Human Pathology (2012) 43, 834–842

www.elsevier.com/locate/humpath

Original contribution

Global histone modification of histone H3 in colorectal

cancer and its precursor lesions☆,☆☆
Tadao Nakazawa MD, PhD a,⁎, Tetsuo Kondo MD, PhD a , Defu Ma MD, PhD a ,
Dongfeng Niu MD a , Kunio Mochizuki MD, PhD a , Tomonori Kawasaki MD, PhD a ,
Tetsu Yamane MD, PhD a , Hiroshi Iino MD, PhD b , Hideki Fujii MD, PhD b ,
Ryohei Katoh MD, PhD a
a
Department of Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Yamanashi 409-3898, Japan
b
Department of Surgery, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi,
Yamanashi 409-3898, Japan

Received 6 April 2011; revised 11 July 2011; accepted 21 July 2011

Keywords: Summary Chromatin remodeling through histone modification is an important mechanism of epigenetic
Global histone gene dysregulation in human cancers. However, little is known about global alteration of histone status
modification; during tumorigenesis and cancer progression. Histone H3 status was examined in benign and malignant
Histone H3; colorectal tumors by immunohistochemistry and Western blotting. For immunohistochemical evaluation,
Immunohistochemistry; 4 anti-histone H3 antibodies, specific to dimethylation at lysine 4 (H3K4me2), acetylation at lysine
Western blotting; 9 (H3K9ac), dimethylation at lysine 9 (H3K9me2), and trimethylation at lysine 27 (H3K27me3), were
Colorectal tumor; used. On immunohistochemistry, H3K4me2, H3K9ac, and H3K27me3 showed no significant changes
Tubular adenoma; between normal and colorectal tumors. On the other hand, the global level of H3K9me2 was distinctly
Adenocarcinoma higher in neoplastic cells (adenoma and adenocarcinoma) than in normal glandular cells. In addition, it was
significantly higher in adenocarcinoma than in adenoma. Correspondingly, Western blotting confirmed
that H3K9me2 expression was significantly higher in adenocarcinomas than in normal colorectal mucosa.
No alteration of H3K9me2 was observed with tumor differentiation and with the histological subtypes of
colorectal cancers. These results suggest that aberration of the global H3K9me2 level is an important
epigenetic event in colorectal tumorigenesis and carcinogenesis involved with gene regulation in
neoplastic cells through chromatin remodeling.
© 2012 Elsevier Inc. All rights reserved.

1. Introduction
☆
Conflict of interest statement: We declare that we have no conflict
of interest. Colorectal cancer is one of the most common malignancies
☆☆
This work was supported by the Ministry of Education, Culture,
in the world and is the major cause of cancer-related death [1].
Sports, Science and Technology, Japan: Grant in-Aid for Young Scientists
(21790348) for T.N. In addition to environmental and alimentary factors,
⁎ Corresponding author. colorectal cancers are thought to occur via a multistep
E-mail address: tadaon@yamanashi.ac.jp (T. Nakazawa). process, resulting in the accumulation of numerous genetic

0046-8177/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.humpath.2011.07.009
Global histone modification of histone H3 in colorectal cancer 835

Table 1 Immunohistochemical results of H3K9 in normal and neoplastic colorectal tissues

Histology n IHC score (H3K9me2) IHC score (H3K9ac)
Negative Low Moderate High Negative Low Moderate High
Normal colon mocusa 85 0 57 (67.1%) 21 (24.7%) 7 (8.2%) ⁎⁎ , ⁎⁎⁎ 0 0 23 (27.1%) 62 (72.9%)
Tubular adenoma 25 0 9 (36%) 10 (40%) 6 (24% ) ⁎ 0 0 9 (36%) 16 (64%)
Adenocarcinoma 60 0 0 11 (18.3%) 49 (81.7%) 0 0 17 (28.3%) 43 (71.7%)
⁎ P b .001 (adenoma vs adenocarcinoma).
⁎⁎ P = .055 (normal vs adenoma).
⁎⁎⁎ P b .001 (normal vs adenocarcinoma).

and epigenetic alterations, including activation of oncogenes 2. Material and methods

and the inactivation of tumor suppressor genes [2-4].
Epigenetic events include DNA methylation and histone 2.1. Human colonic tissues
modifications. Histone protein, around which DNA is
wrapped, can be chemically modified in residues by the
A total of 56 surgically and 33 endoscopically resected
addition of acetyl, methyl, phospholyl, or other groups.
specimens of colorectal tumors from 89 Japanese patients was
Histone modification is mainly accounted for by acetylation
studied. Specimens were collected from the pathological files
and methylation of histone core tails, which mostly occurs at
of Yamanashi University Hospital. Informed consent had
lysine or arginine residues of NH2 termini. Histone been obtained before resection in all patients. The materials
modification plays an important role in reversible regulation
consisted of 25 tubular adenomas and 60 adenocarcinomas,
of chromatin dynamics [5]. In addition, it is known that
including 14 well-differentiated adenocarcinomas, 18 mod-
histone modification acts as an activator or suppressor of
erately differentiated adenocarcinomas, 14 poorly differenti-
gene transcription through chromatin remodeling [6].
ated adenocarcinomas, 14 mucinous adenocarcinomas, and 4
Generally, condensed chromatin (heterochromatin) is asso-
adenocarcinomas accompanied by an adjacent tubular
ciated with gene inactivation, whereas sparse chromatin
adenoma component. Adenocarcinomas limited to the
(euchromatin) promotes gene transcription. This depends on
mucosa were excluded, and the adenocarcinomas showing
the site of modified residues and the type of modification (ie, invasion to layers deeper than the lamina propria were
acetylation, methylation, phosphorylation, and sumoylation),
selected. For these specimens, pathological diagnoses were
that acts on gene transcription [7].
So far, interest in epigenetic involvement in human
epithelial cancers has focused on DNA hypermethylation
of specific genes, resulting in carcinogenesis by silencing
tumor suppressor genes [8]. It has also been shown that
histone modification and DNA methylation are closely
related to each other [9]. The correlation between the
methylation status of CpG islands in the specific genes and
histone modification has been investigated mainly using
the chromatin immunoprecipitation (ChIP) assay [10,11].
Recently, global histone modification levels have been
intensively studied in cancers of various organs using
immunohistochemistry [12-21]. In these reports, global
alteration of histone modification in certain cancers has
been closely associated with patient prognosis and tumor
aggressiveness. With regard to colorectal cancers, there has
been only one report investigating global histone status and
its implications in tumorigenesis and/or carcinogenesis of the
neoplasms [21].
In the current study, to clarify the epigenetic environments Fig. 1 The results of Western blotting for H3K9me2. Pairs of
lanes (1, 5), (2, 6), (3, 7), and (4,8) are the samples obtained from
in colorectal cancers and its precursor lesions, the global
the same patients with moderately differentiated adenocarcinoma,
histone H3 status was studied in individual cell nuclei of 60
respectively. H3K9me2 expression is up-regulated in moderately
adenocarcinomas, 25 adenomas, 4 adenocarcinomas with differentiated adenocarcinomas (lanes 1-4) compared with
adenoma component, and the adjacent nonneoplastic mucosa individual-matched normal tissues (lanes 5-8), as shown by the
by immunohistochemistry with 4 specific histone markers densitometric assessments below (P = .0005). Columns, mean of
(H3K4me2, H3K9ac, H3K9me2, and H3K27me3), along densitometric values of normal colon mucosa (n = 4) or colon
with Western blotting with one histone marker (H3K9me2). carcinomas (n = 4); bars, SE.
836 T. Nakazawa et al.

Fig. 2 Representative photomicrograph and results of immunohistochemical staining for H3K9me2 in adenocarcinoma with adenoma
component. A, The adenocarcinoma component shows submucosal invasion (left side) and the adenoma component is seen continuously (right
side). B, In the adenoma component with low-grade dysplasia, weak immunoreactivity for H3K9me2 is observed in the tumor cell nuclei. C,
The immunoreactivity for H3K9me2 increases in the adenoma component showing severe nuclear overlapping and swelling and loss of
polarity (high-grade dysplasia). D, Diffuse and strong immunoreactivity for H3K9me2 is seen in the adenocarcinoma component.

made based on The World Health Organization Classifica-
tion of Tumours, Pathology and Genetics of Tumours of the
Digestive System [22]. All cases were selected for the
immunohistochemical study on the basis of availability of
routinely processed, paraffin-embedded, formalin-fixed tis-
sue blocks. Four pairs of fresh tumor and normal colorectal
tissues were obtained from the patients with moderately
differentiated adenocarcinoma for Western blotting. The
fresh tissue was immediately frozen with liquid nitrogen and
stored at −80°C immediately after surgical resection.
2.2. Immunohistochemistry
Four-micrometer-thick sections were cut from paraffin
blocks. After deparaffinization, they were steamed for 20
minutes in sodium citrate buffer (diluted to 1× from 10× heat-
induced epitope retrieval buffer). The sections were incubated
with primary rabbit polyclonal antibodies (Cell Signaling
Technology, Boston, MA) at room temperature, for dimethyl
histone H3 Lys9 (H3K9me2) (catalogue number 9753, 1:200
dilution), acetyl histone H3 Lys9 (H3K9ac) (catalogue number
9671, 1:100 dilution), dimethyl histone H3 Lys4 (H3K4me2)
(catalogue number 9725, 1:1500 dilution), and trimethyl
histone H3 Lys27 (H3K27me3) (catalogue number 9733, 1:50
dilution) for 90 minutes. They were then incubated with a goat
antirabbit biotinylated secondary antibody conjugated with
avidin-biotin peroxidase complex for another 90 minutes.
Finally, specific immunostaining was detected with 3′,3′-
diaminobenzidine tetrahydrochloride (Sigma, St Louis, MO)
and counterstained with hematoxylin. Lymph nodes in which
the absence of metastatic tumor was confirmed were stained as
positive controls and concomitantly all sections without the
primary antibodies as negative controls. The stained sections
were examined by 2 pathologists (T.N. and T.K.), who agreed
in all cases.
2.3. Evaluation of immunohistochemistry
For each case, at least 500 cells were counted in both tumor
and adjacent normal colonic mucosa. Only the nuclear
immunopositivity of epithelial components was estimated.
The percentage of immunopositive cells and the intensities of
Global histone modification of histone H3 in colorectal cancer 837

H3K9me2, H3K9ac, H3K4me2, and H3K27me3 were
analyzed. The degree of positive cells was semiquantitatively
evaluated as follows: first, scoring according to intensity
(negative, 0; weak, 1; intermediate, 2; strong, 3); then, scoring
according to the percentage of positive cells (0%, 0; 1%-10%,
1; 11%-50%, 2; N50%, 3). Finally, each sample was divided
into 4 groups depending on the total score (frequency of
immunopositive cells plus staining intensity); total scores of 0,
1 to 3, 4, and 5 to 6 were classified as negative, low expression,
moderate expression, and high expression, respectively.
2.4. Western blotting
Fresh colonic tissue was homogenized using polytron
homogenizer in radioimmunoprecipitation assay buffer with
proteinase inhibitors (1× PBS, 1% Nonidet P-40, 0.5%

Fig. 3 Representative results of immunohistochemical staining for H3K9me2. A, In normal colorectal mucosa, a small number of positive cells
is observed in the nuclei of the nonneoplastic epithelium. B, Positive cells are focally observed in tubular adenoma. Most of the cancer cells
demonstrate strong positivity in well (C), moderately (D), and poorly (E) differentiated adenocarcinoma and mucinous adenocarcinoma (F).
838
Table 2 Immunohistochemical results of H3K9me2 in colorectal adenocarcinomas
Differentiation and histological subtype n IHC score (H3K9me2)
Well-differentiated adenocarcinoma 14
Moderately differentiated adenocarcinoma 18
Poorly differentiated adenocarcinoma 14
Mucinous adenocarcinoma 14
sodium deoxycholate, 0.1% SDS, 1 mmol/L phenyl-
methylsulfonyl fluoride, 12 μg/mL aprotinin, and 1 mmol/L
sodium ovarthovanadate). Equal amounts of protein (40 μg)
solubilized in sample buffer were separated on 10% SDS
polyacrylamide gels and transferred electrophoretically to a
polyvinylidene difluoride membrane. The membrane was
blocked in TBS containing 0.5% Tween 20 plus 5% nonfat
dried milk for 1 hour at room temperature and probed with a
primary antibody (primary antisera: dimethyl histone H3
Lys9, 1:1000; Cell Signaling Technology; anti-actin, 1:1000;
Sigma) at 4°C overnight. The membrane was washed thrice
for 10 minutes in TBS containing 0.5% Tween 20 and
incubated with horseradish peroxidase–conjugated anti-rabbit
secondary antibody (1:2000; Santa Cruz Biotechnology,
Santa Cruz, CA) for 1 hour at room temperature. Targeted
proteins were visualized using an enhanced detection system
(Amersham, Buckinghamshire, UK).
2.5. Statistical analysis
Statistical analysis was carried out using χ 2 tests,
comparing the frequencies of cases that had high expression
of H3K9me2 by immunohistochemistry (normal vs adenoma,
normal vs adenocarcinoma, and adenoma vs adenocarcino-
ma) (Table 1). Student t test was used to compare the Western
blots of H3K9me2 protein in paired colon carcinoma and
normal colon mucosa (Fig. 1). Statistic significance was set at
P b .05. Data analyses were carried out using SPSS version
11.0 for Windows (SPSS, Tokyo, Japan).
3. Results
3.1. H3K9me2 levels in normal, colorectal cancer,
and its precursor lesions
H3K9me2 expression in 4 moderately differentiated
adenocarcinomas was compared with that in the normal
colorectal tissue sampled from the same patients by Western
blotting. After loading an equal amount of protein, parallel
actin immunoblotting was performed, and signal quantifica-
tion was performed by densitometric scanning. The results
showed that H3K9me2 protein expression was significantly
up-regulated in all of the analyzed adenocarcinomas
compared with individual-matched normal colorectal tissues
(P = .0005) (Fig. 1).
T. Nakazawa et al.
Negative Low Moderate High
0 0 2 (14.3%) 12 (85.7%)
0 0 2 (11.1%) 16 (88.9%)
0 0 3 (21.4%) 11 (78.6%)
0 0 4 (28.6%) 10 (71.4%)
In the tubular adenomas, the immunopositivity for
H3K9me2 gradually increased by the degree of nuclear
overlapping and swelling and by loss of polarity. A similar
pattern was also observed in all 4 cases having adenocarci-
noma with adenoma component (Fig. 2). The H3K9me2
immunoreactivity showed a tendency to be higher in the
adenomas with high-grade dysplasia (Fig. 2C) than in those
with low-grade dysplasia (Fig. 2B). As seen in the tumors
consisting of adenocarcinoma alone, diffuse immunoposi-
tivity for H3K9me2 was evident in the adenocarcinoma
components (Fig. 2D).
Fig. 3 shows the immunohistochemical results for
H3K9me2 in the representative sections of the normal
colonic mucosa and the colorectal tumors. The cell nuclei
completely lacked immunoreactivity for H3K9me2 in the
negative controls. The immunohistochemical scores for
H3K9me2 are shown in Table 1. In the positive control
sections, most of the cell nuclei showed low to moderate
expression. In 85 normal colorectal tissue samples, there
were no negative cases. More than half of the cases (57/85,
67.1%) had low expression, 21 (24.7%) of 85 cases had
moderate expression, and 7 (8.2%) of 85 cases had high
expression. In 25 tubular adenomas, moderate expression
was the most frequently observed (10/25 cases, 40%),
whereas 9 (36%) and 6 (24%) of the 25 tubular adenomas
had low and high expression, respectively. In the adenocar-
cinomas, most cases (49/60, 81.7%) had high expression,
and 11 (18.3%) of 60 cases had moderate expression. The
frequency of cases with high expression was significantly
higher in the adenocarcinomas than in normal mucosa (P b
.001). The adenocarcinomas showed a significantly higher
proportion of high expression than the adenomas (P b .001).
H3K9me2 expression tended to be higher in the adenomas
than in the normal mucosa, but the difference was not
significant (P = .055).
The immunohistochemical results for H3K9me2 in
well, moderately, and poorly differentiated adenocarci-
nomas and mucinous adenocarcinoma are shown in
Fig. 1C to F. The immunohistochemical scores of
H3K9me2 in the adenocarcinomas by differentiation and
histological subtypes are shown in Table 2. In the
adenocarcinomas, 12 (85.7%), 16 (88.9%), and 11
(78.6%) cases had high expression in well, moderately
differentiated, and poorly differentiated types, respectively.
Less frequently, moderate expression was demonstrated in 2
well (14.3%), 2 moderately (11.1%), and 3 poorly (21.4%)
Global histone modification of histone H3 in colorectal cancer 839

differentiated adenocarcinomas. Of 14 mucinous adenocar-
cinomas, 4 had moderate expression (28.6%), and 10 showed
high expression (71.4%). In the adenocarcinomas, H3K9me2
was highly expressed regardless of differentiation and
histological subtype.
3.2. H3K9ac, H3K4me2, and H3K27me3 levels in
normal, colorectal cancer, and its precursor lesions
Fig. 4 shows the immunohistochemical results for
H3K9ac, H3K4me2, and H3K27me3 in normal colorectal

Fig. 4 Representative immunohistochemical results for H3K9ac, H3K4me2, and H3K27me3 in the normal and neoplastic nuclei. A,B,
Representative immunohistochemical results for H3K9ac. Diffuse immunopositivity is observed in normal mucosa (A) and moderately
differentiated adenocarcinoma (B). C,D, Representative immunohistochemical results for H3K4me2. Positive cells were focally observed with
mild to moderate intensity in normal colorectal mucosa (C) and moderately differentiated adenocarcinoma (D). E,F, Representative
immunohistochemical results for H3K27me3. Immunopositivity is diffusely observed in normal colorectal mucosa (E) and moderately
differentiated adenocarcinoma (F).
840
Table 3 Immunohistochemical results of H3K4me2 and H3K27me3 in normal and neoplastic colorectal tissues
Histology n IHC score (H3K4me2)
Negative Low Moderate
Normal colon mocusa 85 0 54 (63.5%) 28 (32.9%)
Tubular adenoma 25 0 15 (60%) 9 (36%)
Adenocarcinoma 60 0 37 (61.7%) 20 (33.3%)
mucosa, cancer, and its precursor lesions. The cell nuclei
showed no immunoreactivity for H3K9ac, H3K4me2, and
H3K27me3 in the negative controls. The immunohistochem-
ical scores for H3K9ac in normal colorectal mucosa are
shown in Table 1; moderate expression was observed in 23
(27.1%) of 85, and high expression was seen in 62 (72.9%) of
85. In the positive controls, most of the cell nuclei showed
moderate expression. Negativity and low expression were
found in none of the normal glandular cell nuclei. Similar
patterns were observed both in tubular adenomas and
adenocarcinomas. There was no significant difference
among these 3 groups.
Similarly, no significant differences in the scores of
H3K4me2 and H3K27me3 were seen between the cell
nuclei of the normal and the neoplastic colorectal tissue
(Table 3). The expression levels were equivalent to those of
the positive controls.
There were no significant differences in H3K9ac,
H3K4me2, and H3K27me3 scores among well, moderately,
and poorly differentiated adenocarcinomas, mucinous ade-
nocarcinoma, and adenocarcinoma with adenoma component
(data not shown).
4. Discussion
Histone modification and DNA methylation are
regarded as important epigenetic events that regulate
gene transcription [23]. Recently, there have been several
reports investigating global alteration of histone modifica-
tion in human epithelial cancers by immunohistochemistry
using specific histone markers, but these immunohisto-
chemical analyses do not provide a correlation to specific
gene activities. Nevertheless, they allow us to visualize the
global status of histone modification at a cellular level.
Previous reports studying the global pattern of histone
modification focused on the clinical prognosis and
suggested that global aberration of histone modification
could closely relate to patient prognosis and serve as an
accurate indicator [12-21]. However, little has been said
about how the global state of histone modification can be
changed at a cellular level in tumorigenic and/or
carcinogenic processes in human cancers. Consequently,
to clarify epigenetic events in colorectal cancer and its
precursor lesions, histone H3 status was examined using 4
T. Nakazawa et al.
IHC score (H3K27me3)
High Negative Low Moderate High
3 (3.5%) 0 0 30 (35.3%) 55 (64.7%)
1 (4%) 0 0 6 (24%) 19 (76%)
3 (5%) 0 0 23 (38.3%) 37 (61.7%)
specific histone markers by comparing neoplastic cells to
normal cells.
To the best of our knowledge, there has been only one
report studying global histone modification in colorectal
neoplasms. Ashktorab et al [21] reported that global levels
of H4K12ac and H3K18ac increased in adenocarcinomas
in comparison with those in normal tissue and adenomas
using immunohistochemistry. They also demonstrated that
HDAC2 and H4K12ac expressions in adenocarcinoma
were higher than in adenoma, implying that these
epigenetic changes have a role in the progression from
adenoma to adenocarcinoma.
In the present study, the global level of H3K9me2
expression was higher in neoplastic cells than in normal
glandular cells on immunohistochemistry. Interestingly,
H3K9me2 expression was up-regulated in the adenomas,
which are recognized as precancerous lesions, with high-
grade dysplasia compared with those with low-grade
dysplasia. In addition, H3K9me2 expression was also
significantly increased in the nuclei of adenocarcinoma cells
compared with that in the nuclei of adenoma cells. These
results may suggest that the increased H3K9me2 expression is
associated with progression (adenoma to adenocarcinoma). It
seems preferable to separate adenoma with high-grade
dysplasia from that with low-grade dysplasia and adenoma
with high-grade adenoma from well-differentiated adenocar-
cinoma. However, it is actually difficult to discriminate
between these tumors strictly.
In the present study, it was confirmed that the amount of
H3K9me2 protein was greater in the adenocarcinoma tissue
than in the normal colorectal tissues by immunohistochem-
istry and Western blot. Seligson et al [17] reported that a
low level of H3K9me2 is significantly associated with
aggressive phenotypes of prostatic and renal cancers and
that this can independently predict poor survival. However,
in the current study, the cellular global H3K9me2 levels
were not altered regardless of the tumor differentiation and
the histological subtypes of colorectal adenocarcinomas. It
has been generally recognized that poorly differentiated
adenocarcinoma and mucinous adenocarcinoma are posi-
tively correlated with poor patient clinical outcomes.
Therefore, the global H3K9me2 state seems to be less
relevant in more aggressive colorectal cancers leading to
poor clinical outcomes.
Although it is difficult to interpret the effects, these
findings suggest that global alteration of H3K9me2
Global histone modification of histone H3 in colorectal cancer
expression may correlate with genetic alteration and
instability involving tumorigenesis and/or carcinogenesis
in colorectal adenoma and adenocarcinoma, in conjunction
with other epigenetic events. Previous reports have
demonstrated that methylation of histone H3K9 is asso-
ciated with gene repression through sparse chromatin
(heterochromatin) [7,24]. From this aspect, it is conceivable
that the increased H3K9me2 level may repress transcrip-
tional activity of certain genes that function as tumor
suppressors and/or carcinostasis promoters in colorectal
tumors. Conversely, it can also raise the possibility that
increased H3K9me2 inactivates genes that suppress tumor
cell proliferation after tumorigenesis and/or carcinogenesis
as negative feedback. Quantitative analyses for the
correlation with specific genes using other molecular
methods (ie, ChIP assay in combination with real-time
PCR) will be necessary to determine which hypotheses
provide a correct explanation.
With respect to histone H3 modifications at other
residues, the global states of H3K9ac, H3K4me2, and
H3K27me3 have been investigated with specific histone
markers in a variety of cancers other than colorectal
cancers. In more detail, low H3K9ac has been reported to
be related to a poor prognosis in lung and breast cancer
patients [12,13]. On the other hand, a high level of H3K9ac
has been associated with the aggressive type of prostatic
cancers [14]. Low H3K4me2 was observed in prostatic
cancers with a high risk of recurrence [17]. In kidney
cancers, decreased expression of H3K4me1-3 was corre-
lated with shorter survival [19]. Low expression of
H3K27me3 was positively related to better patient
prognosis in the early stage of esophageal squamous cell
carcinomas [20]. In the present study, there were no
significant differences in the expression levels of H3K9ac,
H3K4me2, and H3K27me3 between normal and neoplas-
tic cell nuclei in the colorectal tumors. In addition, these
levels were stable, irrespective of the degree of differen-
tiation and the histological subtypes of colorectal adeno-
carcinomas (data not shown). As previously reported,
some patterns of global histone modification are interest-
ingly common among cancers of different organ origins
[17]. Nevertheless, the present results show that a similar
pattern could not be observed in the colorectal cancers
for the previously investigated patterns of histone modi-
fication (H3K9ac, H3K4me2, and H3K27me3) in the
other cancers.
In summary, global H3K9me2 expression was verified to
be up-regulated, whereas there were no differences in
H3K9ac, H3K4me2, and H3K27me3 levels in colorectal
tumors. The H3K9me2 expression levels were significantly
higher in adenocarcinoma cells than in adenoma cells.
Among adenocarcinomas, H3K9me2 expression was
unchanged by differentiation and histological subtype.
Aberrant H3K9me2 expression may play a role in tumori-
genesis and progression (adenoma-carcinoma sequence),
rather than in transformation to more aggressive pheno-
841
types of colorectal cancers, although the correlation of this
epigenetic event to specific genes still remains unclear.
From a practical perspective, diffuse H3K9me2 immuno-
positivity can serve as a useful tool for pathological diag-
nosis in differentiating between tubular adenoma and
adenocarcinoma. Furthermore, it raises the possibility that
H3K9me2 is a potential pharmacological therapeutic target
for colorectal cancers.
Acknowledgments
The authors would like to thank Miyuki Ito, Mikiko
Yoda, and especially Yoshihito Koshimizu for their ex-
cellent technical assistance and Kayoko Kono for
executive assistance.
References
[1] Imamura Y, Sobue T. Cancer statistics digest. Mortality trend of colon,
rectal, liver, “gallbladder and biliary tract” and pancreas cancer in
Japan by birth cohort. Jpn J Clin Oncol 2004;34:491-3.
[2] Ashktorab H, Smoot DT, Carethers JM, et al. High incidence of
microsatellite instability in colorectal cancer from African Americans.
Clin Cancer Res 2003;9:1112-7.
[3] Ashktorab H, Smoot DT, Farzanmehr H, et al. Clinicopathological
features and microsatellite instability (MSI) in colorectal cancers from
African Americans. Int J Cancer 2005;116:914-9.
[4] Carethers JM. Racial and ethnic factors in the genetic pathogenesis of
colorectal cancer. J Assoc Acad Minor Phys 1999;10:59-67.
[5] Marusige K. Activation of chromatin by acetylation of histone side
chains. Proc Natl Acad Sci USA 1976;73:3937-41.
[6] Vidali G, Ferrari N, Pfeffer U. Histone acetylation: a step in gene
activation. Adv Exp Med Biol 1988;231:583-96.
[7] Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:
1074-80.
[8] Esteller M. Epigenetic gene silencing in cancer: the DNA hyper-
methylene. Hum Mol Genet 2007;16:R50-9.
[9] Kurdistani SK. Histone modifications as markers of cancer prognosis:
a cellular view. Br J Cancer 2007;97:1-5.
[10] Kondo Y, Shen L, Issa JP. Critical role of histone methylation in tumor
suppressor gene silencing in colorectal cancer. Mol Cell Biol 2003;23:
206-15.
[11] Fujii S, Luo RZ, Yuan J, et al. Reactivation of the silenced and
imprinted alleles of ARHI is associated with increased histone H3
acetylation and decreased histone H3 lysine 9 methylation. Hum Mol
Genet 2003;12:1791-800.
[12] Elsheikh SE, Green AR, Rakha EA, et al. Global histone modifications
in breast cancer correlate with tumor phenotypes, prognostic factors,
and patient outcome. Cancer Res 2009;69:3802-9.
[13] Barlési F, Giaccone G, Gallegos-Ruiz MI, et al. Global histone
modifications predict prognosis of resected non–small-cell-lung
cancer. J Clin Oncol 2007;25:4358-64.
[14] Sligson DB, Horvath S, Shi T, et al. Global histone modification
patterns predict risk of prostate cancer recurrence. Nature 2005;435:
1262-6.
[15] Ellinger J, Kahl P, von der Gathen J, et al. Global levels of histone
modifications predict prostate cancer recurrence. Prostate 2010;70:
61-9.
842
[16] Park YS, Jin MY, Kim YJ, Yook JH, Kim BS, Jang SJ. The global
histone modification pattern correlates with cancer recurrence and
overall survival in gastric adenocarcinomas. Ann Surg Oncol 2008;15:
1968-76.
[17] Seligson DB, Horvath S, McBrian MA, et al. Global levels of histone
modifications predict prognosis in different cancers. Am J Pathol
2009;174:1619-28.
[18] Minardi D, Lucarini G, Filosa A, et al. Prognostic role of global
DNA-methylation and histone acetylation in pT1a clear cell renal
carcinoma in partial nephrectomy specimens. J Cell Mol Med 2009;
13:2115-21.
[19] Ellinger J, Kahl P, Mertens C, et al. Prognostic relevance of global
histone H3 lysine 4 (H3K4) methylation in renal cell carcinoma. Int J
Cancer 2010;127:2360-6.
T. Nakazawa et al.
[20] Tzao C, Tung HJ, Jin JS, et al. Prognostic significance of global
histone modifications in resected squamous cell carcinoma of the
esophagus. Mod Pathol 2009;22:252-60.
[21] Ashktorab H, Belgrave K, Hosseinkhah F, et al. Global histone H4
acetylation and HDAC2 expression in colon adenoma and carcinoma.
Dig Dig Sci 2009;54:2109-17.
[22] Bosman FT, Carneiro F, editors. Pathology and genetics of tumours of
the digestive system. The World Health Organization Classification of
Tumours. Lyon: IARC Press; 2010. p. 131-82.
[23] Gronbaek K, Hother C, Jones PA. Epigenetic changes in cancer.
APMIS 2007;115:1039-59.
[24] Rea S, Eisenhaber F, O'Carroll D, et al. Regulation of chromatin
structure by site-specific histone H3 methyltransferases. Nature 2000;
406:593-9.