Epigenetic modifications, such as DNA hypermethylation and histone deacetylation, are commonly detected in human cancer cells, where they have been shown to downregulate tumour-suppressor genes. Sidransky et al. have been using a combination of techniques to identify additional genes that are silenced in this way, and have subsequently uncovered three new tumour suppressors.

Sidransky's group treated oesophageal squamous-cell carcinoma (ESCC) cells with pharmacological agents to prevent the epigenetic silencing of genes — cells were treated with 5-aza-2′-deoxycytidine to block DNA methylation, and then with tricohstatin A to inhibit histone deacetylase. This treatment reverses the formation of transcriptionally repressive chromatin structures at methylated promoters. Microarray analysis was then used to compare gene-expression patterns between transcriptionally silenced and unsilenced cancer cells. A total of 58 silenced genes were identified by this approach: 44 (76%) were found to contain dense CpG islands — well-known methylation sites — in their promoter regions. Some 13 of 22 tested gene promoters were methylated in the ESCC cell lines, and 10 of these genes were found to be downregulated at the mRNA level.

Three of the genes — CRIP1 , APOD and NMU — had growth-suppressive activity when transfected into ESCC cells, as determined using colony-formation assays. NMU is proposed to be involved in normal oesophageal mucosa integrity, and its receptor, FM3, is a G-protein-coupled receptor that can signal through PI3 kinase-γ — recently shown to block the growth of human colon cancer cells. CRIP1 is believed to be a transcription factor that was shown to induce apoptosis in cancer cell lines, and APOD has been associated with cell growth arrest.

The epigenetically silenced genes were found to cluster in specific chromosomal regions. Many of these loci — such as 3q26, 4q12 and 14q24 — have also been shown to harbour chromosome deletions of loss of heterozygosity in cancer cells. This approach could therefore be used to identify new tumour-suppressor genes and loci in other types of cancer cell.