Immortalized ovarian surface epithelia cells (IOSE) (30) were maintained in a 1:1 mixture of medium 199 (Sigma, St. Louis, MO, USA) and 105 (Sigma) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY, USA), 400 ng/ml hydrocortisone (Sigma), 10 ng/ml EGF and 50 U/ml of penicillin/streptomycin (P/S; Gibco). HeyC2 was maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% FBS and 50 U/ml of P/S, 1X MEM NEAA (Gibco) and 0.01 M of HEPES (Gibco). A2780 and its cisplatin-resistant sublines CP70, MCP2 and MCP3 cells were propagated in RPMI-1640 (Gibco) supplemented with 10% FBS and 50 U/ml of P/S. For demethylation treatment, cells were plated in a 60-mm plate and treated with 0.5 μM 5′-aza-2′-deoxycytidine (5-aza-dC; Sigma) for 3 days. For treatment with the EZH2 inhibitor (GSK126; Cayman Chemical, Ann Arbor, MI, USA), cells were either treated with 10 μM GSK126 alone for 3 days or combined with 5-aza-dC for 3 days. In the control experiment, cells were treated with DMSO. Culture medium and drugs were replaced every 24 h. Cells were then harvested for RNA analysis.

RNA extraction was performed using TRizol reagent (Life Technologies) according to the manufacturer’s protocol. To remove potential contaminating DNA from the complementary DNA, 1 μg of total RNA was treated with DNase I (Amplification Grade; Invitrogen) prior to reverse transcription. First strand cDNA synthesis used MMLV High Performance Reverse Transcriptase (Epicentre, Chicago, IL, USA). The expression of ARNTL in ovarian cancer cell line was examined by RT-PCR analysis. Ten-fold diluted cDNA (4 μl) was amplified in a total volume of 20 μl containing 2X SYBR^® Green Realtime PCR Master Mix (Toyobo, Osaka, Japan), 0.2 μM of each primer (Table I). Samples were first denaturated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, at annealing temperature for 30 sec, and extension at 72°C for 30 sec, followed by a melting curve. The relative gene expression level was determined by comparing the threshold cycle of the test gene against the Ct of GAPDH in a given sample. The qPCR reactions were carried out using ABI StepOne™ Real-Time PCR systems (Applied Biosystems, Foster city, CA, USA).

Before protein extraction, 1×10⁶ cells were seeded into 100-mm dish. When the cells reached 90% confluence, the medium was removed and the dish was washed with 1X PBS at 4°C. The cells were then lysed with 100 μl of PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea) according to the manufacturer’s protocol. Samples and pre-stained marker were loaded to a 12.5% polyacrylamide gel for electrophoresis. The proteins were then electrophoretically transferred from the gel onto a PVDF membrane by the Mini Trans-Blot^® Electrophoretic Transfer Cell system (Bio-Rad Laboratories, Hercules, CA, USA) at 400 mA for ~90 min. After the transfer, the membrane was incubated with 5% non-fat milk in 1X TBST for 1 h at room temperature. Then the first antibodys, ARNTL antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or actin antibody (Santa Cruz Biotechnology) was diluted with 5% non-fat milk in 1X TBST and was added into the membrane followed by incubation for overnight at 4°C. The membrane was then washed in 1X TBST at room temperature 3 times. The secondary antibody conjugated with horseradish peroxidase (IgG-HRP antibody) was also diluted with 5% non-fat milk in 1X TBST and incubated with membrane at room temperature for 1 h. The membrane was washed with 1X TBST at room temperature 3 times. The Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA) was prepared by mixing equal volumes of the HRP substrate luminol reagent as well as the HRP substrate peroxide solution and added onto the membrane. Finally, the light-signal was detected by BioSpectrum^® 2D Imaging System (UVP, Upland, CA, USA).

DNA was bisulfite modified using EZ DNA methylation kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. For COBRA analysis, 4 μl of bisulfite converted DNA was first amplified using specific primers (Table I) targeting promoter region of ARNTL followed by digested with 20 U of BstUI (CGCG) (New England Biolabs, Ipswich, MA, USA). In vitro methylated DNA (IVD) (Merck-Millipore) was used as a positive control for methylation and water was used as negative control. For the un-digested control, water instead of BstUI was added. Digested products were separated on 1.5% agarose gel for visualization. The PCR reactions were carried out using the Veriti^® 96-Well Thermal Cycler (Applied Biosystems).

The CDS of ARNTL was amplified by PCR using specific primers (Table I) from cDNA of IOSE cells which expressed ARNTL. The PCR products were ligated into pIRES2-EGFP vector (Promega, Madison, WI, USA) for sequencing confirmation. ARNTLpIRES was then digested with NheI (New England Biolabs) and BamHI (New England Biolabs), and inserted into multiple cloning site of pIRES2-EGFP expression vector, which was predigested with NheI and BamHI. ARNTL expression vector or empty vector were transfected into CP70 and MCP2 cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. The PCR reactions were carried out using the Veriti^® 96-Well Thermal Cycler (Applied Biosystems).

Following transient transfection, the transfected cells were cultivated with fresh culture medium containing 400 μg/ml geneticin (G418; Sigma) and replaced every 3 days. Each single colony that formed was selected for further culture.

CP70 and MCP2 cells were plated at 1×10⁶ cells/plate in a 100-mm culture dish one day before transfect ARNTL. Transfection was performed as previously described. On the second day after the transfection, cells were plated into 3 plates with fresh culture medium containing 400 μg/ml G418 (Sigma) and replaced every 3 days. Surviving colonies were stained with 0.4% crystal violet (Sigma) in 50% methanol and visible colonies were counted. The colony numbers were counted using Image-Pro 3D Suite software version 5.1.1.38 for windows (Media Cybernetics, Inc., Bethesda, MD, USA).

Cell growth was assessed by MTS assay, as previously described. In brief, for MTS assays, 1×10³ cells were seeded in 96-well plates for 4 days with or without various concentrations of cisplatin (Sigma). Cell growth was determined using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega), according to the manufacture’s protocol. Relative cell numbers were assessed using a 96-well ELISA plate reader (Multiskan^® FC Microplate Photometer; Thermo Fisher Scientific Inc., Waltham, MA) with an absorbance set at 492 nm.

base layer of agar (2.5 ml) (0.5% agar in culture medium) was allowed to solidify in a 6-well flat bottomed plate before the addition of 2 ml of cell suspensions containing 1×10⁴ cells in 0.3% agar in culture medium. The cell-containing layer was then solidified at room temperature for 20 min. Colonies were allowed to grow for 14–21 days at 37°C with 5% CO₂ before imaging. Plates were stained with 1 mg/ml iodonitrotetrazolium chloride (Sigma) overnight at 37°C. The colonies which contained at least 50 cells were counted. The colony numbers were counted using Image-Pro 3D Suite software version 5.1.1.38 for windows (Media Cybernetics).

Cells (1×10⁵) were seeded in 60-mm plates for 2 days with or without 1 mg/ml of cisplatin (Sigma). The supernatant was carefully collected, and then wash in PBS. Followed by centrifugation at 200 × g for 5 min at room temperature. The supernatant was aspirated, the pellet resuspended in ~5 ml cold 1X PBS, and then centrifuged for 5 min at 200 × g. Cells were fixed by adding 4.5 ml of 70% (v/v) cold ethanol to the cell suspension keeping the tubes on ice. The cells were stored in ethanol solution at −20°C at least for 2 days. To remove the ethanol solution, the supernatant was centrifuged at 400 × g for 5 min, the cells were washed in 5 ml of PBS and centrifuged at 400 × g for 5 min. The supernatant was removed and the cells were resuspended in 500 μl of propidium iodide (1 mg/ml), then incubated for at least 30 min at room temperature in the dark. Cells were analyzed by flow cytometry.

Cells were grown to confluence in 100-mm culture dishes in RPMI-1640 media (Invitrogen) supplemented with 10% FBS, followed by culture for 2 days with starvation medium (0.5% serum). At time t=0, the medium was exchanged for 50% serum in RPMI-1640; after 2 h, the medium was replaced with serum-free RPMI-1640. At the indicated times (0, 4, 8, 12, 16 and 20 h) the dishes were prepared for immediate RNA extraction.

cells (1×10⁶) were cross-linked with 1% fresh formaldehyde and then washed with cold 1X PBS in the presence of the protease inhibitor. Cell were homogenized and the chromatin was subjected to ChiP pull down using magnetic Dynabeads (Invitrogen) and antibodies (against H3K4me3; Active Motif, Carlsbad, CA, USA; against H3K27me3; Merck-Millipore; EZH2; Cell Signaling Technology, Danvers, MA, USA; and IgG; Cell Signaling Technology). The quantity of ChIP DNA was measured by quantitative-PCR analysis using 200 pg of pull-down DNA amplified by ARNTL promoter specific primer (Table I), and then quantified as a percentage of the input signal. The PCR reactions were carried out using the Veriti^® 96-Well Thermal Cycler (Applied Biosystems).

Comparison between groups were assessed by the unpaired t-test. All statistical calculations were done using the statistical package SPSS version 13.0 for windows (SPSS, Inc., Chicago, IL, USA). A P-value <0.05 was considered statistically significant.

To investigate genes that are hypermethylated in ovarian cancer cell lines, we performed meDIP-Chip using Agilent 244K CpG island microarray in IOSE and a panel of ovarian cancer cell lines. One of the targets that showed promoter hypermethylation in a sub-set of ovarian cancer cell is ARNTL (also known as BMAL1). To confirm our result, we performed quantitative real-time RT-PCR to examine the mRNA level of ARNTL in these cancer cell lines. Expression of ARNTL was downregulated in A2780, CP70, MCP2 and MCP3 ovarian cancer cell lines as compared with IOSE (Fig. 1A). To examine if epigenetic modifications contribute to this downregulation, we treated the cells with DNA methylation inhibitor, 5-aza and/or the EZH2 inhibitor, GSK126 (31) in CP70, MCP2 ovarian cancer cell lines. The resulted indicated that treatment of 5-aza alone resulted in a partial re-expression of ARNTL in CP70 cells, while treatment with GSK126 resulted in a robust re-expression of ARNTL in these cells, thus, suggesting that ARNTL silencing may occur through epigenetic events (Fig. 1B).

To further confirm if epigenetic modifications contribute to the silencing of ARNTL, we examined DNA methylation in the promoter region of ARNTL in the ovarian cancer cell lines. Combined bisulphite restriction analysis (COBRA) was conducted to determine the methylation status of 342 bp CpG island spanning (+421 bp to +763 bp) region around the transcription start site in the promoter region of ARNTL. Promoter methylation was obvious in MCP2, MCP3 and A2780 cells (Fig. 2). However, promoter methylation was not observed in IOSE and HeyC2 cells which expressed ARNTL. Notably, methylation was not observed in CP70 cells which did not express ARNTL.

Beside DNA methylation, histone modifications also control gene expression through their effects on chromatin structure. We then analyzed the histone modifications for the active and repressive chromatin marks in the ARNTL promoter. Chromatin immunoprecipitation coupled with PCR (ChIP-PCR) was performed using antibodies against trimethylated lysine 4 of histone H3 (H3K4me3), trimethylated lysine 27 of histone H3 (H3K27me3) and EZH2. As expected, active histone mark trimethyl-H3K4 but not repressive mark trimethyl-H3K27 was enriched in ARNTL-expressing HeyC2 cells. On the contrary, trimethyl-H3K27 and EZH2 but not trimethyl-H3K4 were enriched at the promoter region of CP70 and MCP2 cells (Fig. 3B). Hence, histone modifications were also involved in the gene silencing mechanism of ARNTL in ovarian cancer cell lines.

Having demonstrated that ARNTL is epigenetically silenced by DNA methylation and histone modifications, we then investigated the role of ARNTL in ovarian cancer cell lines. By colony formation assay, CP70 or MCP2 cells transfected with ARNTL showed a significant decrease in colony numbers as compared to control cells (Fig. 4).

To investigate the effect of ARNTL to anchorage-dependent growth and drug resistance, we selected cells that stably overexpressed ARNTL in MCP2 ovarian cancer cells. The expression of ARNTL of the stable clones was confirmed by quantitative RT-PCR and western blot analysis (Fig. 5A and B). By soft-agar assay, ARNTL-ovexpressed cells demonstrated a dramatic decrease in colony numbers as compared to control cells (Fig. 5C). Finally, we examined if ARNTL can affect chemosensitivity in these drug-resistant MCP2 cells. Overexpression of ARNTL restored the chemosensitivity to cisplatin in these cells (Fig. 5D). Taken together, these results demonstrated that ARNTL may function as a potential tumor suppressor in ovarian cancer.

To determine the mechanism of ARNTL in restoring chemosensitivity in MCP2 cells, we investigated the effect of ARNTL on apoptosis. Although ARNTL did not increase the number of sub-G1 population under normal condition, overexpression of ARNTL showed a marked increase in the number of cells in the sub-G1 population under cisplatin treatment (P<0.01; Fig. 5E). These results indicated that ARNTL may restore the DNA damage-induced apoptosis in MCP2 ovarian cancer cells.

Finally, we investigated the functional impact of restoring ARNTL expression in ovarian cancer cells. This is particularly interesting because the expression of several mammalian cell cycle genes, such as c-myc (16,32,33), is regulated in a circadian manner. Therefore, we synchronized the ARNTL stable transfectants by serum starvation. RNA was then extracted after serum restoration at the indicated time for gene expression analysis. Notably, expression of c-myc displayed a circadian rhythm in both of the ARNTL-expressing MCP2 clones (Fig. 5F), whereas a relatively constant level of expression without any rhythmicity was observed in the control cells.