Fas expression in metastatic osteosarcoma cells is not regulated by CpG island methylation.

Fas expression in osteosarcoma (OS) cells is inversely correlated with the metastatic potential of OS to the lung. The purpose of this study was to determine whether loss of Fas expression in metastatic OS cells is secondary to DNA methylation of CpG islands in the Fas gene. SAOS-2 cells have high levels of Fas expression and do not form lung metastases when injected intravenously, whereas LM7 cells have low levels of Fas expression and do produce lung metastases. Using the endonucleases HpaII and MspI and a polymerase chain reaction-based methylation assay, we found that all four CpG sites in the CCGG sequence in the Fas promoter region were unmethylated in both SAOS-2 and LM7 cells. We performed detailed analysis of the 28 and 46 CpG sites in the Fas promoter and first intron region, respectively, using bisulfite-modified genomic DNA sequencing. More than 99.8% of the examined CpG sites were unmethylated and there was no difference of CpG methylation in SAOS-2 and LM7 cells as well as LM7 metastatic lung tumor tissue samples. Treatment of LM7 cells and another OS cell line, DLM8 with low levels of Fas expression, with demethylation agent, 5-azadeoxycitidine (AzadC), did not change the Fas expression and did not increase sensitivity of AzadC-treated cells to Fas ligand (FasL) treatment. In conclusion, our data indicate that decreased Fas expression in OS cells is not secondary to DNA methylation of CpG islands in the Fas gene and that Fas expression cannot be increased by using demethylation agents.