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. 2005 Dec 21;97(1):64–71. doi: 10.1111/j.1349-7006.2006.00136.x

Chemical genomic screening for methylation‐silenced genes in gastric cancer cell lines using 5‐aza‐2′‐deoxycytidine treatment and oligonucleotide microarray

Satoshi Yamashita 1, Yoshimi Tsujino 1, Kazuki Moriguchi 1, Masae Tatematsu 2, Toshikazu Ushijima 1,✉
PMCID: PMC11159443  PMID: 16367923

Abstract

To identify novel methylation‐silenced genes in gastric cancers, we carried out a chemical genomic screening, a genome‐wide search for genes upregulated by treatment with a demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐aza‐dC). After 5‐aza‐dC treatment of a gastric cancer cell line (AGS) 579 genes were upregulated 16‐fold or more, using an oligonucleotide microarray with 39 000 genes. From these genes, we selected 44 known genes on autosomes whose silencing in gastric cancer has not been reported. Thirty‐two of these had CpG islands (CGI) in their putative promoter regions, and all of the CGI were methylated in AGS, giving an estimated number of 421 ± 75 (95% confidence interval) methylation‐silenced genes. Additionally, we analyzed the methylation status of 16 potential tumor‐related genes with promoter CGI that were upregulated four‐fold or more, and 14 of these were methylated in AGS. Methylation status of the 32 randomly selected and 16 potential tumor‐related genes was analyzed in 10 primary gastric cancers, and 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one primary gastric cancer. A metastasis suppressor gene, MTSS1, was located in a genomic region with frequent loss of heterozygosity (8q22), and was expressed abundantly in the normal gastric mucosa, suggesting its role in gastric carcinogenesis. (Cancer Sci 2006; 97: 64 –71)

Abbreviations:

5‐aza‐dC

5‐aza‐2′‐deoxycytidine

CGI

CpG island

LOH

loss of heterozygosity

MS‐RDA

methylation sensitive‐representational analysis

MSP

methylation‐specific PCR

PCR

polymerase chain reaction.

Epigenetic alterations are involved in cancer development and progression, and methylation of promoter CGI leads to transcriptional silencing of their downstream genes.( 1 ) In various human cancers, silencing of tumor‐suppressor genes, such as CDKN2A (p16), CDH1 (E‐cadherin) and MLH1, is known to be one of the major mechanisms for their inactivation, along with mutations and LOH. To identify genes silenced by promoter methylation by genome‐wide screenings, various techniques have been developed.( 2 ) Most techniques are based on the methylation status of genomic DNA, including MS‐RDA and restriction landmark genomic scanning. In contrast, Suzuki et al. developed a technique that screens genes re‐expressed after treatment with a demethylating agent, 5‐aza‐dC, using a microarray.( 3 ) The chemical genomic screening technique is simple and is effective in identifying genes silenced in cell lines. It has been applied to colon, bladder, esophageal, pancreatic and prostate cancers.( 3 , 4 , 5 , 6 , 7 )

Gastric cancer is the second most common cause of cancer death in the world.( 8 ) As its molecular basis, deep involvement of aberrant DNA methylation has been indicated by the higher incidences of aberrant DNA methylation of known tumor‐suppressor genes than of mutations.( 9 ) We previously searched for genes silenced in MKN28 and MKN74 cell lines using MS‐RDA,( 10 ) and identified lysyl oxidase as a novel tumor‐suppressor gene.( 11 ) However, the entire picture of methylation‐silenced genes in gastric cancers is still unclear, and further searches for methylation‐silenced genes are necessary.

In the present study, we carried out a chemical genomic screening of methylation‐silenced genes in the human gastric cancer cell line AGS.

Materials and Methods

Tissue samples, cell lines and 5‐aza‐dC treatment

Ten primary gastric cancer samples (male/female = 7/3, aged 38–81 years) and two normal gastric mucosae were obtained from 10 patients undergoing gastrectomy at Aichi Cancer Center (Nagoya, Japan) with informed consent. These samples were frozen and stored at −80°C until extraction of DNA or RNA. Gastric cancer cell lines AGS, MKN28, MKN45 and KATOIII were obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan) and American Type Culture Collection (Manassas, VA, USA). Two gastric cancer cell lines, HSC44 and HSC57, were gifted by Dr Kazuyoshi Yanagihara at the National Cancer Center Research Institute (Tokyo, Japan). AGS cells were seeded at a density of 3 × 105 cells/10 cm dish on day 0 and treated with freshly prepared 1 µM 5‐aza‐dC (Sigma) for 24 h on days 1, 3 and 5. After each treatment, the cells were placed in fresh medium and harvested on day 6. Genomic DNA was extracted by standard phenol/chloroform procedures. Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) and purified using an RNeasy Mini kit (Qiagen, Valencia, CA, USA).

Oligonucleotide microarray analysis

Oligonucleotide microarray analysis was carried out using GeneChip Human Genome 133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA) with 54 000 probe sets and 47 400 transcripts from 39 000 genes. From 8 µg of total RNA, the first‐strand cDNA was synthesized with SuperScript III reverse transcriptase (Invitrogen, Groningen, the Netherlands) and a T7‐(dT)24 primer (Amersham Bioscience, Buckinghamshire, UK), and the double‐stranded cDNA was then synthesized. From the double‐stranded cDNA, biotin‐labeled cRNA was prepared using a BioArray HighYield RNA transcript labeling kit (Enzo, Farmingdale, NY, USA). Labeled cRNA (20 µg) was fragmented, and the GeneChips were hybridized. The arrays were stained and scanned according to the protocol from Affymetrix. The data were processed using GeneChip Operating Software. The signal intensities were normalized so that the average of all of the genes on a GeneChip would be 500. The P‐values for different expression (change P‐value) were calculated in each probe by statistical algorithms based on the Wilcoxon's signed rank test. The change P‐values of 0.003 and 0.997 were used as thresholds to define genes with increased and decreased expression, respectively. Expression data for the normal tissues using GeneChip were obtained from the database RefEXA (http://www.lsbm.org/site_e/database/index.html),( 12 ) with kind permission from Dr H. Aburatani.

Methylation‐specific polymerase chain reaction

DNA (1 µg) digested with BamHI was denatured in 0.3 M NaOH at 37°C for 15 min. Then, 3.6 M sodium bisulfite (pH 5.0) and 0.6 mM hydroquinone were added, and the sample underwent 15 cycles of 30‐s denaturation at 95°C and a 15‐min incubation at 50°C. The sample was desalted with the Wizard DNA Clean‐Up system (Promega, Madison, WI, USA) and desulfonated in 0.3 M NaOH. DNA was ethanol‐precipitated and dissolved in 40 µL of Tris‐EDTA buffer. MSP was carried out with a primer set specific to the methylated or unmethylated sequence (M or U set), using 0.5 µL of the sodium‐bisulfite‐treated DNA. A region 200 bp or less upstream of a putative transcriptional start site was analyzed, except for BDNF (−401 to −214). Primer sequences and PCR conditions are shown in Table 1. DNA methylated with SssI methylase was used to determine specific conditions of PCR for M sets.

Table 1.

Primers for methylation‐specific polymerase chain reaction

Genes M/U Forward primer Reverse primer Annealing(°C) No.cycles
Position † Sequence Position Sequence
ADFP M −169 GGTCGGGTTTTCGTTCGGTTTTC −36 ACCCGAATATCACCCTCGAACACG 55 34
U −170 AGGTTGGGTTTTTGTTTGGTTTTT −36 ACCCAAATATCACCCTCAAACACA 57 34
ALDH1A3 M −108 TCGGTTTCGTAGTTAATTAGGC −18 GACTCGACCCGAACACTACGCA 55 35
U −108 TTGGTTTTGTAGTTAATTAGGT −18 CAACTCAACCCAAACACTACACA 49 34
ANXA5 M −164 TATTTAGGTTCGCGAGATTAGC −48 CCAAAACCCCAACCGCAAACCG 57 34
U −154 TGTGAGATTAGTGGGATAGTTT −48 ACCAAAACCCCAACCACAAACCA 55 34
AREG M −173 TTTTTAGCGAATTTTTACGTAC −22 ATAAAACGACGCGCACCTACCG 55 35
U −165 GAATTTTTATGTATGAGGGAGGT −22 ATAAAACAACACACACCTACCA 55 34
BDNF M −401 TACGTAAATAGCGAGGTTAGTC −214 AACTCCGACGAAACTAAATTCG 55 34
U −411 GTGAGTTGGTTATGTAAATAGT −214 AACTCCAACAAAACTAAATTCA 52 34
CAV1 M −80 TTTCGGGACGTTTTTCGGTGGT −6 TAAAAACGTTTCTCCCGCGCTA 59 34
U −95 GAAAATATTTGTTTTTTTTGGGAT −3 ACAAATAAAAACATTTCTCCCACA 55 35
EEF1a2 M −248 GTTTCGTTTTTCGGGTTCGTC −28 GCCCTACAACACGCCAATACG 57 34
U −250 TTGTTTTGTTTTTTGGGTTTGTT −28 ACACCCTACAACACACCAATACA 58 34
F2R M −187 TTAGGAGGGTCGAGACGGTCGC −96 TCCTCTAAACACCGTTAATTCG 55 34
U −189 TTTTAGGAGGGTTGAGATGGTTGT −98 TCCTCTAAACACCATTAATTCACA 55 35
FADS1 M −234 GTTCGTTTGACGTTAGGAAGTC −34 GCCCAAAACCAACCGCCTACG 55 35
U −234 GTTTGTTTGATGTTAGGAAGTT −34 CACCCAAAACCAACCACCTACA 55 34
FSD1 M −159 AGGGTTTTGGGCGAGGTTAGC −25 AAACTACCTTTACCGCGACCG 56 34
U −158 GGGTTTTGGGTGAGGTTAGTGT −25 CAAACTACCTTTACCACAACCA 58 34
FST M −172 TTTAGATTTAAAGCGCGGTTGC −47 ACGAATAACTCGAACGAACG 55 34
U −173 GTTTAGATTTAAAGTGTGGTTGT −47 ACAAATAACTCAAACAAACA 55 34
GREM1 M −134 CGTCGGTATTTAAACGGGAGAC −35 GAAACTCGACGCGAAATCAACG 55 35
U −134 TGTTGGTATTTAAATGGGAGAT −35 CAAAACTCAACACAAAATCAACA 55 34
IGFBP7 M −195 GGGTCGGTTACGTCGGGTGTTC −18 GACAAAAACGCGAATAAACCG 55 35
U −197 ATGGGTTGGTTATGTTGGGTGTTT −18 CAACAACAAAAACACAAATAAACCA 60 35
IL6R M −117 TTTTTATAGCGTAATTTCGTTTAC  78 AACCGAAACGAATAACGCAACA 48 35
U −124 GGTGTGTTTTTTATAGTGTAATTTT  65 TAACACAACAACCCCACACACCA 60 34
IRS2 M −127 GCGGCGTTAATGCGAGGTAGC −29 TAAATAACACATCGCGCACCG 55 35
U −128 TGTGGTGTTAATGTGAGGTAGT −24 CACACAATAAATAACACATCACA 60 35
KISS1 M −198 AAAGTTTCGTTTCGGAGGGTTC −49 CTTTTATAAAACCCGAAATAACG 58 34
U −198 AAAGTTTTGTTTTGGAGGGTTT −49 CCTTTTATAAAACCCAAAATAACA 55 33
MARK1 M −268 TTTAGACGATCGTAAATTTTGC −26 TCAAAAAAAACGACCCGAACCG 52 34
U −216 GGATAGGTGGGTAAGAGAGTGT −32 AAAACAACCCAAACCAACTACA 55 34
MLF1 M −118 GGGTAGCGGCGTATTGTTTTTC −16 CTCACTCGCCGCGACGCAAACG 55 35
U −120 TAGGGTAGTGGTGTATTGTTTTTT −7 ACAAACAACACCTCACTCACCACA 60 35
MSX1 M −178 CGTCGTTTGGGTTTTGTTTTGC −18 CCGACTCCGAACCCTACCG 55 34
U −160 TTGTGTGTTTTTAGGTTTAGTGT −18 CCAACTCCAAACCCTACCA 58 34
MX1 M −178 GGGTTCGGGTTCGAGAATTTGC −21 TTCGCCTCTTTCACCCCG 55 34
U −179 TGGGTTTGGGTTTGAGAATTTGT −21 ACTTCACCTCTTTCACCCCA 55 36
NT5E M −183 AGTCGATAGTCGCGTTAGGGTC −36 GAACAACTAAAACCGAAACTCG 55 35
U −184 TAGTTGATAGTTGTGTTAGGGTT −41 AACTAAAACCAAAACTCAATACC 53 35
PLAGL1 M −195 GTTCGGGTTTATTTGCGTTAGC −47 AACCCCTAACGAAAACGTCACG 60 33
U −196 GGTTTGGGTTTATTTGTGTTAGT −47 CCCCTAACAAAAACATCACA 60 34
PPIC M −162 GTTTTTCGTATTCGTTTAAGGC −33 AAAATAAAAATCGAACAATCCG 55 35
U −165 GGTGTTTTTTGTATTTGTTTAAGGT −57 AAAAACAAAAACCCAAAACACA 55 34
PYCARD M −186 CGGGGAATCGCGGAGGTTTC −36 AATAAAACCCGAAAAAAAACCG 55 35
U −190 GGTTTGGGGAATTGTGGAGGTTTT −13 ATCACACCCTCCAACTAACCTACA 55 35
RBP4 M −32 TTCGGGTTTCGGTGAGTTAGGGC  69 CCGCTACTTTATAACGCCG 58 34
U −33 GTTTGGGTTTTGGTGAGTTAGGGT  69 ACCCCACTACTTTATAACACCA 60 33
RGS2 M −184 ACGTTAGTAGCGTTTCGGTTTC −37 GTCGCAACATTTATAAAACCTCG 55 35
U −185 GATGTTAGTAGTGTTTTGGTTTT −37 CATCACAACATTTATAAAACCTCA 60 34
SCRN1 M −106 GAGGGTGGGTTCGCGGTTAC −14 CTACAATAACGAAAACGACCG 55 35
U −106 GAGGGTGGGTTTGTGGTTATGT −21 CAATAACAAAAACAACCACCAAACA 60 35
TBX3 M −98 TTGGTTCGAAAGCGTTAAAGAG −22 ACCGAACGTCTACTCGACGACT 53 35
U −110 GTAGTAATATAATTGGTTTGAAAGT −33 CTACTCAACAACTCTAAAAAATCA 55 35
TFAP2C M −146 GCGTTGCGTTAGGTTCGGGTGC  40 CGCGAATATCAAAACCGCTCCG 55 35
U −148 TGGTGTTGTGTTAGGTTTGGGTGT  40 ACCACAAATATCAAAACCACTCCA 60 35
ULBP2 M −213 TGAGTTTGTCGTGGAAGGAATC −89 GTCAAACGAATCATAACGTCACG 55 35
U −193 TTGTGTTTTGGTAGGAGTTGGGT −71 ATCAAACAAATCATAACATCACA 52 34
WIF1 M −131 CGTTCGCGTTTTATTTTTTTGC −27 AACGCGTCGCCTCCCGACCTAA 53 35
U −126 GTGTTTTATTTTTTTGTGTGATTT −21 AACAACTAAACACATCACCTCCCA 55 34
ZNF559 M −147 GGTTCGGGAATTCGAGGTTTC −43 TACCTCAAACGCCAACGAAAACG 58 34
U −149 TGGGTTTGGGAATTTGAGGTTTT −70 CTATTAAAATAACAACCATTATACA 52 34
ABHD9 M −195 CGTGAGTTATCGTATTCGGTTC −115 TCCTATACGAAACTTAAAACCG 59 33
U −197 GGTGTGAGTTATTGTATTTGGTTT −102 ACAAAACCTAACAAATCCTATACA 55 34
BMP7 M −227 GTTTTTTCGTTGTTTTTTCGGC −82 ATACTAACCCCGAACCCCTCG 55 34
U −231 GTTTGTTTTTTTGTTGTTTTTTTGGT −82 AATACTAACCCCAAACCCCTCA 59 34
CDH2 M −217 GCGGTGTCGTTATATAGTAGC −128 ACTCTAAACCTACGCCGCCG 50 34
U −294 GTAAAATTATGAGTTTGAAATTTTGT −114 AAAAAAAAACATATAAACATCTACA 55 34
CDKN2D M −122 GCGGTGTCGTTATATAGTAGC −15 ACTCTAAACCTACGCCGCCG 55 34
U −164 GGTTTTGTGGGTGGAATGTT −15 CTACTCTAAACCTACACCACCA 55 34
CLDN3 M −111 AGGTTTTGGAGAGCGCGGTTTC −47 ACCCTAAACTAAAACCGATACG 50 34
U −105 TGGAGAGTGTGGTTTTGTTTTTATT −47 CTAACCCTAAACTAAAACCAATACA 55 34
CTSL M −182 GATTTTATTTTGCGTCGTTTC −40 ACGCTACGATTAACTATACCG 55 34
U −186 GTTTGATTTTATTTTGTGTTGTTTT −40 ACTACACTACAATTAACTATACCA 48 34
FYN M −228 TCGTACGTATTTTGGGATGTTC −141 CTACGAACCGCAACCATTAACG 55 34
U −229 ATTGTATGTATTTTGGGATGTTT −129 ACCCCTTAAAAACTACAAACCA 55 34
GPR54 M −200 TTATAAACGTTCGGTCGTAGC −54 CAAAATTACGCCCTAACACCG 52 34
U −206 TATGGGTTATAAATGTTTGGTT −54 CAAAATTACACCCTAACACCA 58 34
IGFBP3 M −99 TTTCGGTTTTTATATAGCGGTC −37 AAAAAACGACTAATCCTCAACG 55 34
U −102 TTATTTTGGTTTTTATATAGTGGTT −37 AACAAAAAACAACTAATCCTCAACA 48 34
MTSS1 M −130 GAGAGCGCGTTTTCGTTTGGC −32 CGCCTCCTTTTCACTCCTACG 59 34
U −130 GAGAGTGTGTTTTTGTTTGGT −32 CCACCTCCTTTTCACTCCTACA 55 34
PAX6 M −188 AGGGAGTATTTAATCGGTTGGC −47 CTCCTACGCCTAAACCAAAACG 59 34
U −138 GTAATATTTTGTGTGAGAGTGAGT −47 TCCTCCTACACCTAAACCAAAACA 55 34
PLAU M −177 TTTGTGAGCGTTGCGGAAGTAC −51 ACGATCTCCGCACTATACTACG 55 34
U −153 GGGGTTTGGGTTGTTGAGT −51 CTACAATCTCCACACTATACTACA 50 34
RORA M −213 GGTTGGAGAAGTTTTCGTTAGC −111 GACGAACGAACAAACAAAAACG 55 34
U −215 TTGGTTGGAGAAGTTTTTGTTAGT −123 CAAACAAAAACACAAAAAAACACA 55 34
SNAI1 M −155 ATTTGTTCGGGGAGTGGTTTTC −91 AAAACGAAACCTTATCTACCACG 55 34
U −213 GGAGTTTTTGTTTGGGTTTTTATT −91 AAAAACAAAACCTTATCTACCACA 55 34
TNFSF9 M −198 GTCGAGTTTGGAAGGTCGGAAAC −65 AAAAAACCACGCCCCTCCG 56 34
U −199 GGTTGGAAATGGAAAGGAGAGT −65 AAAAAAAACCACACCCCTCCA 55 34
ZNF177 M −119 GTAGGAGTATTTGCGATGTTTC −12 AAAATAACGAAACGACGAACG 58 34
U −97 GTTTTTAAGTTTTTAGGGTGAATTT −22 AAACAACAAACACCCACTTCCA 55 34
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†

Transcription start site = 0. All primers were designed on the top strand sequences. M, specific to methylated DNA; U, specific to unmethylated DNA.

Results

Oligonucleotide microarray analysis

AGS cells were treated with 1 µM of 5‐aza‐dC, which caused growth suppression at 49%, and upregulated genes were searched for using an oligonucleotide microarray. Among the 39 000 genes (54 000 probe sets) analyzed, 1430 genes (1747 probes) were upregulated four‐fold or more (signal log ratio > 2) and 579 genes (678 probes) were upregulated 16‐fold or more (signal log ratio > 4). To identify silenced genes with known functions from the 579 genes, we excluded genes on chromosome X (95 probes, 70 genes) and genes without known functions (i.e. FLJ genes, KIAA genes, LOC genes, MG genes and Orf genes [149 probes, 141 genes]).

Among the remaining 368 genes (434 probes), we found eight genes (14 probes) whose methylation‐silencing had been reported in gastric cancers (BNIP3,( 13 ) CDKN2A (p16),( 14 ) CHFR,( 15 ) ID4,( 16 ) RBP1,( 17 ) RUNX3,( 18 ) THBD,( 10 ) TIMP ( 19 )). The remaining 360 genes (420 probes) were considered as candidates for novel methylation‐silenced genes in gastric cancers (Table 1).

Methylation analysis of genes upregulated by 5‐aza‐dC treatment

From the 360 genes upregulated 16‐fold or more, we selected 44 genes randomly (Table 2). Among these 44 genes, 32 genes (73%) had CGI in their 5′ regions, which were considered as promoter regions (Table 2). To examine whether the induction of these genes by 5‐aza‐dC treatment was really due to promoter demethylation, the methylation status of these 5′‐CGIs were analyzed by MSP. For all the 32 genes, only methylated molecules were detected before 5‐aza‐dC treatment, and unmethylated DNA molecules were detected after the treatment in AGS, suggesting silencing of the 32 genes by methylation of their 5′‐CpG islands (representative results in Fig. 1).

Table 2.

Genes upregulated after 5‐aza‐dC treatment in the AGS cell line

Probe set Gene title Symbol Fold change CGI
The 44 genes picked randomly from the genes showing greater than 16‐fold upregulation after 5‐aza‐dC treatment
209122_at Adipose differentiation‐related protein ADFP  36.0 Yes
203180_at Aldehyde dehydrogenase 1 family, member A3 ALDH1A3  46.2 Yes
200782_at Annexin A5 ANXA5  32.5 Yes
205239_at Amphiregulin (schwannoma‐derived growth factor) AREG  59.3 Yes
206382_s_at Brain‐derived neurotrophic factor BDNF  29.2 Yes
203065_s_at Caveolin 1, caveolae protein, 22 kDa CAV1  37.2 Yes
210140_at Cystatin F (leukocystatin) CST7  22.1 No
219424_at Epstein–Barr virus induced gene 3 EBI3  18.5 No
204540_at Eukaryotic translation elongation factor 1 alpha 2 EEF1A2  25.0 Yes
203989_x_at Coagulation factor II (thrombin) receptor F2R  59.3 Yes
208962_s_at Fatty acid desaturase 1 FADS1  23.0 Yes
203240_at Fc fragment of IgG binding protein FCGBP   16.8 No
1570515_a_at Filamin A interacting protein 1 FILIP1  34.8 No
219170_at Fibronectin type 3 and SPRY domain containing 1 FSD1  16.0 Yes
226847_at Follistatin FST  29.2 Yes
218469_at Gremlin 1 homolog, cysteine knot superfamily GREM1  59.3 Yes
213620_s_at Intercellular adhesion molecule 2 ICAM2  18.5 No
201162_at Insulin‐like growth factor binding protein 7 IGFBP7  38.4 Yes
205945_at Interleukin 6 receptor IL6R  26.0 Yes
209185_s_at Insulin receptor substrate 2 IRS2  24.0 Yes
205563_at KiSS‐1 metastasis‐suppressor KISS1  54.8 Yes
221047_s_at MAP/microtubule affinity‐regulating kinase 1 MARK1  17.6 Yes
1552456_a_at Methyl‐CpG binding domain protein 3‐like 2 MBD3L2  38.4 No
206560_s_at Melanoma inhibitory activity MIA  23.0 No
204784_s_at Myeloid leukemia factor 1 MLF1  27.0 Yes
205932_s_at Msh homeo box homolog 1 (Drosophila) MSX1  16.0 Yes
202086_at Myxovirus (influenza virus) resistance 1 MX1  18.5 Yes
205581_s_at Nitric oxide synthase 3 (endothelial cell) NOS3  18.5 No
203939_at 5′‐nucleotidase, ecto (CD73) NT5E  53.3 Yes
207002_s_at Pleiomorphic adenoma gene‐like 1 PLAGL1  46.2 Yes
204517_at Peptidylprolyl isomerase C (cyclophilin C) PPIC  23.0 Yes
221666_s_at PYD and CARD domain containing PYCARD  25.0 Yes
219140_s_at Retinol binding protein 4, plasma RBP4  43.6 Yes
202388_at Regulator of G‐protein signaling 2, 24 kDa RGS2  33.6 Yes
201462_at Secernin 1 SCRN1  31.4 Yes
204614_at Serine (or cysteine) proteinase inhibitor, clade B, member 2 SERPINB2  36.0 No
202627_s_at Serine (or cysteine) proteinase inhibitor, clade E, member 1 SERPINE1  26.0 No
208539_x_at Small proline‐rich protein 2 A SPRR2A  22.1 No
224167_at Likely ortholog of mouse spermatogenic Zip 1 SPZ1  18.5 No
219682_s_at T‐box 3 TBX3  36.0 Yes
205286_at Transcription factor AP‐2 gamma TFAP2C  32.5 Yes
221291_at UL16 binding protein 2 ULBP2  29.2 Yes
204712_at WNT inhibitory factor 1 WIF1  31.4 Yes
224518_s_at Zinc finger protein 559 ZNF559  30.3 Yes
Genes showing greater than four‐fold upregulation after 5‐aza‐dC treatment, having CpG islands, and having cancer related function or having chromosomal location in the region of frequent loss in gastric cancer.
220013_at Abhydrolase domain containing 9 ABHD9  8.0 Yes
209591_s_at Bone morphogenetic protein 7 (osteogenic protein 1) BMP7  13.9 Yes
203440_at Cadherin 2, type 1, N‐cadherin (neuronal) CDH2  26.0 Yes
210240_s_at Cyclin‐dependent kinase inhibitor 2D (p19) CDKN2D  4.4 Yes
203953_s_at Claudin 3 CLDN3  45.3 Yes
202087_s_at Cathepsin L CTSL  13.0 Yes
216033_s_at FYN oncogene related to SRC, FGR, YES FYN  78.8 Yes
242517_at G protein‐coupled receptor 54 GPR54  6.3 Yes
210095_s_at Insulin‐like growth factor binding protein 3 IGFBP3  9.8 Yes
203037_s_at Metastasis suppressor 1 MTSS1  6.5 Yes
205646_s_at Paired box gene 6 (aniridia, keratitis) PAX6  18.4 Yes
205479_s_at Plasminogen activator, urokinase PLAU  137.2 Yes
210479_s_at RAR‐related orphan receptor A RORA  55.7 Yes
219480_at Snail homolog 1 (Drosophila) SNAI1  4.6 Yes
206907_at Tumor necrosis factor superfamily, member 9 TNFSF9  4.8 Yes
207417_s_at Zinc finger protein 177 ZNF177  4.4 Yes
Genes reported as silenced genes in gastric cancer and showing greater than 16‐fold upregulation after 5‐aza‐dC treatment
201848_s_at BCL2/adenovirus E1B 19 kDa interacting protein 3 BNIP3  20.3 Yes
207039_at Cyclin‐dependent kinase inhibitor 2 A (p16) CDKN2A  33.6 Yes
223931_s_at Checkpoint with forkhead and ring finger domains CHFR  16.8 Yes
209291_at Inhibitor of DNA binding 4 ID4  57.8 Yes
203423_at Retinol binding protein 1, cellular RBP1  54.8 Yes
204198_s_at Runt‐related transcription factor 3 RUNX3  27.0 Yes
203888_at Thrombomodulin THBD  29.2 Yes
201147_s_at Tissue inhibitor of metalloproteinase 3 TIMP3  65.6 Yes
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Figure 1.

Figure 1

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A representative result of methylation analysis. (A) MLF1; (B) MSX1; and (C) TBX3. The left sides of each panel represent the 5′ CpG islands and regions analyzed by methylation‐specific polymerase chain reaction (MSP). Vertical marks, individual GpC and CpG sites; Open boxes, non‐coding and coding exons; and arrowheads, positions of MSP primers (M sets). The right sides show the results of MSP in gastric cancer cell lines, normal gastric mucosa and primary gastric cancers. 5‐aza‐dC, AGS cells after treatment with 5‐aza‐2′‐deoxycytidine; SssI, genomic DNA methylated with SssI methylase.

Analysis of five additional gastric cancer cell lines (MKN28, MKN45, HSC44, HSC57, KATOIII) showed that five genes (ANXA5, AREG, CAV1, IL6R, TBX3) were methylated only in AGS, and 27 genes were methylated in multiple gastric cancer cell lines (Table 3). The microarray analysis of KATOIII and HSC57 showed that none of the 32 genes were expressed when unmethylated DNA molecules were not present.

Table 3.

Methylation profiles in gastric cancer

Gene Symbol Function Chromo‐ somal location Methylation status in gastric cancer cell line † Expression (signal intensity of GeneChip) Methylation status in gastric cancer ‡
MKN28 MKN45 HSC44 HSC57 KATOIII AGS HSC57 KatoIII +5‐aza‐dC Stomach § Case1 Case2 Case3 Case4 Case5 Case6 Case7 Case8 Case9 Case10 Normal
AGS AGS (RefEXA) tub1 tub2 por2 por tub2 por1 por2 por por2 por2
32 genes selected randomly and with GGI
ADFP Fatty acid transport 9p22.1 M U M/U U M M 2191   74  33 1186 154 U U U U U M U U U U –
ALDH1A3 Vitamin A metabolism 15q26.3 U U U U M M  322   69  16 1435  65 U U U U U M M U M– U –
ANXA5 Apoptosis 4q28‐q32 U U U U U M 1740 4933 144 6960 433 U U U U U M U U U U –
AREG Cell proliferation 4q13‐q21 U U U U U M 9568 9103   7 2677 137 U U U U U M U U U U –
BDNF Growth factor activity 11p13 M M M M/U U M   50   20   5  447  32 M U M M U M M M M M –
CAV1 Cell aging 7q31.1 U U U U U M   24  167  20 1411 102 U U U U U M U U U U –
EEF1A2 Translational elongation 20q13.3 U U M U M M  240   60 112 7234  58 U U U U U M U U U U –
F2R G‐protein signaling 5q13 U U M/U U M M  140    7   4  820  26 U U U U U M M U U U –
FADS1 Fatty acid desaturation 11q12.2‐q13.1 U U M/U U M M  375   19  22  542  40 U U U U U M M– M M U –
FSD1 Microtubule depolymerization 19p13.3 U M/U M/U U M M   49   25  31  362   5 U U U U U M U U M U U
FST Transcription factor 5q11.2 U U M U M M    6    4   7  278 – U U U U U M M U M U –
GREM1 Signal transduction 15q13–15q15 M M M U M M   77   33   5 2128 184 M M M M M M M M M M –
IGFBP7 Angiogenesis 4q12 M U M U M M   13   11   9  934 483 U U M– U U M M U M M– –
IL6R Immune response 1q21 U U U U U M  124  227  12  574  72 U U U U U U U U U U –
IRS2 Signal transduction 13q34 U U U U M M 6602   57  30 1245 274 U U U U U U U U M– U –
KISS1 Metastasis suppressor 1q32 M/U M M U U M   19   26  21 3697   7 U M– M– M– U M– M– M– M– M– U
MARK1 Protein amino acid phosphorylation 1q41 U U U U M M   79   85  45  845  43 U U U U U M U M U U –
MLF1 Cell differentiation 3q25.1 M U U U M/U M  936 1078  22  709  42 M M M M U M M M M M U
MSX1 Transcription factor 4p16.3‐p16.1 U U M/U U M M  524   13  43 2675  32 U U U U U M M– U M U U
MX1 Response to virus 21q22.3 M U M U U M  856 1533  48  996 223 U U U U U U U U U U –
NT5E DNA metabolism 6q14–6q21 U U U U M/U M  115  394   4  821  31 U U U U U U M– U U U –
PLAGL1 Induction of apoptosis 6q24–6q25 M M M M/U M M  456   16   4  668  81 M– M M M M M M M M M U
PPIC Signal transduction 5q23.2 U U U U M M 2724   59  49 1621 186 U U U U U M M– U U U –
PYCARD Signal transduction 16p12–16p11.2 M M M U U M 2739  557   9  420  39 U U U U U U U U U U –
RBP4 Vitamin A metabolism 10q23–10q24 M U M U U M   86  643  10  880  11 U U U M– M– M M M– U M– U
RGS2 G‐protein signaling 1q31 U U U U M/U M/U   27   90  27 1727 575 U U U U U U U U U U –
SCRN1 Exocytosis 7p14.3‐p14.1 M/U U U U M M 1595   57  19 1306 127 U U U U U M U U M U –
TBX3 Morphogenesis 12q24.1 U U U U U M 1280  881  15 1714  38 U U U M M M M M M– M U
TFAP2C Transcription factor 20q13.2 U U M/U U M M 1486   26  27 2446  51 U M– M U U M M U M U –
ULBP2 T cell proliferation 6q25 U U U U M/U M  365  510  29 1286   8 U U U U U M U U U U U
WIF1 Wnt signaling 12q14.3 M M M M/U M M    9   41   7  241   2 U U M M M– M M M M M– U
ZNF559 Transcription factor 19p13.2 M U M/U U M M  609    7   7  532 – M– M M– M– U U U M M– M– U
16 genes with potential tumor‐related functions
ABHD9 Response to chemical substance 19p13.12 M M M U M M   19   18  13  140   3 U M U M– M M M M M M– –
BMP7 Cell proliferation 20q13 U U M U M/U M 4166   19  61 1185  68 U U U M– U M M M– M M– –
CDH2 Cell adhesion 18q11.2 M M M U M M   13   13   9  487  57 M U M M M– M M M M M –
CDKN2D Cdk inhibitor 19p13 U U U U U U   47  339 119  489  28 U U U U U U U U U U –
CLDN3 Cell adhesion 7q11.23 U U M/U U U M 7584   69   7  359   1 U U U U U M M M– M U U
CTSL Protein processing 9q21–9q22 U U M U M/U M 1017  102  50 1753 118 U U U U M– M M M U M –
FYN Proto‐oncogene 6q21 U U U U U M  341 1046   5  797  64 U U U U U M U U U U –
GPR54 G‐protein signaling 19p13.3 U U M U M M   89   85  35  210 – U U U U U M U U U U –
IGFBP3 Induction of apoptosis 7p13–7p12 M/U M/U M/U U M M  106   15  31  277 422 M U M M M– M M M M M –
MTSS1 Cytoskeletal organization 8p22 M/U U M U U M   17  694  73  625 400 M U U U U M U M U U –
PAX6 Transcription factor 11p13 M U M/U U M M   17   21  29  417   6 U U M M U M M M M M– U
PLAU Angiogenesis 10q24 M U U U M M 1060  168  11 2950  80 U U U U U M– U U U U –
RORA Signal transduction 15q21‐q22 U U M U M M  195   49   2  340  20 M M U U M M– M M M M U
SNAI1 Transcriptional repressor 20q13.1‐q13.2 U U U U U U  235   40  52  319   8 U U U U U U U U U U –
TNFSF9 Apoptosis 19p13.3 U U U U U M  615 1133  84  916  23 U U U U U M U U U U –
ZNF177 Transcription factor 19p13.2 M M M M U M   76   59  47  222  14 M M M M M M M M M M U
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From genes upregulated 16‐fold or more, 44 genes were selected randomly, and the methylation status of 32 genes with CGI were analyzed. In addition, 16 genes with potential tumor‐related functions and CGI were selected for methylation analysis. †In cancer cell lines, M: only methylated molecules detected, U: only unmethylated molecules detected, M/U: both methylated and unmethylated molecules detected. ‡In primary cancer samples, ‘M’ and ‘U’ indicate detection and absence, respectively, of methylated molecules. ‘M–’ indicates slight detection of methylated molecules. §Obtained from the RefEXA database. Underlined chromosomal locations, regions of frequent loss in gastric cancer.

We next selected 16 potential tumor‐related genes with promoter CGI and four‐fold or greater upregulation as the above analysis suggested that a considerable number of silenced genes were still present among the genes with upregulation of 16‐fold or less (Table 2). The potential tumor‐related genes were selected based on their tumor‐related function and location in genomic regions with frequent LOH (5q21‐23,( 20 , 21 ) 8p22,( 20 , 21 ) 9p12‐24( 20 , 21 , 22 )) or with DNA loss by comparative genomic hybridization (19p13.12‐p13.3( 23 )) in gastric cancers. MSP showed that 14 of these 16 genes were methylated in AGS before 5‐aza‐dC treatment (Table 3). CDKN2D and SNAI1 were not methylated even before 5‐aza‐dC treatment, suggesting that they were induced as a stress response by 5‐aza‐dC treatment.

Presence of methylation in primary gastric cancers

The methylation status of the above 48 genes (32 selected randomly and 16 tumor‐related genes) were examined in 10 primary gastric cancers. It was shown that 42 genes (ABHD9, ADFP, ALDH1A3, ANXA5, AREG, BDNF, BMP7, CAV1, CDH2, CLDN3, CTSL, EEF1A2, F2R, FADS1, FSD1, FST, FYN, GPR54, GREM1, IGFBP3, IGFBP7, IRS2, KISS1, MARK1, MLF1, MSX1, MTSS1, NT5E, PAX6, PLAGL1, PLAU, PPIC, RBP4, RORA, SCRN1, TBX3, TFAP2C, TNFSF9, ULBP2, WIF1, ZNF177 and ZNF559) were methylated in at least one gastric cancer (Table 3). The numbers of methylated genes in each case ranged from one to 10. Case 6 had a large number of methylated genes, which was similar to AGS (Table 3). The expression levels of the 48 genes in the normal gastric mucosae were obtained from the RefEXA database (Table 3).

There remained a possibility that these silenced genes were normally methylated or were methylated tissue‐specifically. Therefore, we selected 11 genes with relatively high chances of having methylated CGI, based on their low expression in the normal gastric mucosae (CLDN3, FADS1, KISS1, PAX6, PLAGL1, RBP4, RORA, ULBP2, WIF1, ZNF177 and ZNF559). Along with three additional genes (MLF1, MSX1 and TBX3), their methylation status was examined in the normal gastric mucosae. However, none of the 14 genes were methylated.

Discussion

Chemical genomic screening revealed that a considerable number of genes were methylation‐silenced in the AGS gastric cancer cell line. After 5‐aza‐dC treatment of AGS, 579 genes were upregulated 16‐fold or more. When we analyzed 44 selected genes, 32 of them had CGI in their promoter regions, and all of the 32 genes turned out to be methylation‐silenced. Because 32 of the 44 genes selected from 579 genes were silenced, it was estimated that 421 ± 75 (95% confidence interval) genes were silenced in AGS. To avoid overestimation, we randomly selected 44 genes from the 360 genes after excluding: (i) genes on chromosome X, which harbors many normally methylated genes like MAGE; (ii) genes that have not been characterized yet; and (iii) genes whose methylation‐associated silencing was already known in gastric cancers. Among the 16 potential tumor‐related genes, 10 were upregulated 16‐fold or less, and eight of the 10 genes were found to be methylation‐silenced. If genes with relatively small upregulation were analyzed, the number of silenced genes in AGS was expected to be larger.

As for the number of methylation‐silenced genes in a cancer, Costello et al. estimated that an average of 600 CGI in the whole genome were methylated aberrantly in the tumors.( 24 ) However, the number was calculated by analyzing CGI in any location against a gene, and the number of genes silenced, for which methylation of promoter CGI is necessary, was not determined. Using chemical genomic screening, Sato et al. estimated that an average of 140 genes would be methylated aberrantly in pancreatic cancers.( 6 ) Compared with this number, the number of genes silenced in the AGS cell line was considered to be much larger. We recently found that AGS had an increased rate of de novo methylation,( 25 ) and this could be one of the mechanisms.

By methylation analysis of 48 genes (Table 3), 46 genes were found to be methylated in AGS, and 42 genes were methylated in at least one primary gastric cancer. Among the 42 genes, eight genes (CAV1,( 26 , 27 ) IGFBP3,( 28 ) IGFBP7[MAC25/IGFBP‐rP1],( 29 ) PAX6,( 30 ) PLAGL1[ZAC/LOT1],( 31 , 32 ) PLAU[uPA],( 33 , 34 ) RBP4 ( 35 ) and WIF1 ( 36 )) were reported to be silenced with functional relevance in cancers other than gastric cancers. In addition, two genes (CDH2 ( 37 ) and FYN ( 38 )) were reported to be methylated in some cancers, but their functional significance needs clarification.

Also among the 32 genes whose silencing was novel, we were able to find potential tumor‐related genes. To achieve this, some genes were selected based on (i) antioncogenic cellular functions or (ii) location in genomic regions with frequent LOH in gastric cancers. Candidate tumor‐related genes were further selected based on (iii) the presence of methylation of promoter CGI in primary gastric cancers, and (iv) expression in normal gastric mucosae when various tissues were compared. MTSS1/MIM/BEG4 met all of these criteria, and was a good candidate for a novel tumor‐related gene. It mediates Sonic hedgehog signaling by potentiating Gli‐dependent transcription,( 39 ) and is known as a metastasis suppressor gene in bladder cancers.( 40 ) Although LOH was not frequent in their locations, ANXA5, AREG, GREM, IGFBP7, IRS2, BMP7, CTSL and IGFBP3 were expressed in the normal gastric mucosae and had potential antioncogenic functions, such as mediation of SMAD signaling (BMP7)( 41 ) and induction of apoptosis (IGFBP3).( 42 ) There is a possibility that silencing of these genes is causally related to development and progression of gastric cancers. However, considering the large number of methylation‐silenced genes, it was likely that the majority of the genes silenced in AGS did not have causal roles in gastric carcinogenesis.

In summary, we found a considerable number of methylation‐silenced genes in a gastric cancer cell line AGS. Potential tumor‐related genes were selected based on their known functions, chromosomal locations, methylation in primary samples and expression in normal gastric mucosae. The usefulness of chemical genomic screening was confirmed.

Acknowledgments

This work was supported by a Grant‐in‐Aid on Priority Area from the Ministry of Education, Sciences, Culture and Sports (MEXT), Japan.

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