Identification of methylation-silenced genes in colorectal cancer cell lines: genomic screening using oligonucleotide arrays.
OBJECTIVE
Aberrant methylation of promoter CpG islands is associated with the loss of expression of tumor suppressor genes in human cancers. The purpose of this study was to examine methylation-silenced genes in colorectal cancer (CRC) cell lines.


METHODS
Using an oligonucleotide array, we undertook a genome-wide search for genes upregulated following treatment with a demethylating agent (5-aza-2'-deoxycytidine) in two CRC cell lines, DLD-1 and HT29. Promoter methylation status was determined in 12 CRC cell lines and 11 CRC tissues by methylation-specific polymerase chain reaction (MSP).


RESULTS
After treatment, 350 genes were up-regulated 1.5-fold or more. Six genes (PAGE-5, VCX, MAEL, GAGED2, UCHL1, and GAGE7), which contained putative 5' CpG islands in their promoter regions, were confirmed to be silenced in CRC cell lines. UCHL1 (also known as PGP9.5) is involved in regulation of cellular ubiquitin levels, and its promoter methylation was detected in 10 out of 12 CRC cell lines. The level of methylation of UCHL1 was significantly higher in tumors than in corresponding normal mucosae (p = 0.005).


CONCLUSIONS
Chemical genomic screening led to the identification of a specific promoter subject to hypermethylation in CRC. These results suggest that aberrant promoter methylation is the primary mechanism of transcriptional silencing of the UCHL1 gene and that methylation of the UCHL1 gene promoter increases during the development and progression of CRC.
Fukutomi
Satoshi
S
Department of General Surgery, Chiba University Graduate School of Medicine, Chuoh-ku, Chiba, Japan.
Seki
Naohiko
N
Koda
Keiji
K
Miyazaki
Masaru
M
D000368:Aged; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018709:Statistics, Nonparametric; D043222:Ubiquitin Thiolesterase / Q000235:genetics*
C482860:UCHL1 protein, human; D043222:Ubiquitin Thiolesterase