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J. Biol. Chem., Vol. 276, Issue 35, 32506-32514, August 31, 2001
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From the This study was aimed at
identifying the molecular mechanisms by which ceramide inhibits
telomerase activity in the A549 human lung adenocarcinoma cell line.
C6-ceramide (20 µM) 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 C6-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 C6-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 C6-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.
Molecular Mechanisms of Ceramide-mediated Telomerase Inhibition
in the A549 Human Lung Adenocarcinoma Cell Line*
§,,
,, and Departments of Biochemistry and Molecular
Biology and ¶ Pediatrics, Division of Hematology/Oncology, and
** Medicine and Ralph H. Johnson Veteran Administration,
Medical University of South Carolina, Charleston, South Carolina 29425, and the Department of Biochemistry, American University of
Beirut, Beirut, Lebanon
*
This work was supported in part by research grants from the
Department of Energy through the Environmental Biosciences Program at
MUSC, American Cancer Society Grant IRG-97-151-01, and Department of
Defense, Coastal Carolina Program Project project number 2 at Hollings
Cancer Center (to B. O.), and National Institutes of Health Grants
CA87584 and GM43825 (to Y. A. H.) and AG16583 and AG12467 (to
L. M. O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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