© 2000 by Oxford University Press
Journal of the National Cancer Institute, Vol. 92, No. 16, 1303-1307,
August 16, 2000
© 2000 Oxford University Press
ACCELERATED DISCOVERY |
Promoter Methylation and Silencing of the Retinoic Acid Receptor-ß Gene in Lung Carcinomas
Affiliations of authors: A. K. Virmani, A. Maitra, A. F. Gazdar, Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas; A. Rathi, S. Zöchbauer-Müller, Y. Fukuyama, D. Bryant, S. Heda, Hamon Center for Therapeutic Oncology Research; N. Sacchi, Department of Biology, University of Milan, Italy; K. M. Fong, Department of Thoracic Medicine, The Prince Charles Hospital, Brisbane, Australia; F. Thunnissen, Department of Pathology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands; J. D. Minna, Hamon Center for Therapeutic Oncology Research and Departments of Pharmacology and Internal Medicine, University of Texas Southwestern Medical Center.
Correspondence to: Adi F. Gazdar, M.D., Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical School, 5323 Harry Hines Blvd., Dallas, TX 75390-8593 (e-mail: gazdar{at}simmons.swmed.edu).
Background: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-ß (RARß) gene. Because receptor isoforms RARß2 and RARß4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARß gene in human lung tumors and cell lines. Methods: Methylation of the P2 promoter from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell lines and tumor samples was analyzed by the methylation-specific polymerase chain reaction (PCR). Expression of RARß2 and RARß4 was analyzed by reverse transcription–PCR. Loss of heterozygosity (LOH) was analyzed by PCR amplification followed by electrophoretic separation of PCR products. Statistical differences were analyzed by Fisher's exact test with continuity correction. Results: The P2 promoter was methylated in 72% (63 of 87) of SCLC and in 41% (52 of 127) of NSCLC tumors and cell lines, and the difference was statistically significant (two-sided P<.001). By contrast, in 57 of 58 control samples, we observed only the unmethylated form of the gene. Four tumor cell lines with unmethylated promoter regions expressed both RARß2 and RARß4. Four tumor lines with methylated promoter regions lacked expression of these isoforms, but demethylation by exposure to 5-aza-2'-deoxycytidine restored their expression. LOH at chromosome 3p24 was observed in 100% (13 of 13) of SCLC lines and 67% (12 of 18) of NSCLC cell lines, and the difference was statistically significant (two-sided P = .028). Conclusions: Methylation of the RARß P2 promoter is one mechanism that silences RARß2 and RARß4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.
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