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ONCOLOGY REPORTS 11: 617-622, 2004
Loss of estrogen receptor-a expression is associated with hypermethylation near its ATG start codon in gastric cancer cell lines
IN SOOK WOO 1 , MYUNG JAE PARK 2 , SEONG WON CHOI 3 , SUNG JOO KIM 4 , M Y U N G A H L E E 1 ,
J I N H Y U N G K A N G 1 , YOUNG SEON HONG 1 and KYUNG SHIK LEE 1
d e p a r t m e n t of Internal Medicine, College of Medicine, Catholic University, Seoul 137-040; Department of
Internal Medicine, College of Medicine, Kyung Hee University, Seoul 130-701; Department of Pharmacology,
College of Medicine, Seoul National University, Seoul 110-799; Division of Molecular Genetics,
Catholic Research Institutes of Medical Science, Catholic University, Seoul 137-040, Korea
Received September 22, 2003; Accepted November 11, 2003
Abstract. The proportion of gastric cancers positive for
estrogen receptor (ER)-a expression is reported to be between
0-67%, depending upon the study. The role of ER-a in gastric
carcinogenesis is unclear. The E R - a gene is located at
chromosome 6q25.1, and the long arm of chromosome 6 has
been known as a site with frequent loss of heterozygosity
(LOH) in gastric cancer. ER expression is linked to suppression
of cell proliferation in vitro. Epigenetic inactivation might
explain the loss of ER-a gene expression in gastric cancer.
Given there is no information a v a i l a b l e regarding the
methylation status of the ER-a gene promoter region in
gastric cancer, we investigated such methylation in 13 gastric
cancer cell lines. Western blot analysis, reverse transcriptionpolymerase chain reaction (PCR), methylation-specific PCR
(MS-PCR) and bisulfite sequencing analyses were used. ER-a
protein was not detected in any cell line, although ER-a
mRNA was detected in 1 of 13 gastric cancer cell lines. MSPCR and bisulfite sequencing showed all 13 gastric cancer
cell lines had methylated CpG regions in their ER-a gene
promoters. In conclusion, inactivation of ER-a gene expression
in gastric cancer cell lines appears associated with CpG
island methylation near the TGA initiation codon of the ER-a
gene.
Introduction
DNA methylation occurs when a methyl group is added to
the cytosine of a cytosine-guanosine pair (CpG). Aberrant
methylation of CpG islands in promoter regions results in
Correspondence
to: Dr In Sook Woo, Department of Internal
Medicine, Kangnam St. Mary's Hospital, The Catholic University
of Korea, Banpo-dong 505, Seocho-gu, Seoul 137-040, Korea
E-mail: insookwoo@catholic.ac.kr
gene silencing or reduced gene expression, including tumor
suppressor genes (1). Several reports suggest the estrogen
receptor (ER) may mediate inhibition of cell division. The
activated ER gene was reported to suppress growth of a
neuroblastoma cell line (2). Introduction of the ER gene into
ER-negative colon carcinoma cells was found to cause marked
growth suppression (3). Recovery of an epigenetically
inactivated ER gene resulted in growth suppression of colon
cancer cells in vitro and in vivo (4). These data support the
role of the ER gene as tumor suppressor gene in carcino­
genesis. The ER-a gene codes for an isoform of the ER, and
is located on chromosome 6q25.1 (5). Deletions of the long
arm of chromosome 6 are common in gastric carcinoma (6),
suggesting the presence of tumor suppressor genes in this
region. Gastric cancer is a disease with poor prognosis, and
there is still much to u n d e r s t a n d about the genes that
contribute to its progression. The percent of ER-positive
gastric cancers ranges between 0-67% depending on the
method of detection (7). Another ER isoform, ER-6, is highly
expressed in gastric cancer compared with E R - a (8,9).
However, the significance of ER expression and hormone
manipulation in gastric cancer is not established. The loss of
ER-a protein expression in breast cancer can result in hormone
resistance and poorer clinical outcome, and correlates with
methylation of CpG islands in the 5' region of the ER-a gene
(10). Non-sex hormone-dependent tumors, including colon,
kidney, pancreas and liver, are reported to express the ER
(11-13). And methylation of the ER-a gene promoter region
was also reported in esophageal cancer, hematopoietic
n e o p l a s m , b r a i n t u m o r and in c o l o n c a n c e r ( 1 4 - 1 7 ) .
However, epigenetic inactivation of the ER-a gene in gastric
cancer has not been previously reported. The aim of this
study was to assess the expression of ER-a in gastric cancer
cell lines and d e t e r m i n e whether methylation of the 5'
promoter region is associated with loss of ER-a expression in
gastric cancer.
Material and methods
Key words: estrogen receptor-a, methylation, gastric cancer
Cell lines. Thirteen human gastric cancer cell lines (SNU 1,
SNU 5, SNU 16, SNU 484, SNU 520, SNU601, SNU620,
618
WOO et al: ER-a IN GASTRIC CANCER
Figure 1. ER-a gene promoter region. Primer sequences (wild-type) used for methylation-specific PCR (MS-PCR) are shown within the boxed region. The
MS-PCR product size is 123 bp. CAAT box and the ATG codon are in bold within the shadowed box. Nl, nucleotides according to Genbank (accession
no. X03635); N2, nucleotides relative to translation start site. 0, translation start site.
SNU 638, SNU 668, SNU719, AGS, MKN 45, KATO III)
were used. Cells were maintained in RPMI-1640 (GibcoBRL, Rockville, MD, USA) supplemented with 10%
(vol/vol) fetal bovine serum (FBS) at 37°C in an atmosphere
of5%C0 2 .
Western blotting. Sub-confluent cell cultures from 75 cm2
flasks were used for protein extraction. Cells were washed
twice with phosphate-buffered saline (PBS), scraped off the
plates, and lysed in cell lysis buffer [100 mM Tris-HCl
(pH 7.5), 1 M NaCl, 20% Triton X-100, 10% sodium deoxycholate and 20% sodium dodecyl sulfate (SDS)] with protease
inhibitors (2 mM PMSF, 10 mM sodium fluoride, 1 mM
sodium orthovanate, 1 mg/ml leupeptin, 1 mg/ml pepstatin),
pelleted by centrifugation and frozen at -70°C. The protein
concentration was measured by Bradford assay (Bio-Rad
Laboratories, Hercules, C A, USA). Proteins (40 \ig) were
separated by 10% SDS-polyacrylamide gel electrophoresis
(Biocraft, Tokyo, Japan). Proteins were transferred to nitro­
cellulose membranes (Hybond ECL, Amersham Pharmacia
Biotech, Buckinghamshire, UK), membranes blocked with
5% skim milk and 0.2% Tween-20 in Tris-buffered saline
(TBS-T) overnight at 4°C, and then incubated with a mouse
monoclonal antibody against ER-a (1:500 dilution, catalog
no.sc-8002, Santa Cruz Biotechnology, Santa Cruz, CA,
USA). After washing in 0.2% Tween-20 in PBS 3 times
for 10 min at room temperature, a goat anti-mouse IgGHRP (1:2000 dilution) was used as a secondary antibody.
Antibody binding was visualized by chemiluminescence
(Amersham ECL). Restripping of blots was achieved by
immersion in a solution containing 100 mM B-mercaptoethanol, 2% SDS, 62.5 ^iM Tris-HCl, pH 6.7, for 30 min at
60°C with agitation. B-actin detection on blots was carried
out as for ER-a.
RNA extraction and reverse transcription-PCR (RT-PCR).
Total RNA was extracted using Trizol reagent (Invitrogen,
Carlsbad, CA), and 5 jig was converted to first-strand cDNA
using Superscript II Reverse Transcriptase (Invitrogen) with
random hexamers, according to the manufacturer's protocol.
The set of primers used for ER-a was 5'-TGCCAAGGAGA
CTCGCTA-3' and 5'-TCTTGTTCTGGACAGGGATG-3'
(9).
Another set of primers (5'-AAGGTCATCCATCCATGA
CAAC-3' and 5'-CACCCTGTTGCTGTAGCCA-3') was
used for the GAPDH gene to evaluate the efficiency of cDNA
synthesis from each cell line (18). cDNA conversion mixture
(1 |al) was suspended in a total volume of 20 jal of IX PCR
buffer containing 10 mM Tris-HCl, pH 9.0, 50 mM KC1,
1.5 mM MgCl2, 300 nM of each primer, 250 |iM deoxynucleotide triphosphate and 0.5 U TaqDNA polymerase
(Amersham Pharmacia Biotech, Cleveland, OH). PCR
conditions were as follows: for ER-a, 60 sec at 94°C, 60 sec
at 50°C and 2 min at 72°C, 35 cycles; for GAPDH, 60 sec at
95°C, 60 sec at 55°C and 90 sec at 72°C, 30 cycles.
DNA extraction and sodium bisulfite treatment. DNA was
isolated from cultured cell lines using a QIAamp DNA mini
kit (Qiagen, Valencia, CA), according to the manufacturer's
instructions. Genomic DNA (1 ]J,g) was modified with sodium
bisulfate using a CpGenome™ DNA modification kit (Intergen,
Purchase, NY, USA), according to the manufacturer's
recommendations. With bisulfite treatment, cytosine residues
are deaminated and converted to uracil residues, while
methylated cytosine remains unmodified.
Methylation-specific PCR (MS-PCR). The modified DNA
was subjected to MS-PCR around the TGA translation start
site of the ER-a gene. The promoter region used for the
ONCOLOGY REPORTS 11: 617-622, 2004
619
Figure 2. a, Western blot of protein extracts (40 |ig protein) from 12 gastric cancer cell lines probed with antibody against ER-a. MCF-7 lysates were used as a
positive control for ER-a. The same blot was stripped and reprobed with antibody against B-actin. b, RT-PCR analysis of ER-a mRNA expression in 13 gastric
cancer cell lines. GAPDH mRNA levels were determined in order to standardize RNA signals.
MS-PCR is shown in Fig. 1. Each set of unmethylated (U)
and methylated (M) primers were as follows: unmethylated
(forward) 5'-GGATATGGTTTGTATTTTTGTTTGT-3', and
unmethylated (backward) 5'-ACAAACAATTCAAAAACT
CCAACT-3', methylated (forward) 5'-GATACGGTTTGTAT
TTTTGTTCGC-3', and methylated (backward) 5'-CGAACG
ATTCAAAAACTCCAACT-3' (19). The size of product was
123 bp. The PCR mixture contained PCR buffer (10 mM
Tris-HCl, pH 9.0, 50 mM KC1, 1.5 mM MgCl2), 250 nivl
deoxynucleotide triphosphate, 2 pmol/ml of each primer, 0.5
U TaqDNA polymerase (Amersham Pharmacia Biotech) and
1 |al bisulfate-modified DNA in a final volume of 20 |xl.
PCR conditions were as follows: for unmethylated reactions,
30 sec at 94°C, 30 sec at 46°C, 45 sec at 72°C and 8 min at
72°C, 40 cycles; for methylated reactions, 30 sec at 94°C,
45 sec at 49°C, 45 sec at 72°C and 8 min at 72°C, 40 cycles.
Sequencing analysis. To confirm the results of MS-PCR, 40 |il
PCR product was purified using a QIAquick PCR purification
kit (Qiagen) and cloned into the pGEM-T vector using the
TA cloning System (Promega, Madison), according to the
manufacture's instructions. Ten white colonies per sample
were selected and plasmid DNA extracted using a QIAprep®
miniprep plasmid DNA purification kit (Qiagen). The inserted
PCR fragments from individual clones, obtained from each
sample were sequenced with a M13 (-20) primer. DNA
sequences were determined using an ABI PRISM 373 DNA
sequencer (Perkin-Elmer, Norwalk, CT) with the ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer).
Results
Expression of ER-a in gastric cancer cell lines. ER-o
expression was examined in gastric cancer cell lines using
Western blot and RT-PCR analyses. The MCF-7 breast
cancer cell line was used as a positive control. We analyzed
12 gastric cancer cell lines by Western blotting and found
none expressed the 67 kDa ER-a protein (Fig. 2a). Anti-Bactin antibody staining was used to determine equal loading
of lanes and to test the integrity of the protein homogenates
used for Western blot analysis. This staining showed there
was no evidence of protein degradation in the cell line lysates
and there was approximately equal protein loading in each
lane. An RT-PCR-based analysis was performed to determine
ER-a mRNA expression in 13 gastric cancer cell lines. ER-a
mRNA was detected in 1 of 13 gastric cancer cell lines Kato III (Fig. 2b).
Methylation of the promoter region around the translation
initiation codon is associated with gene silencing. We
examined whether loss of ER-a expression in the cell lines
was associated with DNA methylation in the CpG islands of
the ER-a gene. This analysis used MS-PCR and primers that
hybridized around the translation initiation. We found all
13 gastric cancer cell lines, including Kato III, had methylated
sites in the CpG islands of the ER-a gene (Fig. 3). To confirm
methylation of the promoter region, MS-PCR bisulfite genomic
sequencing was performed. Bisulfite modification was
successful, with all cytosines at non-CpG sites being converted
to thymines (Fig. 4a). We found all CpGs within PCR product
were methylated in several sequenced clones derived from
SNU 1. We found all 8 CpGs were methylated in several
sequenced clones derived from SNU 1 cells. However, in
Kato III cells, which expressed a weaker mRNA signal and
showed methylation by MS-PCR, sequencing showed
unmethylation of 2 CpGs (Fig. 4b). This finding suggests
partial methylation near the translation start codon may result
in loss of ER-a protein expression, but not ER-a mRNA
expression.
620
WOO et al: ER-a IN GASTRIC CANCER
Figure 3. Methylation-specific PCR for the ER-a gene was performed after bisulfite modification of DNA from gastric cancer cell lines. M, methylated ER-a
PCR products; U, unmethylated ER-a PCR products. The DU145 prostate cancer cell line was used as a positive control for ER-a gene methylation, while
MCF-7 cells was used as a source of unmethylated ER-a genes. A DNA template negative control (H 2 0) was processed concurrently (-). Molecular markers
(50-2,000 bp) are shown on the left.
Figure 4. Bisulfite genomic sequencing. The positions of primers are underlined. All cytosine (C) are deaminated and converted to thiamine (T) compared with
the sequence in Fig. 1. In cases of methylation, cytosine prior to guanine remains as 5-methylcytosine (*). Unmethylated CpGs are indicated by (•). a, SNU 1
cells; b, Kato III cells. The ATG translation initiation site is marked with an arrow.
Discussion
Gastric cancer is the most common malignancy, and the
second most common cause of cancer-related death in Korea
(20). The majority of gastric cancers show distant metastasis
at the time of diagnosis. Although various combinations of
chemotherapeutic agents have been used in attempts to
improve response rates and survival times for stomach cancer
patients, many patients still suffer from resistance to chemo­
therapy. Our clinical experience showing that gastric cancer
can occur in young women (under the age of 35) and can be
diagnosed in advanced stages in the perinatal period suggests
sex hormones may have an influence on gastric carcinogenesis.
Estradiol at physiological concentrations is reported to
stimulate proliferation in gastric cancer cell lines, and the
active metabolite of the ER inhibitor was shown to enhance
growth in gastric cancer cell lines (21). Despite Tokunaga
et al first reporting ER expression in gastric cancer in 1983
(22), its role in gastric carcinogenesis remains unknown.
Biochemical detection of the ER in gastric cancer tissue is
ONCOLOGY REPORTS 11: 617-622, 2004
similar to that seen in breast and endometrial cancers,
suggesting a hormone connection to gastric cancer (23).
Immunohistochemical and RT-PCR analyses suggest higher
expression of ER-B than ER-cc in gastric cancer (8). Takano
et al found ER-cc mRNA was expressed in 2 of 6 gastric cancer
cell lines (9). They evaluated ER-a and ER-B mRNA in paired
normal and tumor samples and altered mRNA expression was
related to increased metastatic potential in gastric cancers.
The proportion of gastric cancers that are ER-a positive
has been reported to be between 0-67% (7). Several studies
suggest the ER gene might be a tumor suppressor gene (2,4).
The ER-a gene was assigned to chromosome 6q24-27, and
was more precisely mapped to 6q25.1 by fluorescence in situ
hybridization (FISH) (5,24). Frequent loss of heterozygosity
(LOH) in the long arm of chromosome 6 in gastric cancer
and other malignancies suggest a tumor suppressor gene is
present in this region (6,25-28). LOH of the ER gene was
found to occur in about 20% of breast cancers (29,30), but
has not been reported in gastric cancer.
ER expression has been observed in colon cancer, renal
cell carcinoma, pancreatic cancer and hepatoma, suggesting a
role for hormone therapy in treatment of these malignancies
(7,11-13). There are 2 isoforms of the human estrogen receptor,
ER-a and ER-6, which are structurally and functionally
distinct. A report on expression of ER isoforms in pituitary
tumors suggests tumor character and responsiveness to estrogen
and anti-estrogen is dependent on the isoform expressed (31).
In the present study, ER-a protein expression was lost in all
12 gastric cancer cell lines assayed by Western blotting, while
ER-a mRNA was absent in 12 of 13 cell lines tested. Growth
of the Kato III cell line was reported to be suppressed by
tamoxifen or tamoxifen plus 5-fluorouracil. These cells were
said to be ER-negative, and sensitivity to tamoxifen was not
dependent on ER expression (32). However, in the present
study, RT-PCR analysis amplified ER-a mRNA from Kato III
cells, consistent with a report by Takano et al (9). The
mechanism underlying the loss of ER-a expression in gastric
carcinogenesis is not yet understood. Homozygous deletions
or other mutations which could result in loss of the ER-a gene
have not been reported in gastric cancer. One mechanism that
can cause gene inactivation is methylation of CpG islands in
the 5' regulatory region, and treatment with the demethylating
agent 5-aza-2'-deoxycytidine (5-aza-dC) can restore gene
expression. Methylation of the ER-a gene has been reported
in breast cancer and other cancers, including prostate cancer,
esophageal adenocarcinoma, brain tumor, colon cancer and
hematologic neoplasm (14-17,19,33). Loss or mutation of the
ER-a protein in breast cancer results in hormone resistance,
and has a poor clinical outcome (10). Hypermethylation of
the ER gene has been associated with a better prognosis in
AML (33). To date, the methylation status of the ER-a gene
promoter in gastric cancer has not been reported. Using MSPCR we observed methylation of DNA from gastric cancer cell
lines, and this was confirmed by bisulfite genomic sequencing.
Our data suggest methylation in the promoter region CpG
sites around the ATG start codon can cause loss of ER-a
gene expression. Two unmethylated CpG sites were observed
in Kato III cells, unlike SNU 1 cells which did not express
ER-a mRNA and had all 8 CpG sites methylated, including
the primer region.
621
In summary, our data indicate that hypermethylation of
the ER-a promoter may explain the loss of ER-a expression
in gastric cancer cell lines. Treatment with a demethylating
agent, 5-aza-2'-deoxycytidine (5-aza-dC), should be performed
to observe whether the epigenetic alteration to ER-a can be
reversed.
The expression and promoter methylation status of ER-a in
normal gastric mucosa and tissue samples from patients with
gastric cancer should be also investigated to clarify the role of
promoter methylation of ER-a gene in gastric tumorigenesis.
In addition, future studies should examine other possible
mechanisms that may lead to loss of ER-a expression in
gastric cancer. Our findings may ultimately provide the basis
for basic and applied studies exploring the possibility of
hormone treatment for gastric cancer.
Acknowledgment
We acknowledge the financial support of the Catholic
Medical Center Research Foundation in 2003. We thank Dr
Bang YJ (Seoul National University), Park JS (Catholic
University) and Dr Kim SS (Kyung Hee University) for
providing cell lines. Cell lines were also obtained from the
Korean Cell Line Bank (Seoul). We thank Kang GH (Seoul
National University), and Dr Joe YA (Catholic Medical
Center Research Institute) for helpful comments.
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