490 INTERNATIONAL JOURNAL OF ONCOLOGY 41: 490-496, 2012 Genome-wide screening for methylation- silenced genes in colorectal cancer AHMED KHAMAS1, TOSHIAKI ISHIKAWA1,2, KAORU MOGUSHI3, SATORU IIDA1, MEGUMI ISHIGURO1, HIROSHI TANAKA3, HIROYUKI UETAKE1,2 and KENICHI SUGIHARA1 Departments of 1Surgical Oncology, 2Translational Oncology, Graduate School of Medicine, 3Department of Computational Biology, Graduate School of Biomedical Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Received January 20, 2012; Accepted April 9, 2012 DOI: 10.3892/ijo.2012.1500 Abstract. Identification of methylation-silenced genes in suppressor genes, due to epigenetic masking can contribute to colorectal cancer (CRC) is of great importance. We employed cancerous transformation by inducing selective growth advan- oligonucleotide microarrays to identify differences in global tages (3). gene expression of five CRC cell lines (HCT116, RKO, Colo320, Microarray analysis can lead to the identification of changes SW480 and HT29) that were analyzed before and after treatment in gene expression after the treatment of cells with 5-aza- with 5-aza-2'-deoxycitidine. Selected candidates were subjected 2'-deoxycitidine (5-aza-dC), a methyltransferase inhibitor that to methylation-specific PCR and real-time quantitative reverse can cause induction of epigenetically silenced genes (4-7). transcription-PCR using 15 CRC cell lines and 23 paired tumor Profiling of methylated genes is growing rapidly as a and normal samples from CRC patients. After 5-aza-2'-deoxy- powerful diagnostic tool for the early detection, prognosis and citidine treatment, 139 genes were re-expressed in all 5 CRC even prediction of clinical response to treatment of various cell lines collectively with a fold change of more than 1.5 in at cancer types (8). This encourages the search for new candidate least one cell line. These genes include known methylated and genes using global demethylation and microarray analysis that silenced genes in CRC. After applying study selection criteria we could effectively find genes silenced in association with promoter identified 20 candidates. TheGADD45B and THSD1 genes were hypermethylation in several tumor types, including CRC, with selected for further analysis. Among 15 colon cancer cell lines, a role in cell proliferation, tumor progression, apoptosis or methylation was only identified inTHSD1 (27%). THSD1 methy- angiogenesis (8-10). In CRC, the path of epigenetic silencing has lation was subsequently investigated in 23 colorectal tumors not yet been fully explored and further searches for methylation- and methylation was detected in 9% of the analyzed samples; silenced genes are required. Despite the fact that hundreds of the observed promoter hypermethylation was cancer-specific. genes have been identified to be silenced through promoter CpG THSD1 mRNA down-regulation was observed in tumor tissues. island methylation in CRC (11). This genome-wide screening led to the identification of genes In this study, genome-wide demethylation and expression putatively affected by methylation in CRC. The THSD1 gene microarray screening were carried out to discover putative genes may play a role in the tumorigenesis of CRC. inactivated by methylation in CRC. Introduction Materials and methods The transcriptional inactivation of genes through CpG islands Cell lines and 5-aza-dC treatment. Cell lines used in this study hypermethylation at their promoters is a widely reported mecha- were obtained from the Cell Resource Center for Biomedical nism in human malignancies, including colorectal cancer (CRC) Research, Tohoku University (Miyagi, Japan) and the American (1,2). Reduced expression of genes, in particular the tumor Type Culture Collection (Manassas, VA, USA). Fifteen CRC cell lines (CCK81, CoCM1, Colo205, Colo320, ColoTC, DLD1, HCT116, HCT15, HT29, LoVo, RCM1, RKO, SW48, SW480 and WiDr) were cultured in appropriate medium and under conditions described by the providers, with media obtained Correspondence to: Dr Hiroyuki Uetake, Department of Translational Oncology, Graduate School of Medicine, Tokyo Medical from Gibco (Grand Island, NY, USA) or Sigma (St. Louis, MO, and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, USA), supplemented with 10% heat-inactivated fetal bovine Japan serum (FBS; Nichirei, Tokyo, Japan), 100 µg/ml streptomycin, E-mail: [email protected] and 100 U/ml penicillin (Invitrogen, Carlsbad, CA, USA). HCT116, RKO, Colo320, SW480 and HT29 CRC cell lines Key words: oligonucleotide microarray, CpG island, methylation, were treated with 5-aza-dC, each experiment was performed in 5-aza-2'-deoxycitidine, colorectal cancer triplicate. The five cell lines were cultured at 37˚C in 5% CO2. Cells (5x103 per well) were plated in their respective culture KHAMAS et al: PUTATIVE METHYLATED GENES IN COLORECTAL CANCER 491 media on day 1, on day 3 cultured cells were treated with PBS treatment of gDNA converts unmethylated cytosine residues dissolved and filter-sterilized 0.5 µM 5-aza-dC (Sigma) for 72 h (but not methylated cytosine) to uracil, which is then converted with media changed every 24 h, treatments were performed in to thymidine during the subsequent PCR step, giving sequence parallel with drug-free phosphate-buffered saline (PBS) as a differences between methylated and unmethylated DNA. control. The dose of 5-aza-dC was administered on the basis The methylation status of the cyclin-dependent kinase inhi- of its pharmacological dose and the results of our preliminary bitor 2A (CDKN2A/p14ARF), cyclin-dependent kinase inhibitor 2A experiments (12). On day 6, cells were harvested following (CDKN2A/p16INK4A), growth arrest and DNA-damage-inducible incubation with trypsin-EDTA then stained with trypan blue beta (GADD45B), protein-tyrosine phosphatase receptor type O and counted. For the analysis of mRNA expression total RNA gene (PTPRO) and thrombospondin, type I, domain containing 1 was extracted using QIAshreder with RNeasy minikit (Qiagen, (THSD1) genes was determined by methylation-specific poly- Hilden, Germany). Genomic DNA (gDNA) was extracted by merase chain reaction (MSP) with 1 µl of bisulfite-treated DNA proteinase K (Invitrogen) digestion, phenol/chloroform extrac- as template and AmpliTaq Gold 360 Master Mix (Applied tion and ethanol precipitation method for methylation analysis. Biosystems, Foster City, CA, USA) for amplification, as previ- ously described (13). Bisulfite-converted gDNA, methylated Clinical samples. Paired samples from 23 patients, who under- and unmethylated human control DNA (EpiTect control DNA, went surgical treatment for CRC in 2007 at Tokyo Medical Qiagen) was used as a positive control for the methylated and and Dental University Hospital were included in this study. All unmethylated experiments, respectively. specimens (macroscopically resected) were stored at -80˚C until Methylated and unmethylated primer sequences (Life further use. Mean age was 66.3±10.9 years (median 67.0 years; Technologies Inc., Rockville, MD, USA), amplicon lengths and range 38-81 years) and male-to-female ratio was 1.3:1. The PCR conditions have been previously reported (12,14-16). None number of cases for stages I, II, III and IV was six, six, five of the primer sets amplified non-bisulfite-treated gDNA (data and six, respectively. Written informed consent was obtained not shown), this eliminate the possibility of PCR product ampli- from all patients and the Institutional Review Board at Tokyo fication from unconverted DNA. All reactions were performed Medical and Dental University approved the study. All the in duplicate and PCR products were loaded onto a 2.0% agarose samples were used in methylation analysis, of them 14 assigned gel, stained with 0.5 µg/ml ethidium bromide and visualized to gene expression study. under ultraviolet illumination. PTPRO gene was included as a positive control for target Oligonucleotide microarray analysis. RNA was extracted from gene methylation analysis and verified the integrity of gDNA cell lines before and after treatment with 5-aza-dC. Contaminant used in this study (i.e., showed unmethylated bands in both DNA was removed by digestion with RNase-free DNase normal and tumor tissues) (17). Methylation status of p14ARF and (Qiagen). The integrity of the total RNA obtained was assessed p16INK4A gene was used to confirm the efficient demethylation of using an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo CpG-dinucleotides in the treated cell lines (17). Alto, CA, USA). All samples had an RNA integrity number of ≥5.0 prior to gene expression analysis. Complementary RNA Quantitative real-time RT-PCR (qRT-PCR). Total RNA was (cRNA) was prepared from 2 µg total RNA using one-cycle extracted from primary CRC and adjacent normal tissues target labeling and a control reagents kit (Affymetrix, Santa (14 pairs) using RNeasy minikit (Qiagen) and from 14 colon Clara, CA, USA). Hybridization and signal detection of the cancer cell lines. Total RNA (10 µg) was reverse-transcribed Human Genome (HG) U133 Plus 2.0 array (Affymetrix) were into complementary DNA (cDNA) samples using High Capacity performed according to the manufacturer's protocol. cDNA Reverse Transcription Kit (Applied Biosystems) accor- The gene expression data sets were submitted to Gene ding to the manufacturer's protocol. TaqMan gene expression Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/, acces- assays (Applied Bioystems: THSD1, Hs00938785_m1, and sion no. GSE32323). To investigate the difference