more than one
H1 subtype, with the relative amounts of the H1 subtypes varying with cell
type. The expression of the subtypes is differentially regulated throughout
development, through the cell cycle, and during differentiation [125]. Changes
in the relative levels of the H1 subtypes have been observed in normal and neo-
plastic cells [126, 127]. The H1 subtypes differ in their abilities to condense
DNA and chromatin fragments and thus alterations in their composition could
lead to changes in the condensation of chromatin, e.g. ras-transformed mouse
fibroblasts have altered levels of H1 subtype H10and an increase in the nucle-
osome repeat length [16].
Concluding comments
A great deal has been learned about the interplay between genetic and epige-
netic processes in cancer. Pharmacological approaches using DNA methyla-
tion inhibitors, HDAC inhibitors or combinations of both to alter epigenetic
Abnormalities of chromatin in tumor cells 41
programming in cancer cells to effect the reactivation of genes such as tumor
suppressor genes and cell cycle regulating genes are promising approaches that
are currently in clinical trails [5]. A greater appreciation of the mechanisms
involved in genetic alterations influencing epigenetic pathways, the interplay
of epigenetic pathways, and factors such as diet and environment influencing
epigenetic pathways, which may give rise to mutations in the genetic informa-
tion, will provide new strategies to prevent and treat cancer.
Acknowledgements
Research support by a grant from the National Cancer Institute of Canada with funds from the
Canadian Cancer Society, a Canada Research Chair to J.R.D., a K.M. Hunter/CIHR Doctoral Research
Award to K.L.D., a Guardian Angel’s studentship to P.S.E. and a CancerCare Manitoba Foundation
Inc. studentship to B.D. are gratefully acknowledged.
References
1 Khan MA, Walsh PC, Miller MC, Bales WD, Epstein JI, Mangold LA, Partin AW, Veltri RW
(2003) Quantitative alterations in nuclear structure predict prostate carcinoma distant metastasis
and death in men with biochemical recurrence after radical prostatectomy. Cancer 98: 2583–2591
2 Komitowski DD, Hart MM, Janson CP (1993) Chromatin organization and breast cancer progno-
sis: Two- dimensional and three-dimensional image analysis. Cancer 72: 1239–1246
3 Vogelstein B, Kinzler KW (2004) Cancer genes and the pathways they control. Nat Med 10:
789–799
4 Hake SB, Xiao A, Allis CD (2004) Linking the epigenetic ‘language’ of covalent histone modifi-
cations to cancer. Br J Cancer 90: 761–769
5 Egger G, Liang G, Aparicio A, Jones PA (2004) Epigenetics in human disease and prospects for
epigenetic therapy. Nature 429: 457–463
6 Luger K, Mader AW, Richmond RK, Sargent DF, Richmond TJ (1997) Crystal structure of the
nucleosome core particle at 2.8 A resolution. Nature 389: 251–260
7 Zhou YB, Gerchman SE, Ramakrishnan V, Travers A, Muyldermans S (1998) Position and orien-
tation of the globular domain of linker histone H5 on the nucleosome. Nature 395: 402–405
8 Camporeale G, Shubert EE, Sarath G, Cerny R, Zempleni J (2004) K8 and K12 are biotinylated
in human histone H4. Eur J Biochem 271: 2257–2263
9 Zhang L, Eugeni EE, Parthun MR, Freitas MA (2003) Identification of novel histone post-trans-
lational modifications by peptide mass fingerprinting. Chromosoma 112: 77–86
10 Freitas MA, Sklenar AR, Parthun MR (2004) Application of mass spectrometry to the identifica-
tion and quantification of histone post-translational modifications. J Cell Biochem 92: 691–700
11 Zlatanova J, Leuba SH, Van Holde K (1998) Chromatin fiber structure: morphology, molecular
determinants, structural transitions. Biophys J 74: 2554–2566
12 Ridsdale JA, Hendzel MJ, Delcuve GP, Davie JR (1990) Histone acetylation alters the capacity of
the H1 histones to condense transcriptionally active/competent chromatin. J Biol Chem 265:
5150–5156
13 Schubeler D, MacAlpine DM, Scalzo D, Wirbelauer C, Kooperberg C, van Leeuwen F,
Gottschling DE, O’Neill LP, Turner BM, Delrow J et al. (2004) The histone modification pattern
of active genes revealed through genome-wide chromatin analysis of a higher eukaryote. Genes
Dev 18: 1263–1271
14 Herrera RE, Chen F, Weinberg RA (1996) Increased histone H1 phosphorylation and relaxed
chromatin structure in Rb-deficient fibroblasts. Proc Natl Acad Sci USA 93: 11510–11515
15 Chadee DN, Taylor WR, Hurta RAR, Allis CD, Wright JA, Davie JR (1995) Increased phospho-
rylation of histone H1 in mouse fibroblasts transformed with oncogenes or constitutively active
mitogen-activated protein kinase kinase. J Biol Chem 270: 20098–20105
16 Laitinen J, Sistonen L, Alitalo K, Holtta E (1990) c-Ha-ras (val 12) oncogene-transformed
NIH-3 T3 fibroblasts display more decondensed nucleosomal organization than normal fibrob-
lasts. J Cell Biol 111: 9–17
42 B. Drobic et al.
17 Giordano A, Avantaggiati ML (1999) p300 and CBP: Partners for life and death. J Cell Physiol
181: 218–230
18 Roth SY, Denu JM, Allis CD (2001) Histone acetyltransferases. Annu Rev Biochem 70: 81–120
19 Wolffe AP (2001) Chromatin remodeling: why it is important in cancer. Oncogene 20: 2988–2990
20 Mahlknecht U, Ottmann OG, Hoelzer D (2000) When the band begins to play: histone acetylation
caught in the crossfire of gene control. Mol Carcinog 27: 268–271
21 Giles RH, Peters DJ, Breuning MH (1998) Conjunction dysfunction: CBP/p300 in human disease.
Trends Genet 14: 178–183
22 Aguiar RC, Chase A, Coulthard S, Macdonald DH, Carapeti M, Reiter A, Sohal J, Lennard A,
Goldman JM, Cross NC (1997) Abnormalities of chromosome band 8p11 in leukemia: two clini-
cal syndromes can be distinguished on the basis of MOZ involvement. Blood 90: 3130–3135
23 Iyer NG, Ozdag H, Caldas C (2004) p300/CBP and cancer. Oncogene 23: 4225–4231
24 Kojima K, Kaneda K, Yoshida C, Dansako H, Fujii N, Yano T, Shinagawa K, Yasukawa M, Fujita
S, Tanimoto M (2003) A novel fusion variant of the MORF and CBP genes detected in therapy-
related myelodysplastic syndrome with t(10;16)(q22;p13). Br J Haematol 120: 271–273
25 Panagopoulos I, Fioretos T, Isaksson M, Samuelsson U, Billstrom R, Strombeck B, Mitelman F,
Johansson B (2001) Fusion of the MORF and CBP genes in acute myeloid leukemia with the
t(10;16)(q22;p13). Hum Mol Genet 10: 395–404
26 Deguchi K, Ayton PM, Carapeti M, Kutok JL, Snyder CS, Williams IR, Cross NC, Glass CK,
Cleary ML, Gilliland DG (2003) MOZ-TIF2-induced acute myeloid leukemia requires the MOZ
nucleosome binding motif and TIF2-mediated recruitment of CBP. Cancer Cell 3: 259–271
27 Mahlknecht U, Hoelzer D (2000) Histone acetylation modifiers in the pathogenesis of malignant
disease. Mol Med 6: 623–644
28 Ida K, Kitabayashi I, Taki T, Taniwaki M, Noro K, Yamamoto M, Ohki M, Hayashi Y (1997)
Adenoviral E1A-associated protein p300 is involved in acute myeloid leukemia with
t(11;22)(q23;q13). Blood 90: 4699–4704
29 Taki T, Sako M, Tsuchida M, Hayashi Y (1997) The t(11;16)(q23;p13) translocation in myelodys-
plastic syndrome fuses the MLL gene to the CBP gene. Blood 89: 3945–3950
30 Ausio J, Levin DB, De Amorim GV, Bakker S, Macleod PM (2003) Syndromes of disordered
chromatin remodeling. Clin Genet 64: 83–95
31 Agalioti T, Lomvardas S, Parekh B, Yie J, Maniatis T, Thanos D (2000) Ordered recruitment of
chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 103: 667–678
32 Lavau C, Du C, Thirman M, Zeleznik-Le N (2000) Chromatin-related properties of CBP fused to
MLL generate a myelodysplastic-like syndrome that evolves into myeloid leukemia. EMBO J 19:
4655–4664
33 Moe-Behrens GH, Pandolfi PP (2003) Targeting aberrant transcriptional repression in acute
myeloid leukemia. Rev Clin Exp Hematol 7: 139–159
34 Poux AN, Cebrat M, Kim CM, Cole PA, Marmorstein R (2002) Structure of the GCN5 histone
acetyltransferase bound to a bisubstrate inhibitor. Proc Natl Acad Sci USA 99: 14065–14070
35 Zheng Y, Thompson PR, Cebrat M, Wang L, Devlin MK, Alani RM, Cole PA (2004) Selective
HAT inhibitors as mechanistic tools for protein acetylation. Methods Enzymol 376: 188–199
36 Balasubramanyam K, Swaminathan V, Ranganathan A, Kundu TK (2003) Small molecule modu-
lators of histone acetyltransferase p300. J Biol Chem 278: 19134–19140
37 Marks PA, Miller T, Richon VM (2003) Histone deacetylases. Curr Opin Pharmacol 3: 344–351
38 De Ruijter AJ, Van Gennip AH, Caron HN, Kemp S, Van Kuilenburg AB (2003) Histone deacety-
lases (HDACs): characterization of the classical HDAC family. Biochem J 370: 737–749
39 Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang XF, Yao TP
(2002) HDAC6 is a microtubule-associated deacetylase. Nature 417: 455–458
40 Juan LJ, Shia WJ, Chen MH, Yang WM, Seto E, Lin YS, Wu CW (2000) Histone deacetylases
specifically down-regulate p53-dependent gene activation. J Biol Chem 275: 20436–20443
41 Jepsen K, Rosenfeld MG (2002) Biological roles and mechanistic actions of co-repressor com-
plexes. J Cell Sci 115: 689–698
42 Cress WD, Seto E (2000) Histone deacetylases, transcriptional control, and cancer. J Cell Physiol
184: 1–16
43 Timmermann S, Lehrmann H, Polesskaya A, Harel-Bellan A (2001) Histone acetylation and dis-
ease. Cell Mol Life Sci 58: 728–736
44 Huynh KD, Bardwell VJ (1998) The BCL-6 POZ domain and other POZ domains interact with
the co-repressors N-CoR and SMRT. Oncogene 17: 2473–2484
Abnormalities of chromatin in tumor cells 43
45 Kramer OH, Gottlicher M, Heinzel T (2001) Histone deacetylase as a therapeutic target. Trends
Endocrinol MeTab. 12: 294–300
46 de The H (1996) Altered retinoic acid receptors. FASEB J 10: 955 –960
47 Lin RJ, Egan DA, Evans RM (1999) Molecular genetics of acute promyelocytic leukemia. Trends
Genet 15: 179–184
48 Lutterbach B, Westendorf JJ, Linggi B, Patten A, Moniwa M, Davie JR, Huynh KD, Bardwell VJ,
Lavinsky RM, Rosenfeld MG et al. (1998) ETO, a target of t(8;21) in acute leukemia, interacts
with the N-CoR and mSin3 corepressors. Mol Cell Biol 18: 7176–7184
49 Chakrabarti SR, Nucifora G (1999) The leukemia-associated gene TEL encodes a transcription
repressor which associates with SMRT and mSin3A. Biochem Biophys Res Commun 264:
871–877
50 Wang L, Hiebert SW (2001) TEL contacts multiple co-repressors and specifically associates with
histone deacetylase-3. Oncogene 20: 3716–3725
51 Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK (2001) Histone deacetylases
and cancer: causes and therapies. Nat Rev Cancer 1: 194–202
52 Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, Herman JG,
Baylin SB (2002) A genomic screen for genes upregulated by demethylation and histone deacety-
lase inhibition in human colorectal cancer. Nat Genet 31: 141–149
53 Della RF, Criniti V, Della P, V, Borriello A, Oliva A, Indaco S, Yamamoto T, Zappia V (2001)
Genes modulated by histone acetylation as new effectors of butyrate activity. FEBS Lett 499:
199–204
54 Van Lint C, Emiliani S, Verdin E (1996) The expression of a small fraction of cellular genes is
changed in response to histone hyperacetylation. Gene Expr 5: 245–253
55 Butler LM, Zhou X, Xu WS, Scher HI, Rifkind RA, Marks PA, Richon VM (2002) The histone
deacetylase inhibitor SAHA arrests cancer cell growth, up-regulates thioredoxin-binding pro-
tein-2, and down-regulates thioredoxin. Proc Natl Acad Sci USA 99: 11700–11705
56 Lagger G, O’Carroll D, Rembold M, Khier H, Tischler J, Weitzer G, Schuettengruber B, Hauser
C, Brunmeir R, Jenuwein T, Seiser C (2002) Essential function of histone deacetylase 1 in prolif-
eration control and CDK inhibitor repression. EMBO J 21: 2672–2681
57 Sambucetti LC, Fischer DD, Zabludoff S, Kwon PO, Chamberlin H, Trogani N, Xu H, Cohen D
(1999) Histone deacetylase inhibition selectively alters the activity and expression of cell cycle
proteins leading to specific chromatin acetylation and antiproliferative effects. J Biol Chem 274:
34940–34947
58 Richon VM, Sandhoff TW, Rifkind RA, Marks PA (2000) Histone deacetylase inhibitor selec-
tively induces p21WAF1 expression and gene-associated histone acetylation. Proc Natl Acad Sci
USA 97: 10014–10019
59 Yu X, Guo ZS, Marcu MG, Neckers L, Nguyen DM, Chen GA, Schrump DS (2002) Modulation
of p53, ErbB1, ErbB2, and Raf-1 expression in lung cancer cells by depsipeptide FR901228. J
Natl Cancer Inst 94: 504–513
60 Richon VM, Emiliani S, Verdin E, Webb Y, Breslow R, Rifkind RA, Marks PA (1998) A class of
hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases. Proc Natl
Acad Sci USA 95: 3003–3007
61 Scott GK, Marden C, Xu F, Kirk L, Benz CC (2002) Transcriptional repression of ErbB2 by his-
tone deacetylase inhibitors detected by a genomically integrated ErbB2 promoter-reporting cell
screen. Mol Cancer Ther 1: 385–392
62 Kelly WK, Richon VM, O’Connor O, Curley T, MacGregor-Curtelli B, Tong W, Klang M,
Schwartz L, Richardson S, Rosa E et al. (2003) Phase I clinical trial of histone deacetylase
inhibitor: suberoylanilide hydroxamic acid administered intravenously. Clin Cancer Res 9:
3578–3588
63 Qiu L, Kelso MJ, Hansen C, West ML, Fairlie DP, Parsons PG (1999) Anti-tumour activity in vitro
and in vivo of selective differentiating agents containing hydroxamate. Br J Cancer 80:
1252–1258
64 Zelent A, Waxman S, Carducci M, Wright J, Zweibel J, Gore SD (2004) State of the translational
science: summary of Baltimore workshop on gene re-expression as a therapeutic target in cancer
January 2003. Clin Cancer Res 10: 4622–4629
65 Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JCF, Trent JM, Staudt LM, Hudson J Jr,
Boguski MS et al. (1999) The transcriptional program in the response of human fibroblasts to
serum. Science 283: 83–87
44 B. Drobic et al.
66 Brose N, Rosenmund C (2002) Move over protein kinase C, you’ve got company: alternative cel-
lular effectors of diacylglycerol and phorbol esters. J Cell Sci 115: 4399–4411
67 Kazanietz MG (2000) Eyes wide shut: protein kinase C isozymes are not the only receptors for
the phorbol ester tumor promoters. Mol Carcinog 28: 5–11
68 Chang F, Steelman LS, Lee JT, Shelton JG, Navolanic PM, Blalock WL, Franklin RA, McCubrey
JA (2003) Signal transduction mediated by the Ras/Raf/MEK/ERK pathway from cytokine recep-
tors to transcription factors: potential targeting for therapeutic intervention. Leukemia 17:
1263–1293
69 Hazzalin CA, Mahadevan LC (2002) MAPK-regulated transcription: a continuously variable gene
switch? Nat Rev Mol Cell Biol 3: 30–40
70 Soloaga A, Thomson S, Wiggin GR, Rampersaud N, Dyson MH, Hazzalin CA, Mahadevan LC,
Arthur JS (2003) MSK2 and MSK1 mediate the mitogen- and stress-induced phosphorylation of
histone H3 and HMG-14. EMBO J 22: 2788–2797
71 Clayton AL, Mahadevan LC (2003) MAP kinase-mediated phosphoacetylation of histone H3 and
inducible gene regulation. FEBS Lett 546: 51–58
72 Zhong S, Zhang Y, Jansen C, Goto H, Inagaki M, Dong Z (2001) MAP kinases mediate UVB-
induced phosphorylation of histone H3 at serine 28. J Biol Chem 276: 12932–12937
73 Barratt MJ, Hazzalin CA, Zhelev N, Mahadevan LC (1994) A mitogen- and anisomycin-stimulat-
ed kinase phosphorylates HMG-14 in its basic amino-terminal domain in vivo and on isolated
mononucleosomes. EMBO J 13: 4524–4535
74 Chadee DN, Hendzel MJ, Tylipski CP, Allis CD, Bazett-Jones DP, Wright JA, Davie JR (1999)
Increased Ser-10 phosphorylation of histone H3 in mitogen-stimulated and oncogene-transformed
mouse fibroblasts. J Biol Chem 274: 24914–24920
75 Thomson S, Clayton AL, Mahadevan LC (2001) Independent dynamic regulation of histone phos-
phorylation and acetylation during immediate-early gene induction. Mol Cell 8: 1231–1241
76 Clayton AL, Rose S, Barratt MJ, Mahadevan LC (2000) Phosphoacetylation of histone H3 on
c-fos- and c-jun-associated nucleosomes upon gene activation. EMBO J 19: 3714–3726
77 Cheung P, Tanner KG, Cheung WL, Sassone-Corsi P, Denu JM, Allis CD (2000) Synergistic cou-
pling of histone H3 phosphorylation and acetylation in response to epidermal growth factor stim-
ulation. Mol Cell 5: 905–915
78 Bos JL (1989) ras oncogenes in human cancer: a review. Cancer Res 49: 4682–4689
79 Barbacid M (1987) ras genes. Annu Rev Biochem 56: 779–827
80 Andreyev HJ, Norman AR, Cunningham D, Oates JR, Clarke PA (1998) Kirsten ras mutations in
patients with colorectal cancer: the multicenter “RASCAL” study. J Natl Cancer Inst 90: 675–684
81 Lechner S, Muller-Ladner U, Renke B, Scholmerich J, Ruschoff J, Kullmann F (2003) Gene
expression pattern of laser microdissected colonic crypts of adenomas with low grade dysplasia.
Gut 52: 1148–1153
82 Hoshino R, Chatani Y, Yamori T, Tsuruo T, Oka H, Yoshida O, Shimada Y, Ari-i S, Wada H,
Fujimoto J, Kohno M (1999) Constitutive activation of the 41-/43-kDa mitogen-activated protein
kinase signaling pathway in human tumors. Oncogene 18: 813–822
83 Campbell PM, Der CJ (2004) Oncogenic Ras and its role in tumor cell invasion and metastasis.
Semin Cancer Biol 14: 105–114
84 Calipel A, Lefevre G, Pouponnot C, Mouriaux F, Eychene A, Mascarelli F (2003) Mutation of
B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through
the MEK/ERK pathway. J Biol Chem 278: 42409–42418
85 Fischer AH, Chadee DN, Wright JA, Gansler TS, Davie JR (1998) Ras-associated nuclear struc-
tural change appears functionally significant and independent of the mitotic signaling pathway. J
Cell Biochem 70: 130–140
86 Samuel SK, Minish TM, Davie JR (1997) Altered nuclear matrix protein profiles in oncogene
transformed fibroblasts exhibiting high metastatic potential. Cancer Res 57: 147–151
87 Davie JR, Samuel SK, Spencer VA, Holth LT, Chadee DN, Peltier CP, Sun J-M, Chen HY, Wright
JA (1999) Organization of chromatin in cancer cells: role of signalling pathways. Biochem Cell
Biol 77: 265–275
88 Laitinen J, Saris P, Holtta E (1995) DNA methylation is not involved in the structural alterations
of ornithine decarboxylase or total chromatin of c-Ha-rasVal 12 oncogene-transformed NIH-3 T3
fibroblasts. J Cell Biochem 57: 670–679
89 Davie JR, Chadee DN (1998) Regulation and regulatory parameters of histone modifications. J
Cell Biochem 30/31: 203–213
Abnormalities of chromatin in tumor cells 45
90 Berger SL (2002) Histone modifications in transcriptional regulation. Curr Opin Genet Dev 12:
142–148
91 Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H, Sen S, Allis CD, Sassone-Corsi P
(2002) Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora
kinases. Mol Cell Biol 22: 874–885
92 Davie JR (2003) MSK1 and MSK2 mediate mitogen- and stress-induced phosphorylation of his-
tone H3: a controversy resolved. Sci STKE 2003: E33
93 Drobic B, Espino PS, Davie JR (2005) MSK1 activity and histone H3 phosphorylation in onco-
gene-transformed mouse fibroblasts. Cancer Res 64: 9076–9079
94 Chadee DN, Peltier CP, Davie JR (2002) Histone H1(S)-3 phosphorylation in Ha-ras oncogene-
transformed mouse fibroblasts. Oncogene 21: 8397–8403
95 Yamamoto H, Monden T, Miyoshi H, Izawa H, Ikeda K, Tsujie M, Ohnishi T, Sekimoto M,
Tomita N, Monden M (1998) Cdk2/cdc2 expression in colon carcinogenesis and effects of
cdk2/cdc2 inhibitor in colon cancer cells. Int J Oncol 13: 233–239
96 Mihara M, Shintani S, Nakahara Y, Kiyota A, Ueyama Y, Matsumura T, Wong DT (2001)
Overexpression of CDK2 is a prognostic indicator of oral cancer progression. Jpn J Cancer Res
92: 352–360
97 Meraldi P, Honda R, Nigg EA (2004) Aurora kinases link chromosome segregation and cell divi-
sion to cancer susceptibility. Curr Opin Genet Dev 14: 29–36
98 Katayama H, Brinkley WR, Sen S (2003) The Aurora kinases: role in cell transformation and
tumorigenesis. Cancer Metastasis Rev 22: 451–464
99 Goto H, Yasui Y, Nigg EA, Inagaki M (2002) Aurora-B phosphorylates histone H3 at serine28
with regard to the mitotic chromosome condensation. Genes Cells 7: 11–17
100 Wei Y,Yu L, Bowen J, Gorovsky MA, Allis CD (1999) Phosphorylation of histone H3 is required
for proper chromosome condensation and segregation. Cell 97: 99–109
101 Dutertre S, Descamps S, Prigent C (2002) On the role of aurora-A in centrosome function.
Oncogene 21: 6175–6183
102 Bolton MA, Lan W, Powers SE, McCleland ML, Kuang J, Stukenberg PT (2002) Aurora B kinase
exists in a complex with survivin and INCENP and its kinase activity is stimulated by survivin
binding and phosphorylation. Mol Biol Cell 13: 3064–3077
103 Ota T, Suto S, Katayama H, Han ZB, Suzuki F, Maeda M, Tanino M, Terada Y, Tatsuka M (2002)
Increased mitotic phosphorylation of histone H3 attributable to AIM-1/Aurora-B overexpression
contributes to chromosome number instability. Cancer Res 62: 5168–5177
104 Harrington EA, Bebbington D, Moore J, Rasmussen RK, Ajose-Adeogun AO, Nakayama T,
Graham JA, Demur C, Hercend T, Diu-Hercend A et al. (2004) VX-680, a potent and selective
small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo.Nat Med 10:
262–267
105 Davie JR (2004) Histone modifications. In: J Zlatanova, S Leuba (eds): Chromatin structure and
dynamics: state-of-the-art. Elsevier, Amsterdam, 205–240
106 Workman JL, Abmayr SM (2004) Histone H3 variants and modifications on transcribed genes.
Proc Natl Acad Sci USA 101: 1429–1430
107 Cremer M, Zinner R, Stein S, Albiez H, Wagler B, Cremer C, Cremer T (2004) Three dimen-
sional analysis of histone methylation patterns in normal and tumor cell nuclei. Eur J Histochem
48: 15–28
108 Liang G, Lin JC, Wei V, Yoo C, Cheng JC, Nguyen CT, Weisenberger DJ, Egger G, Takai D,
Gonzales FA, Jones PA (2004) Distinct localization of histone H3 acetylation and H3–K4 methy-
lation to the transcription start sites in the human genome. Proc Natl Acad Sci USA 101:
7357–7362
109 Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, Reinberg D, Jenuwein T (2004)
A silencing pathway to induce H3–K9 and H4 –K20 trimethylation at constitutive heterochro-
matin. Genes Dev 18: 1251–1262
110 Li Y, Kirschmann DA, Wallrath LL (2002) Does heterochromatin protein 1 always follow code?
Proc Natl Acad Sci USA 99 Suppl 4: 16462–16469
111 Nielsen SJ, Schneider R, Bauer UM, Bannister AJ, Morrison A, O’Carroll D, Firestein R, Cleary
M, Jenuwein T, Herrera RE, Kouzarides T (2001) Rb targets histone H3 methylation and HP1 to
promoters. Nature 412: 561–565
112 Cao R, Zhang Y (2004) The functions of E(Z)/EZH2-mediated methylation of lysine 27 in his-
tone H3. Curr Opin Genet Dev 14: 155–164
46 B. Drobic et al.
113 Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, Yagyu R, Nakamura Y (2004)
SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat
Cell Biol 6: 731–740
114 Verma M, Srivastava S (2002) Epigenetics in cancer: implications for early detection and pre-
vention. Lancet Oncol 3: 755–763
115 Brown R, Strathdee G (2002) Epigenomics and epigenetic therapy of cancer. Trends Mol Med 8:
S43–S48
116 Kondo Y, Shen L, Issa JP (2003) Critical role of histone methylation in tumor suppressor gene
silencing in colorectal cancer. Mol Cell Biol 23: 206–215
117 Espada J, Ballestar E, Fraga MF, Villar-Garea A, Juarranz A, Stockert JC, Robertson KD, Fuks
F, Esteller M (2004) Human DNA methyltransferase 1 is required for maintenance of the histone
H3 modification pattern. J Biol Chem 279: 37175–37184
118 Stirzaker C, Song JZ, Davidson B, Clark SJ (2004) Transcriptional gene silencing promotes DNA
hypermethylation through a sequential change in chromatin modifications in cancer cells. Cancer
Res 64: 3871–3877
119 Fahrner JA, Eguchi S, Herman JG, Baylin SB (2002) Dependence of histone modifications and
gene expression on DNA hypermethylation in cancer. Cancer Res 62: 7213–7218
120 Moore SC, Jason L, Ausio J (2002) The elusive structural role of ubiquitinated histones. Biochem
Cell Biol 80: 311–319
121 Davie JR, Murphy LC (1990) Level of ubiquitinated histone H2B in chromatin is coupled to
ongoing transcription. Biochemistry 29: 4752–4757
122 Vassilev AP, Rasmussen HH, Christensen EI, Nielsen S, Celis JE (1995) The levels of ubiquiti-
nated histone H2A are highly upregulated in transformed human cells: partial colocalization of
uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. J Cell Sci 108: 1205–1215
123 Okawa Y, Takada K, Minami J, Aoki K, Shibayama H, Ohkawa K (2003) Purification of N-ter-
minally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable
proteolytic products of ubiquitinated H2A. Int J Biochem Cell Biol 35: 1588–1600
124 Parseghian MH, Hamkalo BA (2001) A compendium of the histone H1 family of somatic sub-
types: an elusive cast of characters and their characteristics. Biochem Cell Biol 79: 289–304
125 Lennox RW, Cohen LH (1988) The production of tissue-specific histone complements during
development. Biochem Cell Biol 66: 636–649
126 Tan KB, Borun TW, Charpentier R, Cristofalo VJ, Croce CM (1982) Normal and neoplastic
human cells have different histone H1 compositions. J Biol Chem 257: 5337–5338
127 Giancotti V, Bandiera A, Ciani L, Santoro D, Crane-Robinson C, Goodwin GH, Boiocchi M,
Dolcetti R, Casetta B (1993) High-mobility-group (HMG) proteins and histone H1 subtypes
expression in normal and tumor tissues of mouse. Eur J Biochem 213: 825–832
Abnormalities of chromatin in tumor cells 47
