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 ceramide inhibits telomerase activity in the A549 human lung adenocarcinoma cell line. C(6)-ceramide (20 microm) caused a significant reduction of telomerase activity at 24 h as detected using the telomeric repeat amplification protocol, and this inhibition correlated with decreased telomerase reverse transcriptase (hTERT) protein. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blot analyses showed that C(6)-ceramide significantly decreased hTERT mRNA in a time-dependent manner. Electrophoretic mobility shift and supershift assays demonstrated that the binding activity of c-Myc transcription factor to the E-box sequence on the hTERT promoter was inhibited in response to C(6)-ceramide at 24 h. These results were also confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box deleted sequences cloned upstream of a luciferase reporter gene. Further analysis 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 half-life via increased ubiquitination. Similar results were obtained by increased endogenous ceramide levels in response to nontoxic concentrations of daunorubicin, resulting in the inhibition of telomerase and c-Myc activities. Furthermore, the elevation of endogenous ceramide by overexpression of bacterial sphingomyelinase after transient transfections also induced the inhibition of telomerase activity with concomitant decreased hTERT and c-Myc protein levels. Taken together, these results show for the first time that both exogenous and endogenous ceramides mediate the modulation of telomerase activity via decreased hTERT promoter activity caused by rapid proteolysis of the ubiquitin-conjugated c-Myc transcription factor.

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 growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C(6)-ceramide, but not by the biologically inactive analog dihydro-C(6)-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 microm C(6)-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 microm for 24 h) had no apoptotic effects. Trypan blue exclusion, [(3)H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 microm concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G(0)/G(1) with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not 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 the 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, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 microm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C(6)-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.

Skin fibroblasts from individuals hemizygous for the familial adenopolyposis susceptibility gene show delayed crisis in vitro.

Normal human fibroblast cells have not been reported to escape crisis--that is, they die after about 24 doublings in culture. We have been studying the growth properties of skin fibroblast cells from persons in families with familial adenopolyposis of the colon (FAP). An individual hemizygous at the FAP locus will develop hyperplasia of the colonic epithelium followed by colonic polyps, both at an early age. Polyps themselves still retain a single functional FAP allele. A mutation or deletion in this allele in a polyp is hypothesized to lead to further loss of growth control; thus, a tumor is formed. We found that the in vitro life-span of skin fibroblast cells from FAP individuals and from some asymptomatic children were markedly extended when compared with normal individuals.