Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis

Citation
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
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Onconogenesis & Cancer Research
Journal title
ONCOGENE
ISSN journal
0950-9232 → ACNP
Volume
20
Issue
51
Year of publication
2001
Pages
7505 - 7513
Database
ISI
SICI code
0950-9232(20011108)20:51<7505:SOHARE>2.0.ZU;2-U
Abstract
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.