D. Takai et al., Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis, ONCOGENE, 20(51), 2001, pp. 7505-7513
Aberrantly hypermethylated genes in human lung cancers were searched for by
a genome scanning technique, methylation-sensitive-representational differ
ence analysis (MS-RDA). A total of 59 DNA fragments were isolated as those
methylated more heavily in either/both of two lung squamous cell carcinoma
cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronc
hial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation wa
s confirmed in primary squamous cell carcinomas, were sequenced. By databas
e searches, 17 of them were shown to be located within 2 kb of putative CpG
islands, and five of the 17 DNA fragments had transcribed regions of known
genes in their vicinities. By RT - PCR of the five genes in the carcinoma
cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine rec
eptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite
modification showed that the CpG island in the promoter region of HTR1B was
hypermethylated, while that of EDN1 was not. Demethylation and re-expressi
on of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxy
cytidine. In primary lung cancers, decreased mRNA expression of HTR1B was o
bserved in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohi
stochemical analysis of endothelin-1 confirmed that its immunoreactivity wa
s reduced in squamous cell carcinoma cells compared with that in normal bro
nchial epithelial cells. Considering that endothelin-1 induces apoptosis in
melanoma cells and that silencing of endothelin receptor B is observed in
prostate cancers, its reduced expression was speculated to confer a growth
advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments t
hat are hypermethylated and silenced, such as HTR1B, and those whose expres
sions are altered and the methylation statuses outside the promoter region
are altered, such as EDN1.