Molecular mechanisms of ceramide-mediated telomerase inhibition in the A549 human lung adenocarcinoma cell line

This study was aimed at identifying the molecular mechanisms by which ceram ide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C-6-ceramide (20 mum) caused a significant reduction of telomerase a ctivity at 24 h as detected using the telomeric repeat amplification protoc ol, and this inhibition correlated with decreased telomerase reverse transc riptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C-6-ceramid e significantly decreased hTERT mRNA in a time-dependent manner. Electropho retic mobility shift and supershift assays demonstrated that the binding ac tivity of c-Myc transcription factor to the E-box sequence on the hTERT pro moter was inhibited in response to C.-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic pla smid constructs containing the functional hTERT promoter and its E-box dele ted sequences cloned upstream of a luciferase reporter gene. Further analys is using RT-PCR and Western blotting showed that c-Myc protein but not its mRNA levels were decreased in response to C-6-ceramide at 24 h. The effects of ceramide on the c-Myc protein were shown to be due to a reduction in ha lf-life via increased ubiquitination. Similar results were obtained by incr eased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activitie s. Furthermore, the elevation of endogenous ceramide by overexpression of b acterial sphingomyelinase after transient transfections also induced the in hibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenons and endogenous ceramides mediate the modulation of telomeras e activity via decreased hTERT promoter activity caused by rapid proteolysi s of the ubiquitin-conjugated c-Myc transcription factor.