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

Oncogene volume 20, pages 7505–7513 (2001)Cite this article

Abstract

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference 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 bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database 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 receptor 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-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2′-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial 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 that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

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Acknowledgements

The authors are grateful to Drs T Niki and T Yamada for providing tumor samples and preparing paraffin block sections; to Dr M Nagao for supportive discussions; and to Drs T Kuramoto and K Asada for critical reading of the manuscript. D Takai is a recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare; by a Grant-in-Aid for Human Genome and Gene Therapy; by a Grant-in-Aid from the Ministry of Education, Science, Culture and Sports; and by a grant from the Hishinomi Foundation for Cancer Research.

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Authors and Affiliations

  1. Carcinogenesis Division, National Cancer Center Research Institute 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan

    Daiya Takai, Yukiko Yagi, Kuniko Wakazono, Takashi Sugimura & Toshikazu Ushijima

  2. Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan

    Daiya Takai, Nobuya Ohishi & Yutaka Morita

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  1. Daiya Takai
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Correspondence to Toshikazu Ushijima.

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Takai, D., Yagi, Y., Wakazono, K. et al. Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis. Oncogene 20, 7505–7513 (2001). https://doi.org/10.1038/sj.onc.1204940

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