Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

This study was designed to analyze whether ceramide, a bioeffector of growt h suppression, plays a role in the regulation of telomerase activity in A54 9 cells. Telomerase activity was inhibited significantly by exogenous C-6-c eramide, but not by the biologically inactive analog dihydro-C-6-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 mu M C-6-ceramide at 24 h, Moreover, analysis of phosphatidylserine translocat ion from the inner to the outer plasma membrane by flow cytometry and of po ly(ADP-ribose) polymerase degradation by Western blotting showed that ceram ide treatment (20 muM for 24 h) had no apoptotic effects. Trypan blue exclu sion, [H-3]thymidine incorporation, and cell cycle analyses, coupled with c lonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 muM concentration at 24 h resulted in the cell cycle arrest of th e majority of the cell population at G(0)/G(1) with no detectable cell deat h, These results suggest that the inhibition of telomerase by ceramide is n ot a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing th e full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, show ing that the increased endogenous ceramide is sufficient for telomerase inh ibition. Moreover, treatment of A549 cells with daunorubicin at 1 muM for 6 h resulted in the inhibition of telomerase, which correlated with the elev ation of endogenous ceramide levels and growth arrest. Finally, stable over expression of human glucosylceramide synthase, which attenuates ceramide le vels by converting ceramide to glucosylceramide, prevented the inhibitory e ffects of C-6-ceramide and daunorubicin on telomerase, Therefore, these res ults provide novel data showing for the first time that ceramide is a candi date upstream regulator of telomerase.