Figure 1

Aberrant ROR2 hypermethylation in colon cancer. (A) The ROR2 promoter with its associated CpG island (top). CpG location, transcription start site (TSS) and ATG are indicated. Bisulphite sequencing primers, white arrows; Illumina Infinium probes (465 bp upstream and 322 bp downstream of the TSS), red and green lines, respectively. Lower panels show bisulphite genomic sequencing results of 12 individual clones in healthy colon epithelium, colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO), showing methylated (black squares) or unmethylated cytosines (white squares). (B) DNA methylation analysis of two CpG sites within the ROR2 promoter in healthy colon epithelium, colonocytes and eight colon cancer cell lines using Infinium methylation arrays. Relative methylation for each CpG site (-465 bp and + 322 bp) is represented on a scale from 0.0 to 1.0 (corresponding to a 0% and 100% likelihood of CpG hypermethylation). (C) Methylation-specific PCR (MSP) of ROR2 promoter in healthy colon tissue and colon cancer cell lines. A PCR band in lanes M or U indicates methylated or unmethylated genes, respectively. In vitro-methylated DNA (IVD) was used as a positive control for methylated DNA. (D) Methylation analysis of the ROR2 promoter using MSP of 36 primary colon tumours. Percentage of tumours with ROR2 promoter hypermethylation (unmethylated, white; methylated, red; top) and ethidium bromide staining of an MSP reaction of two representative tumours (unmethylated and methylated) resolved in an agarose gel (bottom).