Investigation of the influence of Fas antigen on Hep G2 cells, an erythropoietin producing cell line

We have already reported that serum levels of soluble Fas (sFas) and Fas-positive mononuclear cells increased concomitantly with deterioration in renal function and the increases were statistically significant. Moreover, the severity of renal anemia in renal failure patients was significantly correlated with serum levels of sFas. Therefore, we investigated whether or not Fas and Fas ligand (FasL) influenced the production of erythropoietin (EPO). Hep G2 cells, an EPO productive human hepatocellular carcinoma cell line, were cultured in MEM medium with 10% of FCS containing 1, 10 or 100 ng/ml of sFas, or sFasL. The EPO concentrations of the supernatants were measured by the ELISA method, Annexin V positive cells were calculated by flow cytometry, H3 leucin uptake was measured by a liquid scintillation counter, an MTT assay was performed using the light absorption method, fragmented nuclei were stained by the TUNEL method and DNA laddering was observed by agarose gel electrophoresis. Their characteristics evaluated at 0, 24, 48 and 72 hrs. Both EPO production and H3 leucin uptake were suppressed in culture with sFas or sFasL, dose-dependently and declines in MTT activities accompanied these changes at 24 hrs. In addition, nuclear fragmentation and DNA laddering were found to be stimulated in culture with sFas or sFasL at 48 hrs. These data suggest that sFas induced apoptosis and had a cytotoxic effect on Hep G2 cells. In conclusion, hyper-sFas-emia observed in chronic renal failure may regulate the production of EPO, which indicates that sFas acts as a uremic toxin.