Figure 1

Methylation and expression of APC, CDKN2A, ID4, MGMT, RBP1, RUNX3, SFRP1, TIMP3 and TMEFF2 in esophageal cancer cell lines OE33 and TE7. The esophageal cancer cell lines OE33 and TE7 were treated with either 1 μmol/L aza-dC or vehicle for 72 hours. The medium was replaced with fresh medium only, and the cells incubated for a further 24 hours before harvesting. Bisulphite modified DNA was amplified using primers and PCR conditions (Table 2) which were specific for bisulphite modified DNA, did not discriminate between methylated and unmethylated sequences, and did not amplify unmodified DNA. The PCR products were melted by increasing the temperature from 60 to 95°C, rising 0.5 or 1°C at each step, waiting 30 seconds on the first step then 5 seconds for each step thereafter. Data was collected and analysed using the Melt Curve Analysis function of the RG-3000 Application Software. The left hand column shows the melt curves for each of the nine genes for OE33, the central column for TE7. Each plot shows the melt curves for the unmethylated (black lines) and methylated (open circles) controls and the cell lines treated with vehicle (red circles) or aza-dC (green circles). The horizontal axis represents temperature and the vertical axis -dF/dT. CDKN2A was not amplified in TE7. Interpretation of the melt curves is described in the Materials and Methods. The right hand column shows the gene expression in cell lines treated with vehicle (red columns) or aza-dC (green columns), as determined by qRT-PCR and normalised to HMBS. Data shown are the means ± SD from three independent experiments. TMEFF2 expression was below detectable limits in either cell line treated with vehicle or aza-dC.