1. ¹Molecular Oncology, IDIBELL-Institut de Recerca Oncològica, Gran Via km 2.7, Hospital Duran i Reynals, L'Hospitalet, Barcelona 08907, Spain
2. ²The Garvan Institute of Medical Research, Darlinghurst NSW 2010, Australia
3. ³IDIBELL-Institut Català d'Oncologia, L'Hospitalet, Barcelona, Spain
4. ⁴Laboratori de Bioestadistica i Epidemiologia, Facultat de Medicina, Universitat Autonoma de Barcelona, Cerdanyola, Barcelona, Spain

Correspondence: MA Peinado, E-mail: mpeinado@iro.es

Received 21 February 2005; Revised 29 April 2005; Accepted 25 May 2005; Published online 11 July 2005.

Abstract

Inactivation of specific tumor suppressor genes by transcriptional silencing associated with hypermethylation of the promoter is a common event in cancer. We have applied the amplification of intermethylated sites (AIMS) technique to a 100 human colorectal cancers and seven cell lines to identify recurrent alterations that may unveil silenced tumor suppressor genes. Bisulfite sequencing was used to confirm differential DNA methylation results. Gene expression analysis was performed by real-time RT–PCR. An AIMS band recurrently displayed in tumors but not in normal tissues was isolated and identified as part of the CpG island of the prostacyclin synthase (PTGIS) gene promoter. PTGIS promoter was hypermethylated in 43 out of 100 colorectal cancers and in all cell lines. Bisulfite sequencing and clonal analysis confirmed the results obtained by AIMS and demonstrated biallelic hypermethylation of PTGIS promoter. Hypermethylation of the PTGIS promoter was associated with diminished gene expression, that was restored after treatment with demethylating and histone deacetylases inhibitor agents. PTGIS hypermethylation was associated with aneuploidy and p53 mutations. In the adjusted model, PTGIS methylation, but not p53 mutation, maintained the association with aneuploidy. We conclude that epigenetic inactivation of the PTGIS gene is a recurrent alteration in colorectal carcinogenesis.