1. ¹Department of Otolaryngology, Head and Neck Cancer Research Division, Johns Hopkins University, Baltimore, MD, USA
2. ²Department of Genetic Medicine, Institute of Cell Engineering, Johns Hopkins University, Baltimore, MD, USA
3. ³Karmanos Cancer Institute, Department of Pathology, Wayne State University, Detroit, MI, USA
4. ⁴Department of Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Tsurumibaru, Beppu, Japan

Correspondence: Dr D Sidransky, The Department of Oncology and Otolaryngology, The Division of Head and Neck Cancer Research Institute, Johns Hopkins University School of Medicine, Johns Hopkins Cancer Research Building II, 1150 Orleans Street, Baltimore, MD 21231, USA. E-mail: dsidran1@jhmi.edu

Received 5 September 2007; Revised 22 October 2007; Accepted 1 December 2007; Published online 28 January 2008.

Abstract

To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription–PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65% , 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3% , 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.