Spreading of methylation within RUNX3 CpG island in gastric cancer

RUNX3 is a novel tumor suppressor gene that is frequently silenced by promoter hypermethylation in gastric cancer. The methylation status of multiple regions within the RUNX3 promoter CpG island (3478 bp) was examined in gastric cancer cell lines, primary gastric cancers and non-neoplastic gastric mucosa to clarify how methylation spreads within the CpG island. The critical regions for RUNX3 silencing were evaluated by analysis of cell lines. The most 5' region of the CpG island was methylated in 90% (9/10) of gastric cancer cell lines, 96% (43/45) of primary gastric cancers and in 96% (43/45) of non-neoplastic gastric mucosa. The frequencies of methylation were less near the transcription start site and were 40% (4/10) in cell lines, 53% (24/45) in primary gastric cancers and 11% (5/45) in non-neoplastic gastric mucosa, where methylation was proven to be critical for gene silencing. Thus, hypermethylation initially occurs at the most 5' region of the RUNX3 CpG island and spreads to the transcription start site before ultimately shutting down RUNX3 mRNA expression. The detection of hypermethylation at multiple regions within the RUNX3 CpG island may be useful in the diagnosis and risk assessment of gastric cancer.