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: firstname.lastname@example.org 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. 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