An ovarian adenocarcinoma cell line, BG-1, is well known for the expression of ERα [20]. This cell line was obtained from Dr. K.S. Korach (National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA). It was cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 mixture (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 100 U/mL penicillin G and 100 µg/mL streptomycin (Life Technologies, Rockville, MD, USA) at 37℃ in a humidified atmosphere of 5% CO₂-95% air.

After 70% confluence, BG-1 cells were treated with a concentration of E₂ (10^-7 M; Sigma-Aldrich) or BPA (10^-5 M; Sigma-Aldrich) at different time points (1, 8 and 24 h) before harvest. Ethanol was added to the control media in the same final solvent concentration (typically 0.1%).

Western blot assay was performed to identify the expression of ERα and ERβ in BG-1 cells. Thirty µg of total protein was run on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The membrane was Western-blotted using a mouse monoclonal antibody for ERα (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ERβ (Abcam, Cambridge, MA, USA). Briefly, 1×10⁶ cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in 100 µL of in ice-cold RIPA buffer [containing 1× PBS (pH 7.4), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/mL phenylmethylsulfonyl fluoride, 30 mg/mL aprotinin, and 10 mg/mL leupeptin] for 15 min on ice. The protein concentration was determined using Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). The protein solution was then subjected to electrophoresis on a 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane (Hybond C; Amersham Pharmacia, Oakville, ON, Canada). After washing three times with tri-buffered saline-Tween (TBS-T; 0.1% Tween-20 in TBS) for 15 min, the signals were detected with horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia) and visualized using the ECL chemiluminescent system (Amersham Pharmacia).

Total RNA was extracted with TriZol (Invitrogen Life Technologies, Carlsbad, CA, USA) according to manufacturer's suggested procedure, and purified using RNeasy total RNA isolation kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. DNA was digested using an RNase-free DNase set (Qiagen) during RNA purification. Total RNA was quantified by spectrophotometer and its integrity was assessed by running on a 0.8% agarose gel contained diethyl pyrocarbonate (DEPC). To make cDNA from total RNA for microarray analysis, the same quantity of each RNA sample from the treated groups (n=4) or control groups (n=3) was pooled. A cDNA microarray consists of approximate 8,000 cDNA spot including Incyte clones, housekeeping genes and Arabidopsis thaliana (A. thaliana) DNA as controls. The PCR was prepared according to the standard protocol and reaction mixtures were subjected to be amplified at 35 cycles. The primer pair used for amplification was overlap primer-1 (5'-AAT TAA CCC TCA CTA AAG GG-3') and overlap primer-2 (5'-GTA ATA CGA CTC ACT ATA GGG C-3'). The size and amount of the PCR products were verified on 1% agarose gel. The PCR products were purified by ethanol precipitation, then resuspended in 15 µL of hybridization solution (GenoCheck, Sangrokgu, Korea), and spotted onto CMT-GAPS II silane slide glass (Corning Life Sciences, Corning, NY, USA) with a pixsys 5500 arrayer (Cartesian Technologies Inc., Irvine, CA, USA) using 16-Stealth Micro spotting pins. The printed slides were processed according to CMT-GAPS II silane slide protocol. Briefly, the spots were re-hydrated with 1× SSC for 1 min and then DNA linked using a UV crosslinker (Stratagene Corp., La Jolla, CA, USA). The slides were soaked in the succinic anhydride/sodium borate solution for 15 min with gentle agitation and then transferred to a 95℃ water bath for 2 min. The slides were quickly transferred to 95% ethanol for 1 min and then dried using a centrifuge at 3,000 rpm for 20 sec.

Eighty mL of probe mixture was heated at 95℃ for 3 min, and kept at 65℃ until use. Prior to hybridization, array slide was incubated with prehybridization buffer containing 5× SSC, 0.1% SDS and 1% bovine serum albumin (BSA) at 48℃ for 45 min. The slide was washed by dipping in deionized water for 30 sec and dipped in isopropanol for 10 sec and spun dried in a centrifuge for 2 min. To hybridize probe with array slide, probe was added to the denatured array slide and cover slip was put on this slide. This slide was transferred to hybridization chamber and incubated overnight at 42℃. The slide was washed twice with wash buffer containing 1× SSC and 0.2% SDS at 42℃ and washed two times with 1× SSC at room temperature. Slide was then spun dried for 5 min. This microarray was scanned as described by the scanner's manufacturer. Microarray results were categorized into several functional groups related with apoptosis, cancer, cell cycle, growth factor/hormone, and signal transduction.

Hybridized slides were scanned with the Axon Instruments GenePix 4000B scanner and the scanned images were analyzed with the software program GenePix Pro 5.1 (Axon Instruments, Foster, CA, USA) and GeneSpring 7.1 (Sillicon Genetics, Redwood, CA, USA). In order to allow algorithm to eliminate all bad spots, no data points were eliminated by visual inspection from the initial GenePix image. For signal normalization, housekeeping genes (β-actin) and positive control genes (A. thaliana genes) were spotted onto each slide. The signals of these spots were used for normalization. To filter out the unreliable data, spots with signal-to-noise (signal-background-background SD) below 100 were not included in the data. Data were normalized by global, lowess, print-tip and scaled normalization for data reliability. Data were sorted of above 2-fold altered genes using GeneSpring 7.1 (Sillicon Genetics, Redwood, CA, USA) and a hierarchical clustering analysis was performed using Pearson correlation. The statistical significance of differential expression was assessed by computing a q-value for each gene. To determine the q-value, we used a permutation procedure, and for each permutation a two-sample t-statistic was computed for each gene. The result was considered significant when the logarithmic gene expression ratio of three independent hybridizations was more than twofold the difference in the expression level. The accuracy of microarray analysis in this study was confirmed by real-time PCR.

The standard curve was generated for a standard RNA preparation by serial dilution (1, 1/10, 1/100, 1/1000, and 0). A real-time PCR reaction was carried out in a 25 µL final volume containing 12.5 µL of 2× premix (TaKaRa Bio, Otsu, Japan), 0.3 µL of each of forward and reverse primers, 1 µL of cDNA, and distilled water up to 25 µL. The oligonucleotide sequences of primers were employed to detect various genes as shown Table 1. PCR using the Smart Cycle System (TaKaRa Bio) began with an initial denaturation at 95℃ for 30 sec. Each of the 35 amplification cycles consisted of denaturation at 95℃ for 5 sec, annealing at 55℃ for 15 sec, and extension at 72℃ for 15 sec. Relative expression levels of each sample were calculated based on the cycle threshold (Ct) and monitored for an amplification curve. The PCR amplification curves were evaluated by fluorescence of the double-stranded DNA-specific dye, SYBR Green, versus the amount of standardized PCR product. All gene expressions were normalized to the expression of β-actin mRNA as a control.

Data were presented as the mean±SD. Data of real-time PCR were analyzed by one-way ANOVA, followed by Dunnettos test for two-pair comparisons. P<0.05 was considered statistically significant.

We confirmed the expression of ERα and ERβ by immunoblot analysis using ERα and ERβ specific antibodies in ovarian cancer cells (BG-1). Both ERα and ERβ proteins were expressed in BG-1 cells and detected in 68 kDa and 55 kDa, respectively (Figure 1).

Following individual treatment of BG-1 cells with E₂ or BPA, total RNAs from the cells were isolated and processed by microarray analysis as described. Although the number of genes whose expression was altered by E₂ or BPA tested is modest, there is substantial change in the gene expression altered or modified by their exposure time. Increased expression levels were shown in red and decreased expression levels shown green (Figure 2). The intensities of each spot in this study were calculated and plotted. A clustering analysis of typical of the three self vs. self comparisons was performed in Figure 2, which showed that a more than two-fold change would be necessary to overcome the intrinsic variability introduced by the hybridization procedure. For this reason, the data was further filtered to select for genes that showed an absolute differential of at least two-fold or greater. The change in the mRNA expressions of 51 genes was listed in Table 2 and Table 3. Table 2 showed the common 33 genes related apoptosis, cancer and cell cycle induced by E₂ or BPA treatment at different time points (1, 8, and 24 h). In addition, altered gene expressions involved in signal transduction and nucleic acid binding were listed following treatment with E₂ or BPA in Table 3. These genes might be universal estrogen-responsive genes among various tissues. This compound exhibited a reasonable hierarchical clustering; BPA resembled to E₂.

The expression patterns of several genes up-regulated by E2 or BPA were confirmed to verify the results of the microarray analysis using real-time PCR analysis. The expression levels of altered genes including RAB31_MEMBER RAS ONCOGENE FAMILY (U59877), CYCLIN D1 (X59798), CYCLIN-DEPEND ENT KINASE 4 (U37022), INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 4 (U20982), and ANTI-MULLERIAN HORMONE (NM_000479) were measured by microarray analysis following treatment with E2 or BPA at 24 h in BG-1 cells. In parallel with their microarray results, the mRNA levels of these genes were significantly induced by E2 or BPA at 24 h as shown in Figure 3. Among them, the most increased gene was RAB31_MEMBER RAS ONCOGENE FAMILY (U59877) as demonstrated. These results indicate that BPA in parallel with E₂ induced the transcriptional levels of E₂-responsive genes in an ER-positive BG-1 cells.