Yonago Acta medica 2010;53:1–7

Expression of DNA Methyltransferase (DNMT) 1, 3a and 3b
Proteins in Human Hepatocellular Carcinoma
Takanori Miyake, Kanenori Endo, Soichiro Honjo, Yasuaki Hirooka and Masahide
Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8504, Japan

Alteration of aberrant DNA methylation is one of the most consistent epigenetic changes
found in human cancers. DNA methylation is catalyzed by DNA methyltransferase(DNMT). In this study, we examined DNMT protein expression by immunohistochemistry in surgically resected hepatocellular carcinomas (HCCs). Sections of paraffin-embedded specimens were obtained from 95 patients with HCC between 1989 and 2002. The
specimens were stained with anti-DNMTs (DNMT1, DNMT3a and DNMT3b) antibodies.
There were statistically significant associations between DNMT protein expression andtumor differentiation (P < 0.05) and intrahepatic metastasis (P < 0.05). DNMT3a protein
expression was significantly correlated with portal vein involvement of tumors (P < 0.05).
The overall survival rates of patients with DNMT3a-positive HCCs and DNMT3b-positive
HCCs were significantly lower than those of patients negative for these proteins (P < 0.005,
respectively). To further evaluate the correlation between DNMT protein expression andpatient survival, we classified patients into 3 groups: Group 1, DNMT1(+), 3a(–) and 3b(–);
Group 2) DNMT1(+), 3a or 3b(+); and Group 3) DNMT1(+), 3a(+) and 3b(+). The overall survival rate of patients in Group 3 was significantly lower than those of patients inGroups 1 and 2 (P = 0.0009). In conclusion, the results of this study suggest that DNMT1,
DNMT3a and DNMT3b are cooperatively involved in determining the extent of HCCs,
and that DNMT protein overexpression in HCCs may be a predictive factor for poor survival.
Key words: DNA methyltransferase; hepatocellular carcinoma; immunohistochemistry; prognosis

DNA methylation plays an important role in theregulation of gene expression, and gene silencing
by aberrant hypermethylation of CpG islands ingene promoter regions frequently occurs in humancancers. DNA methyltransferases are involvedin the transfer of methyl groups to promoter CpG
islands, and many tumor suppressor genes canbe silenced by promoter CpG island methylation.
Alteration of aberrant DNA methylation is one of
the most consistent epigenetic changes found in hu-

man cancers (Girault et al., 2003; Ding et al., 2008;
Nosho et al., 2009).
DNA methyltransferases have been reported
to be encoded by 3 distinct families of DNMT
genes: DNMT1, DNMT2, and DNMT3. DNMT1
is the best known DNMT involved in the maintenance of methylation, while DNMT3 is involved
in de novo methylation and is encoded by 2 distinct genes, DNMT3a and DNMT3b (Łuczak andJagodzinski, 2006). The function of DNMT2

Abbreviations: CH, chronic hepatitis; DNMT, DNA methyltransferase; HCC, hepatocellular carcinoma; LC, liver cirrhosis; siRNA, small interfering RNA

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T. Miyake et al.

Table 1. Relationship between DNMTs and tissue specimensPatientsn		Immunostaining score
DNMT3a

DNMT1

DNMT3bAge (year)							
< 6039 2.179 ± 0.721
NS
1.897 ± 0.882
NS
1.205 ± 0.656
NS≥ 60
56 2.357 ± 0.586		
1.786 ± 0.847		
1.232 ± 0.831
Sex							
Male76 2.263 ± 0.640
NS1.776 ± 0.873
NS
1.145 ± 0.905
NS
Female19 2.368 ± 0.684		
2.053 ± 0.800		
1.526 ± 0.905
Background of cancerous liver tissue							Chronic hepatitis
27 2.370 ± 0.655
NS
1.556 ± 1.013
NS
1.000 ± 0.679
NS
Liver cirrhosis
68 2.250 ± 0.629		
1.941 ± 0.770		
1.309 ± 0.778
Tumor differentiation							Well differentiated19 1.842 ± 0.765 P < 0.05
1.421 ± 0.769 P < 0.05
0.895 ± 0.657 P < 0.05
Moderately differentiated
59 2.339 ± 0.633		
1.814 ± 0.880		
1.254 ± 0.779
Poorly differentiated
17 2.471 ± 0.624		
2.235 ± 0.831		
1.412 ± 0.870
Portal vein involvement of tumor							Positive
51 2.314 ± 0.678
NS2.000 ± 0.849 P < 0.05
1.294 ± 0.782
NS
Negative
44 2.250 ± 0.615		
1.636 ± 0.838		
1.136 ± 0.734
Pathological findings Fc-inf							Positive
68 2.319 ± 0.675
NS1.870 ± 0.873
NS
1.188 ± 0.791
NS
Negative
27 2.192 ± 0.567		
1.731 ± 0.827		
1.308 ± 0.679
Intrahepatic metastasis							Positive
28 2.571 ± 0.504 P < 0.05
2.143 ± 0.803 P < 0.05
1.464 ± 0.693 P < 0.05
Negative
67 2.164 ± 0.665		
1.701 ± 0.853		
1.119 ± 0.769
Tumor size							≤ 20 mm
22 2.136 ± 0.640
NS1.864 ± 0.774
NS
1.273 ± 0.767
NS> 20 mm or < 50 mm
50 2.400 ± 0.670		
1.800 ± 0.948		
1.180 ± 0.800≥ 50 mm
23 2.087 ± 0.733		
1.783 ± 0.723		
1.217 ± 0.736
n, number of patients.
Fc-inf, infiltration to the capsule; NS, not significant.

remains to be determined. Interactions among
DNMTs and methyl CpG-binding domain proteinsshould also be considered as a mechanism for
maintaining DNA methylation patterns using theproofreading or remethylation function (Hattori etal., 2004).
Aberrant hypermethylation of CpG islands
has recently been implicated in hepatocarcinogenesis, and it has been reported that tumor suppressor
genes in hepatocellular carcinomas (HCCs) are
affected by such silencing (Saito et al., 2001; Yanget al., 2003). To study the significance of aberrant
DNMT (DNMT1, 3a and 3b) expression in human
HCC, we evaluated the correlation between DNMT
protein expression and clinicopathologic featuresand prognosis in patients with HCC.

Materials and Methods
Patients and samples
Stored HCC and corresponding non-cancerous tissue specimens from our laboratory were obtainedfor 95 patients from 1989 to 2002. The protocol was approved by the ethics committee of the
School of Medicine, Tottori University (1040). Informed consent was obtained from all patients.
The clinicopathologic features of the 95 patients are shown in Table 1. A mean age was 61± 10 years (range, 17–80 years). For comparison,
normal liver tissues were also obtained from 3
patients who had undergone surgical resection forhepaticolithiasis.
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DNMT protein expression in HCC

Immunohistochemistry

Statistical analysis

Formalin-fixed, paraffin-embedded tissues were cut
into 4-μm-thick continuous sections and mounted
on poly-L-lysine-coated glass slides. Slides weredeparaffinized in xylene, rehydrated in graded alcohols and washed in tap water. Endogenous peroxidase activity was blocked by incubating sections
in 3% H2O2. Slides were heated in a microwave at
600 W for 15 min with 10 mM sodium citrate buffer (pH 6.0) and treated with polyoxyethylene sorbitan monolaurate (Tween 20) for 30 min for antigen
retrieval. Rabbit polyclonal antibodies for DNMT1(H-300, sc-20701, dilution 1:200), DNMT3a (H-295,
sc-20703, dilution 1:200) and DNMT3b (H-230,
sc-20704, dilution 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) were applied overnight, followed by incubation with biotinylated secondary
antibodies [anti-rabbit IgG, N-Histofine, Simple
Stain MAX PO (MULTI)] at room temperature for
30 min. Antigen-antibody complexes were visualized using a streptavidin-horseradish peroxidase
conjugate (LSAB kit; Dako, Carpinteria, CA) withdiaminobenzidine as a chromogen. Slides werecounterstained with Harris’ hematoxylin and methylgreen for 3 to 5 s.

Statistical analysis was performed using Prism software (GraphPad Software, La Jolla, CA). Spearman’s rank correlation test and Student’s t-test wereused to study the relationships between DNMT immunoreactivities and various clinicopathologic parameters. Overall survival rates were determinedby the Kaplan-Meier method, and the significanceof differences between survival rates was studied
using the log-rank test. Differences or correlations
with P values < 0.05 were considered significant.
Positive DNMT expression was defined as moderate (2+) or strong (3+), and negative DNMT expression as absent (–) or weak (1+) expression. To further evaluate the correlation between DNMT protein expression and patient survival, we subdivided
the patients into 3 groups: Group 1) DNMT1(+),3a(–) and 3b(–); Group 2) DNMT1(+), 3a or 3b(+);
and Group 3) DNMT1(+), 3a(+) and3b (+).

Results
The immunohistochemical staining results areshown in Table 2 and Fig. 1. The non-cancerous
liver tissues showed no expression of nuclear
DNMT proteins by immunohistochemistry. There
were no differences in cytoplasmic DNMT expres-Scoring of immunoreactivity
Two independent observers (T.M. and K.E.) independently evaluated the immunostaining without
knowledge of the clinical outcomes; the concordance ratio was > 90%. When differences in scoring were encountered, a consensus of two was used
to obtain a final decision.
Nuclear positivities for the 3 proteins were
evaluated using the scoring system previously reported by Choi et al. (2003), according to staining
intensity (0, negative; 1, weak; 2, moderate; and 3,
severe) and proportion of positive cells (0, negative;
1, positive in ≤ 10%; 2, positive in > 10% and ≤33%; 3, positive in > 33% and ≤ 66%; 4, positive in> 66% of cells). The 2 scores were added in eachcase, and the expression was graded as – (0), 1+ (1or 2), 2+ (3 to 5) and 3+ (6 or 7).

Table 2. Expression of DNMT 1, 3a and 3b proteins in patients with hepatocellular carcinoma
–
n (%)
DNMT1
0 (0)
DNMT3a 8 (8.4)
DNMT3b 14 (14.7)

DNMT protein expression
1+
2+
3+
n (%)
n (%)
n (%)
10 (10.5)
20 (21.1)
51 (53.7)

50 (52.6)
48 (50.5)
25 (26.3)

35 (36.8)
19 (20.0)
5 (5.2)n, number of patients: total number, 95.
Nuclear positivities for the 3 proteins were evaluated using
the scoring system, according to staining intensity (0, negative; 1, weak; 2, moderate; and 3, severe) and proportion of
positive cells (0, negative; 1, positive in ≤ 10%; 2, positive in> 10% and ≤ 33%; 3, positive in > 33% and ≤ 66%; 4, positive in > 66% of cells). The 2 scores are added in each case,and the expression is graded as – (0), 1+ (1 or 2), 2+ (3 to 5)
and 3+ (6 or 7).

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T. Miyake et al.

sion between patients with CH and LC (data notshown). None of the 3 normal control livers demonstrated nuclear expression of DNMTs.
We indicated the immunostaining scoresaccording to the clinicopathological findings inTable 1. There were significant associations between DNMT protein expression and tumor differentiation or intrahepatic metastasis. DNMT3a
protein expression was significantly correlated withportal vein involvement in the tumor. On the otherabcFig. 1. Immunohistochemical staining results for (a) DNMT1,(b) DNMT3a and (c) DNMT3b proteins in hepatocellular
carcinomas. Strong nuclear expression and faint cytoplasmic
expression are seen in these 3 cases. a and b: counterstainedwith Harris’ hematoxylin. c: conterstained with methylgreen.
Bar = 100 µm.

4Fig. 2. Kaplan-Meier survival curves for patientswith hepatocellular carcinomas according to DNMT
protein expression levels. [ ], number of patients.
a: DNMT 1.
b: DNMT 3a.
c: DNMT 3b.

DNMT protein expression in HCC

Fig. 3. Kaplan-Meier survival curves
were plotted for 94 patients according to the correlation with expressionof the DNMT protein complex. [ ],number of patients.
Group 1: DNMT1(+), 3a(–) and 3b(–).
Group 2: DNMT1(+), 3a or 3b(+).
Group 3: DNMT1(+), 3a(+) and 3b(+).

hand, no significant correlations were noted between DNMT1 or 3b and portal vein involvement.
There were no significant associations between
DNMT expression and age, sex, tumor size, infiltration to the capsule or underlying liver disease.
Follow-up information was available for all
95 patients, covering periods ranging from 0 to 183
months (mean, 63.7 months). Kaplan-Meier survival curves were plotted for the 95 patients according
to expression of each DNMT.
The overall survival rates of DNMT3apositive and DNMT3b-positive HCC patients weresignificantly lower than those of patients negative
for these proteins (Fig. 2b, P = 0.003, log-rank
test; Fig. 2c, P = 0.0034, log-rank test). However,
the overall survival rate of DNMT1-positive HCC
patients was not significantly different from that of
DNMT1-negative HCC cases (Fig. 2a, P = 0.1038,
log-rank test).
The overall survival rate of patients in Group
3 [DNMT1(+), 3a(+) and 3b(+)] was significantly
lower than those of patients in Groups 1 and 2(Fig. 3, P = 0.0009, log-rank test).

and frequent event, and that stepwise progressionof methylating events contributes to multistep hepatocarcinogenesis (Roncalli et al., 2002; Lee et al.,
2003; Yang et al., 2003). The levels of DNMT1,
DNMT3a and DNMT3b mRNA in HCCs are significantly higher than in noncancerous liver tissues,
including normal, chronic CH and LC tissues (Saito
et al., 2001; Oh et al., 2007).
In the present study, we used an immunohistochemical technique to directly examine the protein expression of 3 DNMTs in HCCs. The results
demonstrated that DNMT protein expression in
HCCs was significantly higher than in noncancerous liver tissues including normal, CH and LC tissues. In addition, there was no significant relationship between DNMT expression, CH and LC.
Immunohistochemical staining showed verystrong nuclear expression and faint cytoplasmic
expression of DNMT1, and strong nuclear and faintcytoplasmic expression of DNMT3a and DNMT3b.
Some immunohistochemical staining of these
DNMTs has previously been performed in a variety of cancers (Girault et al., 2003; Lin et al., 2007;
Ding et al., 2008) and HCC (Saito et al., 2003; Ohet al., 2007). Although nuclear and cytoplasmic
DNMT3b expression has been detected in othercancers, no results of immunohistochemical staining of DNMT3b have previously been reported in
HCC.
The present study showed a significant relationship between DNMT3a protein expression
and portal vein involvement, and significant rela-

Discussion
DNA methylation is well known to be involvedin the early developmental stages of HCC with abackground of chronic liver disease, including CH
and LC. It has recently been suggested that aberrant DNA methylation of CpG islands is an early5T. Miyake et al.

tionships between DNMT protein expression andtumor differentiation or intrahepatic metastasis.
However, a previous report found no significant
relationship between DNMT mRNA levels andportal vein involvement and tumor differentiationin HCCs (Saito et al., 2001), but the same researchgroup reported a significant relationship between
DNMT1 protein expression and portal vein involvement or tumor differentiation (Saito et al.,2003). Reverse transcriptase-polymerase chain
reaction is sufficiently sensitive to detect small elevations in DNMT mRNA levels, while immunohistochemistry is only able to detect elevations inprotein expression when they reach a certain levelin more malignant HCCs. We suggest that DNMT
protein expression may be more strongly correlatedwith progression of the cancerous stage of HCCs,
rather than the precancerous stage. To the best of
our knowledge, there have been no reports of any
significant relationship between DNMT protein
expression and intrahepatic metastasis, and there
have been some reports of a lack of any significant
relationship between DNMT protein expression
and lymphogenous metastasis in other cancers(Ding et al., 2008; Nosho et al., 2009). However,
the relationship between DNMT protein expression and hematogenous metastasis has not beenpreviously reported. E-cadherin, an intercellularadhesion-related protein, is coded for by a tumor
suppressor gene. E-cadherin gene methylation has
been associated with reduced expression of the protein, resulting in a loss of intercellular adhesion andtissue destruction in HCCs (Kanai et al., 1997). In
a recent study, E-cadherin expression was reported
to be significantly increased after concomitantsiRNA-mediated depletion of DNMT1, 3a and 3bin endometrial epithelial carcinoma cells (Rahnama
et al., 2009). DNMTs are thought to be involved in
E-cadherin methylation. In addition, reduction ofE-cadherin may also be related to the acquisition ofcell motility, tumor cell invasion and vascular invasion. E-cadherin gene methylation in HCCs has
been shown to be associated with a poor prognosis(Kozyraki et al., 1996; Lee et al., 2003). DNMT

protein expression may therefore contribute to hematogenous metastasis in HCC.
We investigated the relationship between
expression of the 3 DNMTs and overall patient
survival. This is the 1st report to reveal poor overall survival in HCC patients with positive immunoreactivity for DNMT3a and DNMT3b protein
expression. No significant relationship between
DNMT1 protein expression and overall patient survival was noted, though some studies on HCC andlung cancers have shown a significant relationshipbetween DNMT1 protein expression and overall
survival (Saito et al., 2003; Lin et al., 2007). The
reasons for this discrepancy remain to be determined. DNMT1 is referred to as a “maintenance”
methyltransferase, and DNMT1 protein expression
may thus not contribute to overall survival. However, our study demonstrated a significant relationship between protein expression of the 3 DNMTsand poor overall survival in HCCs. It was recently
reported that DNMT1 and DNMT3a/3b may associate directly and function as a complex for DNA
methylation of CpG islands (Kim et al., 2002; Rheeet al., 2002; Hattori et al., 2004). The results of thepresent study suggest that DNMT1, DNMT3a and
DNMT3b may be cooperatively involved in determining the extent of HCCs. Thus each DNMT
could affect survival in patients with HCC.
DNA methylation is known to be a reversible
process. In a previous study, promoter methylation
by DNMT was shown to be reversible using the
DNMT inhibitor 5-aza-2’-deoxycytidine, which induced re-expression of promoter-methylated genes
in a cell line in vitro (Juttermann et al., 1994; Yanget al., 2001; Arai et al., 2006). 5-Aza-2’-deoxycytidine and 5-azacytidine in non-toxic doses havebeen approved by the United States Food and Drug
Administration as DNMT inhibitors for hematological malignancies (Mulero-Navarro and Esteller,2008). The relationship between DNMT protein
expression and patient prognosis demonstrated in
the present study suggests a potentially importantrole for DNMT inhibitor therapy in HCC patientswith an increased DNMT protein expression.

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DNMT protein expression in HCC

In conclusion, the present study suggests thatoverexpression of DNMT proteins in HCCs may be
a predictive factor for poor survival. In addition,
DNMTs may represent new prognostic markers
and potential therapeutic targets in HCC.

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Received December 23, 2009; accepted January 6, 2010
Corresponding author: Takanori Miyake

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