Epigenetic silencing of the imprinted gene ZAC by DNA methylation is an early
event in the progression of human ovarian cancer
Tetsuya Kamikihara1,2, Takahiro Arima1, Kiyoko Kato1, Takao Matsuda1, Hidenori Kato1, Tsutomu Douchi2,
Yukihiro Nagata2, Mitsuyoshi Nakao3and Norio Wake1*
1Department of Molecular Genetics, Division of Molecular and Cell Therapeutics, Medical Institute of Bioregulation,
Kyusyu University, Oita, Japan
2Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan
3Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, Kumamoto, Japan
ZAC is a paternally expressed, imprinted gene located on chromo-
some 6q24, within a region known to harbor a tumor suppressor
gene for several types of neoplasia, including human ovarian can-
cer (HOC). We have failed to identify genetic mutations in the
ZAC gene in tumor material. Many imprinted genes contain dif-
ferentially allele-specific-methylated regions (DMR) and harbor
promoter activity that is regulated by the DNA methylation. Aber-
rant DNA methylation is a common feature of neoplasia and
changes in DNA methylation at the ZAC locus have been reported
in some cases of HOC. We investigated the DNA methylation and
ZAC mRNA expression levels in a larger sample of primary HOC
material, obtained by laser capture microdissection. ZAC mRNA
expression was reduced in the majority of samples and this corre-
lated with hypermethylation of the ZAC-DMR. Treatment of
hypermethylated cells lines with a demethylating agent restored
ZAC expression. Our studies indicate that transcriptional silencing
of ZAC is likely to be caused by DNA methylation in HOC. Forced
expression of ZAC resulted in a reduction in proliferation and
marked induction of apoptotic cell death. The ZAC-mediated
apoptosis signal is p53-independent and eliminated by inhibitors
of caspase 3, 8 and 9. Reduced expression of ZAC would therefore
favor tumor progression. As there were no significant differences
in either DNA methylation or expression of ZAC mRNA between
localized and advanced tumors, our data indicates that loss of
ZAC is a relatively early event in HOC. (Supplementary material
for this article can be found on the International Journal of Cancer
' 2005 Wiley-Liss, Inc.
Key words: genomic imprinting; human ovarian cancer; ZAC;
DNA methylation; tumor suppressor gene
Genomic imprinting plays an important role in mammalian
development, growth and cell differentiation.1Mutations that
affect the epigenetic status of imprinted loci underlie a number of
diseases, including developmental abnormalities, congenital dis-
eases and malignant tumors.2Alterations in the expression of
imprinted genes is one of the most common changes seen in can-
cer.3,4Several imprinted genes including ARH1,5PEG36and
ZAC7,8function as tumor suppressor genes suggesting a direct link
between loss of imprinting, either by epigenetic changes or chro-
mosomal deletions, and failure of tumor suppressor mechanisms.
Global changes in DNA methylation occur during carcinogene-
sis. Although there is an overall decrease in DNA methylation,
some CpG island sequences become hypermethylated.9Hyperme-
thylation of CpG islands seems to be responsible for the transcrip-
tional silencing of critical genes, including caretaker genes and
suppressor genes. Transcriptional silencing of these genes may be
selected during the development and progression of a variety of
Methyl-CpG binding domain (MBD) proteins have been identi-
fied as candidate mediators for silencing methylated DNA.10
MeCP2 is postulated to form a5-mCpG-dependent transcriptional
repression complex with Sin3a and the histone deacetylase
(HDAC).11MBD2, which also binds methylated DNA, is part of
the NuRD (nucleosome remodeling and deacetylation) complex
containing HDAC, MBD3 and Mi-2.12The role of HDAC in
transcriptional silencing in cancer is unclear. For some genes,
treatment with trichostatin A (TSA), a HDAC inhibitor, is suffi-
cient to reverse repression associated with CpG island hyperme-
thylation, whereas for other genes, TSA treatment alone is unable
to restore gene expression.13Combined treatment with TSA and
an inhibitor of DNA methylation has been reported to trigger the
expression of silenced cancer genes carrying somatic CpG island
ZAC was identified originally, along with p53, in a functional
screen by their common ability to induce expression of the
PACAP (pituitary adenylate cyclase activating polypeptide)
Type I receptor gene.14ZAC and p53 are both pleiotropic regula-
tors that have a number of similar activities; both regulate cell
cycle, apoptosis and nuclear receptor functions and both interact
physically and functionally with CBP and p300 that serve as inte-
grators of multiple signaling pathway.15,16P53 has a pro-apoptotic
activity and recent experiments indicate that this activity mainly
involves the mitochondrial pathway that is dependent on the activ-
ity of ApafI and caspase 9.17ZAC functions to enhance the activ-
ity of p53 on ApafI and may itself be activated by p53.18,19
We identified ZAC in a screen for imprinted genes8and pro-
posed that deregulation of expression of ZAC may be a factor in
neonatal diabetes mellitus (TNDM). ZAC encodes an imprinted
zinc finger protein that localizes to the nuclear compartment and
functions as a transcription factor with anti-proliferative activity.14
ZAC is expressed only from the paternal allele and maps to human
chromosome 6q24. This region is involved frequently in allelic
losses in many tumors.20–22Loss of ZAC expression has been
reported in a number of tumor types.23–26Lot1 (Lost on transfor-
mation), the rat orthologue of ZAC, was cloned from rat ovarian
surface epithelial cells transformed spontaneously in vitro27sug-
gesting an association of ZAC with ovarian cancer. The frequent
LOH of 6q24 in ovarian cancer28and some preliminary data29
suggest that loss of ZAC expression may play a role in the initia-
tion and/or progression of human ovarian cancer.
In mice, imprinting of Zac1 may be regulated by a differentially
methylated CpG island (DMR) that partially overlaps the Zac1
and Hymai genes.29–31This region shows gamete-specific DNA
methylation that persists throughout pre- and post-implantation
development. Within this DMR, there is a region that exhibits a
high degree of homology between mouse and human that acts as a
strong transcriptional repressor when DNA methylated. We have
Grant sponsor: Ministry of Health and Welfare of Japan.
The first two authors contributed equally to this paper.
*Correspondence to: Department of Molecular Genetics, Division of
Molecular and Cell Therapeutics, Medical Institute of Bioregulation,
Kyusyu University, 4546, Tsurumihara, Beppu, Oita 874-0838, Japan.
Received 20 August 2004; Accepted after revision 29 November 2004
Published online 4 March 2005 in Wiley InterScience (www.interscience.
Int. J. Cancer: 115, 690–700 (2005)
' 2005 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
proposed that this DMR is an imprint control region (ICR) that
regulates expression of the imprinted genes within this domain,
including ZAC .32
Hypermethylation of the human ZAC locus has been reported in
some human ovarian cancers in a previous study.29Only 4 pri-
mary samples were examined and ZAC mRNA expression levels
not tested in these samples. ZAC mRNA expression levels was
found to be reduced in ovarian cancer cell lines but the authors did
not find a correlation between the increased methylation and
decreased ZAC mRNA expression levels in these samples. The
CpG rich region examined is not within the region we have pro-
posed as the DMR. We have now examined the DNA methylation
status of the DMR in a larger sample of ovarian cancer materials
and correlated its methylation status with ZAC mRNA expression
levels. We firstly examined human ovarian cancer (HOC) cell
lines. Our initial results suggested an association between
increased DNA methylation and loss of ZAC mRNA expression
levels in HOC. Because epigenetic changes can occur as a conse-
quence of in vitro culturing33we also carried out our analysis on
28 primary cancer tissues obtained by laser capture micro dissec-
tion (LMD) from surgically removed tissues. This method enables
small samples of tissue to be obtained from specific tissue sections
for RNA and DNA analysis. We report that the ZAC DMR, which
contains the ZAC promoter, is subject to epigenetic changes of
ovarian cancers and that the observed increase in DNA methyla-
tion correlates with loss of expression of ZAC mRNA in both
localized and advanced tumor groups. We suggest that the changes
in ZAC mRNA expression are relative early event in the progres-
sion of HOC. Furthermore, we have used 2 HOC cell lines to
experimentally address whether DNA methylation or histone ace-
tylation plays a role in repressing ZAC expression.
The function of ZAC in ovarian cells is unknown. We have
examined the effect of exogenous ZAC expression in HOC cell
lines that lack the endogenous ZAC. We examined tumorigenicity,
cell cycle regulation and apoptosis. Our studies suggest that
silencing of ZAC mRNA expression could contribute to ovarian
cancer development by allowing increased cellular proliferation
with a simultaneous escape from apoptosis.
Material and methods
Human ovarian cancer (HOC) cell lines (KK, TYK-nu, PA-1,
MH, KF, HTOA, SKOV-3, MCAS, HAC2, RMG and KM) were
used in our study. The source of these cells is as described.34They
were grown in either DMEM or RPMI1640 supplemented with
10% FBS before the isolation of DNA and RNA.
Northern blot analysis
We prepared total RNA from HOC cell lines using ISOGEN
(Nippon Gene, Tokyo, Japan). The ZAC cDNA (GenBank acces-
sion number AA463204) was used as a probe. Northern blot anal-
ysis was carried out as described.32The same membrane was
reprobed with a GAPDH probe (Clontech, Tokyo, Japan) as a con-
trol for loading of RNA.
Cancerous tissues and adjacent non-cancerous tissues were
excised from the patients during surgery, after informed consent had
been obtained. Frozen sections (8 ?m) of the samples were made
and mounted on glass slides covered for the micro dissection system
(Leica Microsystems, Tokyo, Japan) essentially as described previ-
ously.35Total RNA from these sections was extracted with RNeasy
mini kit (Qiagen, New York, NY) according to the manufacturer’s
protocol. The amplification of mRNA was made from total RNA
using Agilent Low RNA Input Fluorescent Linear Amplification kit
(Agilent Technologies, NY, USA). Amplified RNAs were treated
with DNAseI (Roche, Mannheim, Germany) and reverse transcribed
to single-stranded cDNAs using oligo (dT) primer and the RNA
PCR core kit (Roche, Piscataway, NJ). We prepared appropriate
dilutions of each cDNA for PCR amplification by monitoring
the GAPDH transcript. Primer sequences were as follows:
FIGURE 1 – Expression analysis
of human ZAC in normal human
ovary and human ovarian cancer
cell lines and tissue. (a) Expression
of ZAC in human ovary by in situ
expressed in the ovarian surface
epithelium (left). This is the devel-
opmental origin of most ovarian
cancers. It is also expressed in the
follicle epithelial cells (right). (b)
ZAC expression in 11 human ovar-
ian cancer cell lines by Northern
blot analysis. The level of expres-
sion of ZAC was reduced in 4 lines
(Lanes 4, 6, 8, 9) and completely
absent in 7 lines. Lanes: KK (1),
TYK-nu (2), PA1 (3), MH (4), KF
MCAS (8), HAC2 (9), RMG (10)
and KK (11). C1 and C2 are con-
GAPDH cDNA probe was used as
a control for RNA levels.
EPIGENETIC SILENCING OF ZAC IN HOC
CTGACACGTTG-30for GAPDH, and 50-AGGAAGGTGTGA-
GAAGCAAAGC-30and 50-CCATTTTGTTGGGGTCGTGG for
ZAC . Real-time PCR analysis was carried out using the iCycler
iQ Multi-Color Detection System (Bio-Rad, Tokyo, Japan) and
the Master Mix from a Quantitect SYBR Green PCR kit (Qia-
gen). PCR cycles were 958C for 30 sec, 578C for 30 sec and
extension at 728C for 30 sec for ZAC and GAPDH. The amplifi-
cation plots of ZAC and GAPDH in each sample were analyzed
at the appropriate cell cycle number and amplification curve to
obtain stable quantitative results for the experiments. To confirm
that we amplified the correct fragment, the PCR products were
resolved on 3% agarose gels. Statistical significance was deter-
mined with a Spearman’s and Pearson’s rank correlation.
Bisulphite PCR methylation assay
DNA (0.1 ?g) obtained using LMD system was used for bisulphite
treatment as described previously.36Bisulphite-treated DNA was
amplified by the PCR of the CpG island in the ZAC gene. Primer
sequences for ZAC: BS1F (ZAC), 50-GGGGTAGTYGTGTTT-
ATAGTTTAGTA-30and BS1R (ZAC), 50-CRAACACCCAAA-
CACCTACCCTA-30. PCR conditions were as follows: after an
initial standard denaturation step, 30 cycles of denaturation at
958C for 30 sec, annealing at 598C for 30 sec and extension at
728C for 30 sec was carried out. The amplified fragments were
cloned into the TOPO TA vector (Invitrogen). Individual clones
were sequenced using a T7 primer or M13 reverse primer.
In situ hybridization analysis
Part of the human cDNA (GenBank accession number
AJ006354; 18-621nt) for ZAC was amplified by PCR and used to
prepare sense and antisense RNA by in vitro transcription using
the DIG RNA labeling kit (Roche, Mannheim, Germany). Eight
micrometer sections from normal human ovary were used for in
situ hybridization as described previously.37Sections were counter
stained with 4% eosin. Mounted sections were photographed.
Analysis of tumorigenicity using Tet-on system
pTet-on regulator plasmid (pTet-on), pTRE2 response plasmid,
containing hygromycin-resistance gene, was purchased from
Clontech. Plasmid DNA was introduced into SKOV and PA1 HOC
cells by Lipofectin (Gibco BRL, Tokyo, Japan), carried out accord-
ing to the manufacturer’s instructions. Selection of pTet-on clones
started 24 hr after transfection using 500 ?g/ml hygromycin
(Merck, Tokyo, Japan). A second selection of clones expressing
Zac1 vector, kindly provided by Spengler (pPURDM [tetr0] 5.
mZac) was carried out at 5.0 ?g/ml puromycin. Stably transfected
lines were generated. The cotransfected cells (PA1Z and SKOVZ)
were treated with DOX (tetracycline derivative doxycycline) at the
The cells were seeded in a 24-well microplate at a density of 1 ?
104cells/well. After incubation, cells were trypsinized and sus-
pended in PBS. Cells (1 ? 107) were suspended in 100 ?l of culture
medium. The PA1Z and SKOVZ cell suspension was injected dor-
sal-subcutaneously (s.c.) into the nude mice. After 3 days, half of
animals were injected once with the 200 ?l DOX (500 pM for
PA1Z and 10 nM for SKOVZ) and the remaining half were injected
with saline. Tumor formation was monitored 3 times a week for up
to 8 weeks. These animals were sacrificed and the tumors were
examined by H&E staining.
Treatment of cells with 5-AzaC or TSA
HOC cell lines (PA1 and SKOV) were plated at a density of 5 ?
105cells/60 mm2dish. Twenty-four hours later, they were treated
with 100 ng/ml TSA (Wako, Tokyo, Japan) or 1 ?M 5-AzaC
(Sigma) for the times stated. Total RNA was prepared and analyzed
by Northern blot. The methylation status of the ZAC CpG island/
DMR was examined by the bisulphite PCR methylation assay
FIGURE 2 – Association of the ZAC expression and the methylation
status of ZAC CpG island in ovarian cancers. (a) Primers used for
PCR amplification of bisulphite-treated genomic DNA are indicated.
In total 17 CpG sites within ZAC CpG island were analyzed. Filled
circles represent methylated residues. (b) Methylation status of ZAC is
shown in 4 HOC cell lines and representative 6 ovarian cancer tissues
obtained by LMD and analyzed by bisulphite-sequencing. Individually
sequenced clones are shown. The methylation status of the normal,
adjacent tissue for each sample is expressed as a percentage and given
in parentheses. The difference in expression level of ZAC between the
non-cancerous and cancerous material determined by real-time RT-
PCR is given above the samples.
KAMIKIHARA ET AL.
SiRNA ‘‘Knock-down’’ experiments and transient
SiRNA encoding MBD2 and MeCP238were transfected into
PA1 and SKOV cells using oligofectamine (Invitrogen). An addi-
tional siRNA transfection was undertaken 48 hr later to increase
the efficiency of the knock-down. The effectiveness of the knock-
down was monitored by Northern blot analysis of the target
ZAC promoter-luciferase reporter constructs (pGL3 vector,
Promega) were treated with SssI methylase (New England Bio-
labs, UK) and then transfected into the cell lines using Lipofect-
amine (Invitrogen). After 48 hr, the transfected cells were lysed
using passive lysis buffer (Promega, Madison, WI). Luciferase
reporter activity was assayed using a Dual-Luciferase reporter
assay system (Promega) and luminometer. Each experiment was
repeated 3 times.
Analysis of the ZAC -induced apoptosis
To inhibit the activation of caspases, cells were pretreated with a
general caspase inhibitor 20 ?M zVAD-FMK (ICN Biomedicals
Inc., Costa Mesa, CA), a caspase 9-specific inhibitor Z-LEHD-
FMK (R&D System, Minneapolis, MN), a caspase 8-specific inhibi-
tor Z-IETD-FMK (R&D System) and a caspase 3-specific inhibitor
Z-DEVD-FMK (R&D System). Apoptosis was determined by a
Tunel assay using the ApoTaq Kit (Invitrogen). Treated cells (2 ?
105) were analyzed by flow cytometry using FACScan flow cytom-
eter (Becton Dickinson, San Jose, CA).
Expression of ZAC in human ovary
Loss of heterozygosity (LOH) has been reported at high fre-
quency in human ovarian cancer28in the 6q24 region where the
putative tumor suppressor gene, ZAC is located. Mouse Zac1 is
expressed strongly in many regions including lung, tongue, sclero-
tome and the periphery of pancreas.30The expression profile of
human ZAC is less well defined. We investigated whether ZAC
was expressed in adult human ovary by in situ hybridization.
Strong signals were observed in the ovarian surface epithelium
and the follicular epithelium (Fig. 1a; antisense probe; data not
shown for sense probe). Exclusive expression of ZAC in the ovar-
ian surface epithelium but not the ovarian stroma is significant
because this region is the developmental origin of almost all ovar-
Expression of ZAC is lost or highly reduced in ovarian cancer
and correlates with DNA hypermethylation
Reduced ZAC function could result from at least 2 mechanisms,
either a genetic mutation inactivating the gene or an epigenetic
TABLE I – CLINICAL CHARACTERISTICS OF PATIENTS WITH OVARIAN CANCERS, ZAC EXPRESSION AND % METHYLATION OF ZAC PROMOTER1
CaseAge Histopathology StagingZAC expression in cancer % Methylation (cancer) %Methylation (non-cancer)
Localized tumors (Stage I, II)
Advanced tumors (Stage III, IV)
1Clinical stages were determined according to the classification of the International Federation of Gynecology and Obstetrics (FIGO). Patients
were devided into 2 groups, those with localized tumors (Stage I, II) and advanced tumors (Stage III, IV). SCA, serous cystadenocarcinoma;
MCA, mucinous cystadenocarcinoma; E, endmetrioid adenocarcinoma; C, clear cell carcinoma; SCC, squamous cell carcinoma in mature cystic
teratoma; NT, not tested.
TABLE II – SUMMARY OF TUMOR METHYLATION AND ZAC EXPRESSION
Tumor type%Methylation (mean)
cancerous/normal ratio (mean)
Non-cancer (n ¼ 12)
Localized tumor (n ¼ 10)
Advanced tumor (n ¼18)
Total, mean (n ¼28)
EPIGENETIC SILENCING OF ZAC IN HOC
mutation, such as DNA methylation or histone deacetylation, lim-
To assess the ZAC genetic status, we screened for mutations by
single strand conformation polymorphism (PCR-SSCP). No muta-
tions or polymorphisms were detectable in 11 HOC and 28 pri-
mary ovarian cancer samples tested (data not shown).
To assess ZAC mRNA expression, we first analyzed ZAC
mRNA levels in the 11 HOC cell lines by Northern blotting
(Fig. 1b). In all 11 lines, the level of ZAC mRNA expression was
reduced significantly (4 lines) or completely absent (7 lines) as
compared to normal ovarian tissues.
We have identified previously a fragment from within the CpG
island at the HYMAI / ZAC locus that behaves as a transcriptional
repressor when DNA methylated. This 480 bp PX fragment (NheI-
SmaI) contains 47 CpG sites and is highly homologous to the
DMR proposed as the imprint control region at the mouse locus. It
also contains the ZAC promoter. We determined the DNA methyl-
ation status in 17 CpG sites within this region in the 11 HOC cell
lines. The bisulphite PCR methylation assay showed dense meth-
ylation of 17 CpG sites within the PX fragment, demonstrating a
correlation between increased DNA methylation of the ZAC DMR
and decreased mRNA expression of ZAC (Fig. 2 b and supplemen-
Tumor cell lines sometimes have genetic or epigenetic changes
due to their growth in culture that can hinder accurate character-
ization. Quantitative evaluation of gene expression in vivo is
essential but can be complicated by variable contamination with
normal tissues. The recent development of the laser micro dissec-
tion (LMD) system enables the precise isolation of specific parts
from tissue sections for RNA and DNA analysis.39,40We com-
bined the LMD technique and quantitative reverse-transcription
polymerase chain reaction (RT-qPCR) to analyze gene expression
in primary HOC tissues. First, frozen sections of ovarian cancer-
ous tissue and adjacent non-cancerous tissue was stained with tol-
uidine blue solution and dissected in the LMD system. cDNA and
genomic DNA was prepared from these samples. ZAC and
GAPDH were amplified from the cDNA by real-time PCR. RT-
PCR bands were checked and were of the expected sizes (data not
shown). We examined 28 primary ovarian cancer cases, repeating
the PCR 3 times on each sample. The ratio of the level of the
mRNA of ZAC to the level of GAPDH was significantly decreased
in 24 primary ovarian cancer tissues, compared to the non-cancer-
ous counterparts (Table I). The reduction of ZAC transcription
level was modest in 2 cancer tissues and no data was obtained for
the remaining 2 samples.
We tested the cytosine methylation status of the PX fragment in
the same primary ovarian cancer tissues (Table I). Genomic DNA
from the cancerous tissues and 12 control samples from adjacent tis-
sue was treated with bisulphite and PCR was carried out. PCR prod-
ucts were subcloned and independent clones were sequenced. In the
FIGURE 3 – Reactivation of ZAC
expression by treatment with 5-
AzaC or TSA. (a) Expression level
of ZAC mRNA. HOC cell lines
PA1 and SKOV have reduced lev-
els of expression of ZAC and DNA
methylation of the closely linked
CpG island (DMR). Treatment
with 100 ng/ml TSA for 24 hr
slightly increased the expression of
5-AzaC for 72 hr led to an almost
2-fold increase in expression. A
combination of 5-AzaC and TSA
led to a 2-fold increase in line PA1
and a 3.65-fold increase in line
SKOV. (b) Methylation status of
ZAC promoter. The ZAC CpG
island was amplified from bisul-
phite-modified genomic DNA and
digested with a restriction enzyme
RsaI that distinguishes between
material. After 48 hr of 5-AzaC
treatment, the ZAC promoter was
partially demethylated. At 72 hr,
no detectable DNA methylation
was present at the sites examined.
This correlates with the gradual
increase in expression of the tran-
script over time.
KAMIKIHARA ET AL.
12 non-cancerous samples, the percentage of methylation of the
individually sequenced PCR products showed that nearly 50% of
the residues were methylated. Importantly, the individual clones
analyzed were mostly either heavily methylated or unmethylated
suggestive of allele-specific methylation. In contrast, the 17 CpG
sites in the ZAC promoter were heavily methylated in most individ-
ual clones from primary ovarian cancer tissues (average ¼ 78.4%),
compared to the non-cancerous tissues (average ¼ 46.5%). There is
a tendency for primary ovarian cancer samples with the densely
methylated DMR to show reduced ZAC mRNA expression in both
the localized and advanced tumor groups. We found that this was
statistically significant by Spearman’s rank correlation (good rank
correlation ¼ 0.724, p < 0.01). We did not observe a significant dif-
ference in either DNA methylation or mRNA expression between
the localized tumor groups and the more advanced tumors
(Table II). This suggests that the changes in ZAC occur as an early
event in the progression of ovarian cancer
Reactivation of ZAC in HOC cell lines by 5-AzaC and TSA
Earlier studies have suggested that DNA methylation or histone
modifications may be involved in regulating mRNA expression of
ZAC .23,29,41In addition, the link between reduced mRNA expres-
sion of ZAC and increased DNA methylation in most ovarian can-
cer tissues suggests that aberrant DNA methylation may be
responsible for the reduced ZAC mRNA expression in ovarian
cancer. To examine this link, we carried out an in vitro study on 2
of the ovarian cancer cell lines, PA1 and SKOV, where we had
found both reduced mRNA expression of ZAC and hypermethyla-
tion of the ZAC CpG island. They were treated with the demethy-
lating agent 5-AzaC (5-aza-20-deoxycytidine) or the HDAC
inhibitor, TSA (trichostatin A) and ZAC mRNA expression levels
were compared to untreated cells (Fig. 3a). Treatment of 2 HOC
cell lines with 100 ng/ml TSA for 24 hr only slightly induced the
mRNA expression of ZAC consistent with a previous study.29
Treatment with 1 ?M 5-AzaC for 48 and 72 hr lead to an 1.8-fold
increase in expression. A combined treatment with both 5-AzaC
and TSA contributed to a 2.1- and 3.65-fold increase, respectively.
This result suggests that DNA methylation and deacetylation act
synergistically to repress ZAC mRNA expression in HOC cell
lines. To confirm that demethylation had occurred at the ZAC
locus, we examined the DNA methylation status of the DMR in
the treated cells using our methylation-sensitive assay30(Fig. 3b).
At 48 hr, some DNA methylation was detectable but by 72 hr,
when we observe maximal derepression, DNA methylation at this
site was completely removed.
Methylation-dependent ZAC promoter repression
It is possible that upregulation of ZAC by 5-AzaC and TSA in
culture may be an indirect effect due to global demethylation and
deacetylation. Methyl-CpG binding domain (MBD) family pro-
teins, MeCP2 and MBD2 are involved in the transcriptional
repression11,42,43therefore, we tested whether MBD2, MeCP2 or
both MBD2 and MeCP2 were directly responsible for repression
of the ZAC promoter activity. The levels of MBD proteins were
reduced in HOC cells by treatment with specific siRNA.38We
used this system to determine the effect of MBD on ZAC promoter
activity using a reporter system. The promoter construct contains
the 480 bp PX NheI-SmaI fragment from the ZAC CpG island
adjacent to a luciferase sequence. This region was demonstrated to
have promoter activity.31First, a comparison was made between
luciferase activity from the unmethylated reporter and from the
methylated reporter ( SssI-methylated) in the 2 cell lines, PA1 and
SKOV, in the absence of the siRNA (Fig. 4a). This confirmed
DNA methylation-dependent repression of the promoter activity.
In the presence of siRNA, methylation dependent repression of the
promoter activity was partially abrogated with the greatest effect
seen with a combined knock-down of MBD2 and MeCP2 in PA1
cells. The effectiveness of MBD knock-down with a specific
SiRNA was confirmed by Northern blotting (Fig. 4 b ). These data
suggest that DNA methylation of the DMR could suppress ZAC
transcription by recruiting HDAC and methyl-CpG binding
domain family proteins to the ZAC promoter region. These 2
repressors are not thought to act in the same complex and there-
fore these preliminary observations must be explored further.
Tumor suppressor activity of ZAC in HOC cell lines
We examined the tumor suppressor activity of ZAC by stably
transfecting the 2 ovarian cancer cell lines (PA1 and SKOV) with
a DOX-sensitive expression vector linked to the Zac1 cDNA.14
We tested the toxic affect of different DOX concentrations on the
cell lines. PA1 was not significantly effected by DOX at up to
500 pM and SKOV was not significantly effected up to 10 nM
(Fig. 5a). Subsequent analysis was carried out under non-toxic
conditions. Exogenous Zac1 mRNA expression was confirmed by
Northern blot analysis in 2 randomly chosen sub lines, PA1Z and
SKOVZ. We found that over expression of Zac from the Zac1
cDNA negatively affected cell growth in the 2 ovarian cancer cell
lines (Fig. 5a). Significant prolongation of population doubling
time was demonstrated in the HOC cells with induced Zac1
mRNA expression (Table III).
We examined the effect of Zac1 mRNA expression on growth
rate and tumor formation in nude mice (Table III). Two clones,
the slow growing PA1Z clone and the fast growing SKOVZ clone,
were tested for tumorigenicity by hypodermic injection into nude
FIGURE 4 – Loss of repression of the methylated ZAC promoter
after targeted reduction of MBD2 or MeCP2. (a) MBD2 or MeCP2
were reduced in HOC cells by SiRNA knock-down. SssI-methylated
ZAC promoter-luciferase plasmids were transfected into SiRNA
treated HOC cells. Luciferase levels are given as a percentage of the
luciferase levels in the absence of in vitro DNA methylation. Both
MBD proteins were found to contribute to repression of promoter
activity. Lane 1,2, no siRNA; lane 3, SiRNA to MBD2; lane 4,
SiRNA to MeCP2; lane 5, SiRNA to both MBD2 and MeCP2. (b)
Effectiveness of SiRNA knock-down of MBD proteins was monitored
by Northern blotting with MBD2 and MeCP2 cDNA probes.
EPIGENETIC SILENCING OF ZAC IN HOC
mice. It was found that the parent PA1 and SKOV lines and the
non-DOX treated PA1Z and SKOVZ lines formed progressively
growing tumors. No tumors were detected after the injection of
PA1Z and SKOVZ treated with DOX after 7 weeks (Table III),
demonstrating that ZAC possesses a tumor suppressing activity in
HOC cell lines in vivo.
Induction of apoptosis by ZAC in HOC cell lines
ZAC was reported to arrest cell cycle and induce apopto-
sis.14Therefore, we examined these functions in the 2 HOC
cell lines. The PA1 cell line carries a wild-type (WT) p53
gene whereas the SKOV cells have a mutant p53 gene.44To
test for cell cycle regulation, we analyzed the cells by flow
FIGURE 5 – Tumor suppressor activity of ZAC in ovarian cancer cells. (a) Growth curve using the Tet-on system of ZAC cDNA in 2 HOC cells
PA1Z (p53 wild-type) and SKOVZ (p53 mutant). PA1 and SKOV are the original, untransfected cell lines. (b) Flow cytometry analysis for cell
cycle regulation of ZAC in 2 cell lines. The number of cells in Sub G1 phase increased after exogenous ZAC expression in both cell lines.
(c) TUNEL analysis shows that apoptosis accompanies ZAC-induced growth arrest.
KAMIKIHARA ET AL.
FIGURE 5 – CONTINUED.
EPIGENETIC SILENCING OF ZAC IN HOC
cytometry. Expression of Zac1 in the p53-WT and mutant
(mt) cell lines markedly increased the number of cells in the
sub G1 phase (40.2%, 55.6%) (Fig. 5b). A TUNEL immuno-
biochemical analysis also demonstrated the high incidence of
apoptotic cell death in the 2 ovarian cancer cell lines express-
ing Zac1 exogenously (Fig. 5c). We conclude that ZAC regu-
lates cell proliferation through inducing apoptosis, even in the
absence of p53.
TABLE III – IN VITRO AND IN VIVO PROPERTIES OF THE OVARIAN CANCER CELLS BEFORE AND AFTER
ACTIVATING EXOGENOUS EXPRESSION OF ZAC
doubling time (hr)
Tumour forming ability
(no. of tumors/no. of incubation sites)
PA1Z (DOX 500pM)
SKOVZ (DOX 100nM)
FIGURE 6 – ZAC induces apop-
tosis in p53-independent pathway.
PA1Z (a) and SKOVZ (b) were
analyzed by flow cytometry after
induction of ZAC expression in the
absence (none) and presence of the
general caspase inhibitor z-VAD-
FMK (20 mM) and the individual
(Z-IETD-FMK) and Caspase 3
(Z-DEUD-FMK). The number of
cells in SubG1 phase decreased
after caspase inhibition in both the
p53 wild-type line, PA1, and the
p53 mutant line, SKOV, demon-
strating that escape from apoptosis
KAMIKIHARA ET AL.
Effect of caspase inhibitors on ZAC -induced apoptosis
We assessed the effect of a general caspase inhibitor on ZAC-
induced apoptosis in the HOC cell cultures. The 2 HOC cell lines
expressing exogenous Zac1 were exposed to caspase inhibitor and
analyzed by FACS. Cells (1 ? 106) (PA1Z, SKOVZ) cells were
grown in a 60-mm2culture dishes and treated with DOX with the
caspase general inhibitor, z-VAD-fmk (20 ?M). After 3 days, we
collected the cells and measured the cell cycle distribution. We
observed significant reduction of both PA1Z and SKOVZ cells in
the sub G1 fraction (Fig. 6). We additionally examined the Zac1-
induced apoptosis in the presence of three more specific caspase
inhibitors (3, 8, 9). All inhibitors interrupted Zac1-induced apopto-
sis in both cell lines. These results support the finding that the apop-
totic signal is p53-independent. The result that apoptosis was
abolished completely by the pretreatment with general or individual
3, 8 and 9 caspase inhibitors, indicates that the ZAC-induced apop-
tosis is transmitted from a signal upstream of caspase 8 cascade.
Among the different epigenetic modifications involved in
imprinting, DNA methylation is particularly relevant as the great
majority of imprinted genes examined so far contain methylated
regions that are allele-specific (DMR). These DMR have variable
locations within different imprinted genes, and their functional
role in the silencing of the imprinted allele is not fully understood.
Indeed, it may be that not all DMR within imprinted regions func-
tion in the same way. Some DMR harbor a promoter activity that
is regulated by DNA methylation. This suggests a direct role in
the repression of transcription by DNA methylation. Previous data
supports such a function for the differentially methylated CpG
island linked to the HYMAI / ZAC locus.30,31
The chromosomal location, expression profile and preliminary
functional data are all consistent with a role for ZAC in the pro-
gression of ovarian cancer and there is data to support this idea.29
We have extended and refined the previous analysis by examining
the methylation status of 17 CpG sites located within the proposed
DMR for the locus in large sample of normal and ovarian cancer
material. Normal, non-cancerous ovarian tissues show partial
methylation of this sequence. As the individual clones sequenced
were mostly either heavily methylated or entirely unmethylated,
this suggests that the methylation is allele-specific consistent with
previous reports.29In all 11 ovarian cancer cell lines and 28 pri-
mary ovarian cancer tissues, we found increased methylation of
this sequence. Importantly, we also found a significant reduction
in the level of expression of the ZAC transcript in almost all of the
samples. When we examined the clinical characteristics of the
tumor types, we found more fully methylated clones in the
advanced tumor group and a lower average level of expression of
the transcript. There was still significant changes in methylation
and expression in the localized tumor group suggesting that the
changes in ZAC mRNA expression are a relatively early event in
the progression of HOC.
To demonstrate the importance of the DMR methylation in the
regulation of ZAC transcription, we used two human ovarian can-
cer cell lines in which we had found hypermethylation and loss of
expression the ZAC . Treatment of these cell lines with 5-AzaC, a
potent inhibitor of DNA methylation, partially released ZAC from
silencing. We found that the level of derepression correlated with
the progressive loss of methylation at individual CpG dinucleoti-
des. A much milder affect was seen when the cells were treated
with the histone deacetylase inhibitor, TSA, similar to that previ-
ously reported.29We did, however, observe derepression with a
combination of 5-AzaC and TSA that was greater than with
We and others29have observed repression of a methylated
reporter sequence containing the ZAC DMR suggesting that DNA
methylation is directly linked to repression of transcription.
MBD2 and MeCP2 are proteins that have a binding-specificity for
methylated DNA and have been shown to repress transcription.
When siRNA was used to knock down transcripts for these pro-
teins, we observed loss of repression of the methylated reporter.
The identification of ovarian cancer cell lines with markedly
reduced mRNA expression of ZAC provided an in vitro system to
test the function of ZAC in this cell type. Forced expression of a
mouse Zac1 cDNA resulted in the negative regulation of prolifera-
tion of ovarian cancer cells and marked induction of apoptotic cell
death. This ZAC-mediated apoptosis signal is p53 independent as
ZAC has the capability to elicit apoptosis in cells with mutant p53.
In addition, all of inhibitors of caspase 3, 8 and 9 completely elim-
inated the ZAC-mediated apoptosis. Although the detailed signal
transduction remains unknown, the signal that activates caspase 8
may be involved in this ZAC-mediated apoptosis. Further demon-
stration of molecular mechanisms of ZAC-mediated apoptosis may
allow us to establish a new type of ovarian cancer treatment.
In conclusion, we report that hypermethylation and loss of
mRNA expression of ZAC is a frequent occurrence in ovarian can-
cer. Current data,29and this report, suggest that CpG island meth-
ylation and recruitment of HDAC and MBD family proteins is
critical in the repression of this locus. We have demonstrated that
ZAC can function to regulate cellular proliferation and apoptosis.
Our finding that ZAC mRNA expression is reduced even in early
stage samples suggests that the deregulation of these functions
may be an important factor in the progression of ovarian cancer.
We would like to thank Dr. Y. Yoshikawa, Mr. Komatsu and
Ms. Hachisu H. for technical assistance and all members of the
laboratory for their support and valuable suggestion. In particular,
Dr. R. John for the comments on manuscript.
1.Surani MA, Barton SC, Norris ML. Development of reconstituted
mouse eggs suggests imprinting of the genome during gametogenesis.
Hall JG. Genomic imprinting: nature and clinical relevance. Annu
Rev Med 1997;48:35–44.
Joyce JA, Schofield PN. Genomic imprinting and cancer. Mol Pathol
Feinberg AP. DNA methylation,
Curr Top Microbiol Immunol 2000;249:87–99.
Yu Y, Xu F, Peng H, Fang X, Zhao S, Li Y, Cuevas B, Kuo W,
Gray JW, Siciliano GM, Mills GB, Bst RC. NOEY2 (ARHI), an
imprinted putative tumor suppressor gene in ovarian and breast carci-
nomas. Proc Natl Acad Sci USA 1999;96:214–9.
Kohda T, Asai A, Kuroiwa Y, Kobayashi S, Aisaka K, Nagashima G,
Yoshida MC, Kondo Y, Kagiyama N, Kirino T, Kaneko-Ishino T,
Ishino F. Tumor suppressor activity of human imprinted gene PEG3
in a glioma cell line. Genes Cells 2001;3:237–47.
4. genomic imprinting and cancer.
7. Varrault A, Ciani E, Apiou F, Bilanges B, Hoffmann A, Pantaloni C,
Bockaert J, Spengler D, Journot L. hZAC encodes a zinc finger pro-
tein with antiproliferative properties and maps to a chromosomal
region frequently lost in cancer. Proc Natl Acad Sci USA 1998;95:
Kamiya M, Judson H, Okazaki H, Kusakabe M, Muramatsu M,
Takada S, Takagi N, Arima T, Wake N, Kamimura K, Satomura K,
Hermann R,et al. The cell cycle control gene ZAC/PLAGL1 is
imprinted for a strong candidate gene for transient neonatal diabetes.
Hum Mol Genet 2000;9:433–60.
Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev
10. Nan X, Meehan RR, Bird A. Dissection of the methyl-CpG binding
domain from the chromosomal protein MeCP2. Nucleic Acids Res
11. Nan X, Ng H-H, Johnson CA, Laherty CD, Turner BM, Eisenman
RN, Bird A. Transcriptional repression by the methyl-CpG-binding
EPIGENETIC SILENCING OF ZAC IN HOC
protein MeCP2 involves a histone deacetylase complex. Nature 1998;
12. Zhang Y, Ng H-H, Bromage HB, Tempst P, Bird A, Reinberg D.
Analysis of the NuRD subunits reveals a histone deacetylase core
complex and a connection with DNA methylation. Genes Dev 1999;
13. Cameron EE, Bachman KE, Myohanen S, Herman JG, Baylin SB.
Synergy of demethylation and histone deacetylase inhibition in the re-
expression of genes silenced in cancer. Nat Genet 1999;21:103–7.
14. Spengler D, Villalba M, Hoffmann A, Pantaloni C, Houssami S,
Bockaert J, Journot L. Regulation of apoptosis and cell cycle arrest by
Zac1, a novel zinc finger protein expressed in the pituitary gland and
the brain. EMBO J 1997;16:2814–25.
15. May P, May E. Twenty years of p53 research: structural and func-
tional aspects of the p53 protein. Oncogene 1999;18:7621–36.
16. Huang SM, Stallcup MR. Mouse Zac1, a transcriptional co-activator
and repressor for nuclear receptors. Mol Cell Biol 2000;20:1855–67.
17. Henis-Korenblit S, Shani G, Sines T, Marash L, Shohat G, Kimchi A.
The caspase-cleaved DAP5 protein supports internal ribosome entry
site-mediated translation of death proteins. Proc Natl Acad Sci USA
18. Huang SM, Schonthal AH, Stallcup MR. Enhancement of p53-
dependent gene activation by the transcriptional coactivator Zac1.
19. Rozenfeld-Granot G, Krishnamurthy J, Kannan K, Toren A,
Amariglio N, Givol D, Rechari G. A positive feedback mechanism in
the transcriptional activation of Apaf-1 by p53 and the coactivator
Zac-1. Oncogene 2002;21:1469–76.
20. Taguchi T, Jhanwar S, Siegfried J, Keller S, Testa J. Recurrent dele-
tions of specific chromosomal sites in 1p, 3p, 6q, and 9p in human
malignant mesothelioma. Cancer Res 1993;53:4349–55.
21. Fujii H, Zhou W, Gabrielson E. Detection of frequent allelic loss of
6q23–q25.2 in microdissected human breast cancer tissues Genes
Chromosomes and Cancer 1996;16:35– 9.
22. Theile M, Seitz S, Arnold W, Jandrig B, Frege R, Schlag P, Haensch W,
Guski H, Winzer K-J, Barrett J, Scherneck S. A defined chromosome
6q fragment (at D6S310) harbors a putative tumor suppressor gene for
breast cancer. Oncogene 1996;13:677–85.
23. Bilanges B, Varrault A, Basyuk E, Rodriguez C, Mazumdar A,
Pantaloni C, Bockaert J, Theillet C, Spengler D, Journot L. Loss of
expression of the candidate tumor suppressor gene ZAC in breast can-
cer cell lines and primary tumors. Oncogene 1999;18:3979–88.
24. Pagotto U, Arzberger T, Theodoropoulou M, Grubler Y, Pantaloni C,
Saeger W, Losa M, Journot L, Stalla GK, Spengler D. The expression
of the antiproliferative gene ZAC is lost or highly reduced in nonfunc-
tioning pituitary adenomas. Cancer Res 2000;60:6794–9.
25. Singhal S, Amin KM, Kruklitis R, DeLong P, Friscia ME, Litzky LA,
Putt ME, Kaiser LR, Albelda SM. Alterations in cell cycle genes in
early stage lung adenocarcinoma identified by expression profiling.
Cancer Biol Ther 2003;2:291–8.
26. Koy S, Hauses M, Appelt H, Friedrich K, Schackert HK, Eckelt U.
Loss of expression of ZAC/LOT1 in squamous cell carcinomas of
head and neck. Head Neck 2004;26:338–44.
27. Abdollahi A, Bao R, Hamilton TC. LOT1 is a growth suppressor gene
down-regulated by the epidermal growth factor receptor ligands and
encodes a nuclear zinc-finger protein. Oncogene 1999;18:6477–87.
28. Colitti CV, Rodabaugh KJ, Welch WR, Berkowitz RS, Mok SC. A
novel 4cM minimal deletion unit on chromosome 6q25.1–q25.2 asso-
ciated with high grade invasive epithelial ovarian carcinomas Onco-
gene 1998;16:555– 9.
29. Abdollahi A, Pisarcik D, Roberts D, Weinstein J, Cairns P,
Hamilton TC. LOT1 (PLAGL1/ZAC1),
pressor gene at chromosome 6q24–25, is epigenetically regulated
in cancer J Biol Chem 2003;278:6041– 9.
30. Arima T, Drewell RA, Arney KL, Inoue J, Makita Y, Hata A,
Oshimura M, Wake N, Surani MA. A conserved imprinting control
region at the HYMAI/ZAC domain is implicated in transient neonatal
diabetes mellitus. Hum Mol Genet 2001;10:1475–83.
31. Varrault A, Bilanges B, Mackay DJ, Basyuk E, Ahr B, Fernandez C,
Robinson DO, Bockaert J, Journot L. Characterization of the methyla-
tion-sensitive promoter of the imprinted ZAC gene supports its role in
transient neonatal diabetes mellitus. J Biol Chem 2001;276:18653–6.
32. Arima T, Drewell RA, Oshimura M, Wake N, Surani MA. A novel
imprinted gene, HYMAI, is located within an imprinted domain on
human chromosome 6 containing ZAC. Genomics 2000;67:248–55.
33. Antequera F. High levels of de novo methylation and altered chroma-
tin structure at CpG islands in cell lines. Cell 1990;62:503–14.
34. Ueoka Y, Kato K, Kuriaki Y, Horiuchi S, Terao Y, Nishida J, Ueno
H, Wake N. Hepatocyte growth factor modulates motility and inva-
siveness of ovarian carcinomas via Ras-mediated pathway. Br J Can-
35. Inoue K, Sakurada Y, Murakami M, Shirota Y, Shirota K. Detection
of gene expression of vascular endothelial growth factor and flk-1 in
the renal glomeruli of the normal rat kidney using the laser microdis-
section system. Virchows Arch 2003;442:159–62.
36. Olek A, Walter J. The pre-implantation ontogeny of H19 methylation
imprint. Nat Genet 1997;17:275–6.
37. Wilkinson DG, Nieto MA. Detection of messenger RNA by in situ
hybridization to tissue sections and whole mounts. Methods Enzymol
38. Bakker J, Lin X, Nelson WG. Methyl-CpG binding domain protein 2
represses transcription from hypermethylated-class glutathione S-
transferase gene promoters in hepatocellular carcinoma cells. J Biol
39. Kohda Y, Murakami H, Moe OW, Star RA. Analysis of segmental
renal gene expression by laser capture microdissection. Kidney Int
40. Kolble K. The LEICA microdissection system: design and applica-
tions. J Mol Med 2000;78:B24–5.
41. El Kharroubi A, Piras G, Stewart CL. DNA demethylation reactivates
a subset of imprinted genes in uniparental mouse embryonic fibro-
blasts. J Biol Chem 2001;276:8674–80.
42. Jones PL, Veenstra GJC, Vermaak D, Kass SU, Landsberger N,
Strouboulis J, Wolffe AP. Methylated DNA and MeCP2 recruit his-
tone deacetylase to repress transcription. Nat Genet 1998;19:187–91.
43. Hendrich B, Guy J, Ramsahoye B, Wilson VA, Bird A. Closely
related proteins MBD2 and MBD3 play distinctive but interacting
roles in mouse development. Genes and Dev 2001;15:710–23.
44. Terao Y, Nishida J, Horiuchi S, Fengnian R, Ueoka Y, Matsda T,
Kato H, Furugen Y, Yoshida K, Kato K, Wake N. Sodium butyrate
induces growth arrest and senescence-like phenotypes in gynecologic
cancer cells. Int J Cancer 2001;94:257–67.
the candidate tumor sup-
KAMIKIHARA ET AL.