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INTRODUCTION |
Telomerase is a multisubunit DNA polymerase that elongates
telomeres at the end of chromosomes comprising long tandem repeats of
hexanucleotide 5'-TTAGGG-3' (1-3). Telomeres function to protect the
end of chromosomes from degradation and ligation. Telomere length is
primarily controlled by telomerase, a ribonucleoprotein composed of a
catalytic protein subunit, telomerase reverse transcriptase (4-8), telomerase-associated protein (TEP1) (9), and a stably associated RNA moiety (hTR), which acts as an intrinsic template for
the elongation of telomeres (1, 2). In vitro reconstitution experiments show that human telomerase reverse transcriptase and hTR
may be sufficient for the activity of the enzyme (10, 11). Recently,
alternatively spliced variants of human telomerase reverse transcriptase mRNA were described, showing the existence of a variety of telomerase holoenzymes with diverse biological functions (12). It has been well established that telomerase is not active in
most somatic tissues. Conversely, it is activated in the majority of
cancer-derived cell lines and malignant tumors, suggesting that
telomerase plays an important role in cell immortalization and
tumorigenesis (13, 14). This is further supported by studies showing
that the expression of telomerase activity results in telomere
stabilization and immortality of germ line cells when further oncogenic
signals are present (15). Moreover, it has been reported that the
disruption of telomere maintenance by inhibiting telomerase limits
proliferation of human cancer cells due to excessive telomere erosion
(16, 17).
Lung cancer remains the leading cause of cancer-related deaths in the
United States, with a less than 5% overall survival rate and no
successful therapeutic strategies. Clinical studies have demonstrated
that the presence of telomerase activity in tumors of non-small cell
lung cancer patients correlates with a high cell proliferation rate and
advanced pathologic stage (18). Telomerase activity is also correlated
with shorter survival in non-small cell lung cancer patients as
compared with those with a telomerase-negative tumor (19). Therefore,
telomerase, which is up-regulated in about 85% of lung cancers and not
detected in normal lung tissues, is regarded as one of the pivotal
elements in cellular proliferation and tumor development (20-22).
Thus, it is believed that the modification of telomerase activity may be a potential therapeutic modality for the treatment of lung and other cancers (20). Several proteins such as p53, retinoblastoma (Rb),1 c-Myc, Bcl-2, protein
kinase C, and protein phosphatase 2A have been shown to potentially
regulate telomerase activity with transcriptional and
posttranscriptional mechanisms (23). Therefore, identifying intracellular factors and their signaling pathways involved in the
control of telomerase activity is extremely important for both cancer
research and therapy.
The sphingolipid ceramide has been proposed as a regulator of
antiproliferative biological responses (24). The generation of
endogenous ceramide is induced by various agents (such as anticancer drugs, ionizing radiation, Fas ligand, tumor necrosis factor-
, interleukin 1-
, nerve growth factor, and
-interferon), which cause apoptotic cell death, growth arrest, or differentiation (24).
Ceramide has also been shown to activate Rb, c-Jun
NH2-terminal kinase, and effector caspases and to
down-regulate c-Myc and other targets involved in proliferative
responses such as phospholipase D and Akt (25).
Because protein phosphatase 2A, Rb, and c-Myc are downstream targets of
ceramide and are also involved in the regulation of telomerase
activity, we hypothesized that ceramide might play a functional role in
mediating signal transduction pathways involved in the control of
telomerase activity. Therefore, this study was designed to investigate
this hypothesis in the A549 human lung adenocarcinoma cell line. We
present data that show for the first time that both exogenous
C6-ceramide and endogenous ceramides are involved in
telomerase modulation in vivo, and this modulation is not
due to toxicity or apoptotic cell death but rather correlates with the
inhibition of cell proliferation. This study also provides evidence
that the attenuation of endogenous ceramide levels caused by
overexpression of glucosylceramide synthase, which converts ceramide to
glucosylceramide, prevents C6-ceramide- and
daunorubicin-induced telomerase inhibition, revealing an important role
of ceramide in telomerase regulation.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Culture Conditions--
The A549 human lung
carcinoma cells were obtained from Dr. Alice Boylan (Medical University
of South Carolina, Charleston, SC). Cells were maintained in growth
medium containing 10% fetal calf serum and 100 ng/ml each penicillin
and streptomycin (Life Technologies, Inc.) at 37 °C in 5%
CO2. Cell-permeable and biologically active short chain
ceramide (C6-ceramide) and its biologically inactive analog
dihydro-C6-ceramide were obtained from the Synthetic Lipid
Core at the Department of Biochemistry and Molecular Biology, Medical
University of South Carolina. Daunorubicin (DNR) was purchased from Sigma.
Determination of Telomerase Activity--
Telomerase activity in
cell extracts was measured by the PCR-based telomeric repeat
amplification protocol (TRAP) using the TRAPeze kit (Intergen,
Gaithersburg, MD), which includes a 36-base pair internal control to
allow quantitation of activity. Briefly, the cells, grown in 6-well
plates, were washed in phosphate-buffered saline and homogenized in
CHAPS lysing buffer containing 10 mM Tris-HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM benzamidine, 5 mM
-mercaptoethanol, 0.5%
3-[3-cholamidopropyl-dimethyl-amino]-1-propane-sulfonate, and 10%
glycerol for 30 min on ice. Then, 50-100 ng of protein from each cell
extract were analyzed in the TRAP reaction containing 20 mM
Tris-HCl, pH 8.3, 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM EGTA, 0.01%
bovine serum albumin, 1 µl of dNTPs, 1 µl of telomerase
substrate primer (6 × 105 cpm), 1 µl of
reverse primer mix, and 0.5 µl (2 units) of
Taq-polymerase. After the extension of telomerase
substrate primer at 30 °C for 30 min, the extended telomerase
products were then amplified by two-step PCR (94 °C for 30 s,
60 °C for 30 s) for 27 cycles. The PCR products were separated
on 10-12.5% polyacrylamide gels and analyzed by autoradiography. The
telomerase activity in each sample was quantitated by measuring the
ratio of the 36-base pair internal standard to the extended telomerase
products in three independent experiments as described by the
manufacturer using ChemiImager (Alpha Innotech Corp., San Leandro, CA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Bromide (MTT) Assay--
The concentrations of
C6-ceramide or DNR that inhibited cell growth by 50%
(IC50) were determined from cell survival plots obtained by
MTT assay as described elsewhere (26). In short, the cells were plated
into 96-well plates containing 100 µl of the growth medium in the
absence or presence of increasing concentrations of
C6-ceramide or DNR at 37 °C in 5% CO2 for
24-48 h. They were then treated with 25 µl of MTT for 4-5 h. After
lysing the cells in 100 µl of the lysis buffer, the plates were read
in a micro plate reader (Dynatech, Chantilly, VA) at 570 nm. After
that, the IC50 concentrations of the compounds were
determined from cell viability plots as we described (26). Triplicate
wells were used for each treatment. The final concentration of
Me2SO (a solvent for ceramide analogs and DNR) in
the growth medium was less than 0.1% (v/v), which has no effect on
cell growth and survival.
Annexin V Binding Assay--
After treatments with
C6-ceramide or DNR, the cells that remained attached to the
growth flasks were used for determining the translocation of
phosphatidylserine (PS) to the outer plasma membrane using the
human vascular phospholipid-binding protein, annexin V, conjugated with
fluorescein (Molecular Probes) by flow cytometry as described by the manufacturer.
Western Blotting--
The degradation of poly(ADP-ribose)
polymerase (PARP) protein levels in cells was detected by Western blot
analysis. In short, total proteins (50 µg/lane) were separated by
4-15% SDS-polyacrylamide gel electrophoresis and blotted onto an
Immobilon membrane, and PARP protein was detected using 1 µg/ml
rabbit polyclonal anti-PARP antibody (Santa Cruz Biotechnology) and
peroxidase-conjugated secondary anti-rabbit antibody (1:2500). The
proteins were visualized using the ECL protein detection kit (Amersham
Pharmacia Biotech) as described by the manufacturer. Equal
loading was confirmed by Coomassie Blue staining of SDS-polyacrylamide
gel electrophoresis strips cut from gels containing 50 µg of
protein/lane of each sample prior to blotting.
Trypan Blue Exclusion Method--
The effects of
C6-ceramide on cell growth were determined by the trypan
blue exclusion method as described previously (27). In short, cells,
seeded as 80 × 103 cells/well in 6-well
plates, were treated in the absence or presence of various
concentrations of C6-ceramide for 24 h. Then, cells were trypsinized and diluted in phosphate-buffered saline. The floating
dead cells in the medium and cells that remained attached to the
plates were then counted using a hematocytometer in the presence of
trypan blue solution at a 1:1 ratio (v/v) (Sigma) as described by the
manufacturer. The final concentration of Me2SO in the
growth medium was less than 0.1% (v/v).
Analysis of Growth by [3H]Thymidine
Incorporation--
The effects of ceramide on the inhibition of cell
proliferation were examined by detection of trichloroacetic
acid-precipitable [3H]thymidine incorporation as
described previously (28). Cells were treated in the absence or
presence of 20 µM C6-ceramide for various
time periods. Then, cells were washed and pulsed with the
presence of 1 µCi/ml [3H]thymidine in the growth medium
for 1 h, and after washes with phosphate-buffered saline, they
were incubated with 2 ml of 0.5% trichloroacetic acid on ice for 20 min. Then, the trichloroacetic acid precipitates in the wells were
washed twice with absolute EtOH and dissolved in 2 ml of 0.1 M NaOH, 2% Na2CO3, and 400 µl of the trichloroacetic acid precipitates were neutralized with 144 µl of 1 M HCl and counted in a scintillation counter.
Analysis of Cell Cycle Profiles--
The effects of 20 µM C6-ceramide on the cell cycle profiles of
A549 cells at 24 h were analyzed in the presence of DNase-free RNase and propidium iodine by flow cytometry as described previously (29). Untreated cells were used as controls.
Clonogenic Cell Survival Assay--
The anchorage-independent
growth of A549 cells treated in the absence or presence of 5, 10, 20, and 50 µM C6-ceramide for 24 h was
examined by clonogenic cell survival on soft agar as described
previously (30). In short, after treatments, cells were trypsinized,
and various dilutions of the cells were resuspended in 2 ml of the
growth media containing 12% serum and 0.3% agarose and then
plated in 6-well plates containing 2 ml of growth media with 12% serum
and 0.6% agarose. The plates were incubated at 37 °C at 5%
CO2 for 2 weeks, and colonies were counted as described (30).
Measurement of Total Endogenous Ceramide Levels--
Total
endogenous ceramide levels were measured using the diacylglycerol
kinase method as described previously (31). In short, after total
cellular lipids were extracted using the standard Bligh and Dyer
protocol, they were dried under N2. The dried lipids were
then resuspended, and duplicate aliquots were used for phosphate measurements and the Escherichia coli diacylglycerol kinase
assay as modified for ceramide. This assay depends on the
phosphorylation of ceramide and diacylglycerol, generating ceramide
phosphate and phosphatidic acid, respectively, by diacylglycerol kinase in the presence of radiolabeled ATP. The radiolabeled products were
visualized by thin layer chromatography, and the phosphorylated products of ceramide and diacylglycerol were identified by comparison with known standards run on the same plate. In addition, the amounts of
products that were scraped from the plates were quantitated by
scintillation counting and normalized to internal phosphate levels.
Plasmids and Stable Transfections--
The full-length
glucosylceramide synthase (GCS) fragment was cloned into the
pcDNA3 mammalian expression vector system (Invitrogen, San Diego,
CA) as described (32). The full-length bacterial sphingomyelinase
(b-SMase) cDNA fragment was cloned into the pEGFPN1 mammalian
expression vector system (Invitrogen) upstream of the green fluorescent
protein (GFP) sequence as described (33). The stable and transient
transfections of A549 cells were performed using Effectene transfection
reagent as described by the manufacturer (Qiagen). The purified b-SMase
was purchased from Sigma.
In Vitro GCS Enzyme Activity Assays--
The conversion of
NBD-C6-ceramide to NBD-C6-glucosylceramide was
detected in cell extracts at 37 °C for 60 min in the presence of
UDP-glucose as described (34). The final products were separated on TLC
plates and observed under a UV source.
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RESULTS |
Effects of Exogenous C6-Ceramide on Telomerase
Activity--
To examine the effect of ceramide on telomerase
activity, A549 cells were treated in the absence or presence of
increasing concentrations of C6-ceramide for various time
periods. The dead cells (always less than 2% of the cell
population) that detached from the culture flasks were removed by
phosphate-buffered saline washes, and telomerase activity was measured
in viable cells (confirmed using trypan blue exclusion) by the
PCR-based TRAP. First, serial dilutions of proteins (1-100 ng)
obtained from untreated A549 cell extracts were used in the TRAP assay
to establish a linear response range of the assay for reliable
quantification of telomerase activity. Telomerase activity in each
sample was quantitated by measuring total density of the bands present
in the characteristic DNA ladder of the TRAP assay using ChemiImager.
The 36-base pair internal standard PCR product was used as a control
for quantification. Treatment of the cell extracts with RNase or
proteinase K eliminated telomerase activity, showing the specificity of
the assay (data not shown). Linear amplification was obtained using
50-100-ng proteins, which were used in subsequent TRAP assays (data
not shown). Fig. 1A shows that
treatment of A549 cells with 5-50 µM C6-ceramide for 6 h resulted in a
dose-dependent decrease in telomerase activity compared
with untreated controls (lanes 2-6 and 1,
respectively). The concentration of C6-ceramide that
inhibited 50% telomerase activity was 20 µM after 6 h treatment (Fig. 1A, lane 4). Moreover, C6-ceramide had no effect on telomerase activity when added
directly to the TRAP assay in vitro (data not shown),
demonstrating that ceramide is indirectly involved in the inhibition of
telomerase.

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Fig. 1.
Analysis of telomerase activity in response
to C6-ceramide in A549 cells. A, A549 cells
grown in 6-well plates (about 80 × 103 cells/well)
were treated in the absence or presence of 0-50 µM
C6-ceramide for 6 h (lanes 1-6),
and then telomerase activity in the cell extracts was determined by the
TRAP assay using 50-100 ng of protein/sample as described under
"Experimental Procedures." The telomerase products were separated
by 12.5% polyacrylamide gel electrophoresis and visualized by
autoradiography. IS, internal standard.
B, the time dependence of telomerase inhibition was analyzed
by treating cells in the absence or presence of 20 µM
C6-ceramide for 0-24 h (lanes 1-4) using the
TRAP assay as described. C, the specificity of
C6-ceramide on telomerase inhibition was analyzed by
growing the cells in the absence or presence of 20 µM
C6-ceramide or its biologically inactive analog
dihydro-C6-ceramide for 24 h (lanes 1-3).
Telomerase activity was measured by the TRAP assay in these cell
extracts as described above, and the samples were separated on 12.5%
acrylamide gels. The 36-base pair PCR product was used as the internal
standard in the TRAP assay. The figures presented are representatives
of at least three independent experiments.
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To examine whether the inhibition of telomerase by
C6-ceramide is time-dependent, A549 cells were
treated in the presence of 20 µM C6-ceramide
for 0, 3, 6, and 24 h (Fig. 1B, lanes 1-4, respectively). As seen in Fig. 1B, telomerase activity was
slightly decreased after 3 h of treatment with
C6-ceramide. However, treatment of the cells with
C6-ceramide for 6 and 24 h resulted in a significant inhibition of telomerase activity (about 50 and 85%, respectively). Moreover, as shown in Fig. 1C, treatment of cells with 20 µM dihydro-C6-ceramide, a biologically
inactive analog of C6-ceramide, for 24 h had no effect
on telomerase activity (lane 3). These results show that the
inhibition of telomerase activity by C6-ceramide is specific.
Detection of the Effects of C6-Ceramide on Cell
Survival and Apoptosis--
To determine whether the effective
concentration of C6-ceramide (20 µM) for the
inhibition of telomerase activity in A549 cells is due to its growth
inhibitory effects, we performed an MTT assay. A549 cells were grown
with increasing concentrations of C6-ceramide for 24 h, and then the IC50 concentration of
C6-ceramide that inhibited cell viability or growth by 50%
was determined from cell survival plots (Fig.
2A) and found to be 36 µM. In independent experiments it was also observed that
around 70% of the cells had successfully taken up the MTT reagent and
converted tetrazolium salt to a colored formazan following treatment
with 20 µM C6-ceramide for 24 h (data
not shown). The MTT is cleaved to formazan by the succinate-tetrazolium
reductase system, which belongs to the respiratory chain of the
mitochondria and is active only in viable cells. Therefore, the MTT
assay detects viable cells, and decreased MTT-positive cells (around
30-40%) in response to 20 µM C6-ceramide at
24 h can be due to either death or inhibition of growth.

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Fig. 2.
Determination of the effects of
C6-ceramide on growth and apoptosis in A549 cells.
A, the effects of increasing concentrations of
C6-ceramide on cell viability were determined by the MTT
assay, and the concentration of C6-ceramide that inhibited
the growth by 50% (IC50) was determined using cell
survival plots as described under "Experimental Procedures."
Triplicate samples were used in MTT assays in at least two independent
experiments. B, the translocation of PS from the inner to
the outer plasma membrane in cells that remained attached to the flasks
after treatment with C6-ceramide at 0, 20, and 50 µM for 24 h was determined by flow cytometry using
the human vascular phospholipid-binding protein annexin V conjugated
with fluorescein. FITC, fluorescein isothiocyanate.
C, the degradation of PARP in A549 cells following
treatments with 0, 20, and 50 µM C6-ceramide
(lanes 1-3, respectively) for 24 h was analyzed by
Western blotting using the rabbit polyclonal anti-PARP antibody as
described under "Experimental Procedures." The molecular
mass markers are indicated on the left. D, the
effects of various concentrations of C6-ceramide on cell
growth at 24 h were analyzed by the trypan blue exclusion method
as described under "Experimental Procedures." Results are
representative of three independent experiments. Error bars represent
standard deviations. If not visible, error bars are smaller than the
diameters of the points.
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To analyze whether telomerase modulation by ceramide is related to its
effects on apoptotic cell death, the translocation of PS from the inner
to the outer leaflet of the plasma membrane, one of the early stages of
apoptotic changes, was analyzed in cells treated with 0, 20, and 50 µM C6-ceramide for 24 h using an annexin
V-fluorescein isothiocyanate antibody binding assay and detected by
flow cytometry. As Fig. 2B shows, translocation of PS to the
outer plasma membrane was detected in only about 2% of the cells
treated with 20 µM C6-ceramide, whereas it
was detected in about 42% of the cells in the presence of a toxic concentration of C6-ceramide (50 µM). We have
confirmed these results also by the absence of PARP cleavage in cells
treated with C6-ceramide at 20 µM for 24 h by Western blotting, whereas the cleavage of PARP was apparent in the
presence of 50 µM C6-ceramide in these cells
compared with untreated controls (Fig. 2C, lanes 2, 3, and 1, respectively). Therefore, these
results show that the inhibition of telomerase by ceramide precedes
caspase activation and apoptotic cell death, and more importantly this
inhibition is not due to apoptotic or other toxic effects of ceramide
in A549 cells.
Analysis of the Effects of C6-Ceramide on Growth
Inhibition and Cell Cycle--
To rule out the possibility that a
decreased number of viable cells in response to 20 µM
C6-ceramide at 24 h observed in MTT assays, which was
not reflected in the annexin-V staining and PARP cleavage, might be due
to death via non-apoptotic mechanisms, we performed the trypan blue
exclusion method as described under "Experimental Procedures."
Trypan blue is taken up by cells with damaged plasma membranes due to
apoptosis or necrosis, and therefore its exclusion reflects viability.
As shown in Fig. 2D, after treatment with 0, 1, 5, 10, 20, and 50 µM C6-ceramide for 24 h, cell
viability was about 205, 198, 175, 163, 122, and 51% of the
pretreatment value (80 × 103 cells/well),
respectively. These results show that ceramide at 5-20
µM at 24 h causes growth inhibition but not cell
death. However, after treatment with 50 µM
C6-ceramide, the number of cells decreased to about 51% of
the pretreatment value, which indicates cell death, as observed in the
annexin-V staining and PARP cleavage. In addition, the number of
floating cells in the growth medium was also determined by the trypan
blue exclusion method and found to be around 1-2% of the total cell
number in the absence or presence of C6-ceramide at 20 µM for 24 h.
The inhibition of cell proliferation by 20 µM
C6-ceramide was further analyzed by examining the
incorporation of [3H]thymidine into trichloroacetic
acid-precipitable macromolecules at various time points (Fig.
3A). It was observed that
[3H]thymidine incorporation was decreased around 40 and
90% in response to 20 µM C6-ceramide at 6 and 24 h, respectively, showing the effects of ceramide on the
inhibition of cell growth.

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Fig. 3.
Analysis of the effects of
C6-ceramide on growth arrest and survival on soft agar in
A549 cells. A, the inhibition of growth in response to
20 µM C6-ceramide at various time points was
examined by detection of [3H]thymidine incorporation into
trichloroacetic acid-precipitable macromolecules as described under
"Experimental Procedures." Cell cycle profiles of the A549 cells in
the absence or presence of 20 µM C6-ceramide
at 24 h (B and C, respectively) were
analyzed by flow cytometry as described. D, the
anchorage-independent growth of A549 cells grown in the absence or
presence of various concentrations of C6-ceramide at
24 h was examined by a clonogenic survival assay on soft agar as
described. The results shown represent at least two independent
experiments. Error bars represent standard deviations. If not visible,
error bars are smaller than the diameters of the points.
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Moreover, cell cycle profiles of A549 cells grown in the absence or
presence of C6-ceramide (20 µM) at 24 h
were examined by flow cytometry (Fig. 3, B and C,
respectively) as described under "Experimental Procedures." Results
show that about 85% of the cell population was arrested in the
G0/G1 phase of the cell cycle in response to
C6-ceramide (Fig. 3C). It should be noted also
that no apoptotic (sub-G0/G1) cell population
was detectable by flow cytometry in the absence or presence of 20 µM C6-ceramide.
To confirm MTT and trypan blue exclusion assays, we performed a
clonogenic cell survival assay on soft agar as described under "Experimental Procedures." As shown in Fig. 3D, the
growth of cells treated in the presence of 5, 10, 20, and 50 µM C6-ceramide for 24 h was inhibited
about 17, 25, 36, and 56% compared with controls, similar to MTT and
trypan blue results. More importantly, the ability of these cells to
grow on soft agar following treatment with 20 µM
C6-ceramide for 24 h showed that these cells were
still viable, although the majority of the cell population was arrested in the G0/G1 phase of the cell cycle after
ceramide treatment. Taken together, these results show that the
inhibition of telomerase by C6-ceramide precedes apoptosis
but correlates with the effects of ceramide on growth arrest in the
G0/G1 phase of the cell cycle in A549 cells.
The Effects of Transient Expression of b-SMase on Telomerase
Activity--
To extend the results above and demonstrate whether
endogenous ceramide is involved in regulating telomerase activity, we subcloned the full-length cDNA of b-SMase, which is shown to
elevate endogenous ceramide levels via the hydrolysis of sphingomyelin, into an expression vector upstream of the GFP for transient
transfections and then determined the effects of its overexpression on
telomerase activity in A549 cells after 48 h of growth. As seen in
Fig. 4A, b-SMase-GFP (around
70 kDa) expression was successful, causing usually about 35-40%
increased ceramide generation (around 1.65 pmol/nmol Pi)
compared with GFP controls, as determined by the diacylglycerol kinase
assays (data not shown), compared with control transfectants with the
vector alone, as detected by Western blotting (lanes 2 and
1, respectively). Importantly, b-SMase expression resulted
in a significant telomerase inhibition (around 76%) in these cells
compared with A549-GFP and A549 parental cells used as controls
(Fig. 4B, lanes 3, 2, and
1, respectively). However, when A549 cells were treated with
purified b-SMase exogenously (100 milliunits/ml for 0-24 h), which is
known to cause the elevation of ceramide in the outer leaflet of the
plasma membrane but not in the cytoplasmic compartments of the cell
(33), telomerase levels remained similar to those of untreated cells
(Fig. 4C). Therefore, these results imply that the elevation
of an endogenous pool of ceramide is sufficient to inhibit
telomerase.

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Fig. 4.
Analysis of the effects of b-SMase on
telomerase activity in A549 cells. A, the full-length
b-SMase cDNA was cloned in an expression vector upstream of GFP.
A549 cells were transiently transfected with b-SMase-GFP or the
vector control using the Effectene transfection reagent as described
under "Experimental Procedures." The overexpression of b-SMase-GFP
protein at 48 h in A549-b-SMase transfectants was confirmed by
Western blotting using the mouse monoclonal anti-GFP antibody
(lane 2) compared with A549-GFP control transfectants
(lane 1) as described. B, telomerase activity in
A549, A549-GFP, and A549-b-SMase transfectants was determined by the
TRAP assay as described above (lanes 1-3, respectively).
C, the effects of treatment of A549 cells exogenously with
the purified b-SMase at 100 milliunits/ml for 0-24 h on telomerase
activity were examined by the TRAP assay (lanes 1-4). The
samples were separated on 10% acrylamide gels, and telomerase activity
levels were normalized to the internal standard (IS) levels
of the assay as described under "Experimental Procedures."
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Effects of Daunorubicin on Telomerase Activity and Endogenous
Ceramide Levels--
It has been shown previously that DNR at subtoxic
concentrations increases the accumulation of endogenous ceramide via
activating either neutral sphingomyelinase or the de novo
pathway in some human cancer cells (35, 36). Therefore, we examined the
effects of DNR on ceramide and telomerase activity. First, as seen in Fig. 5A, after treatment of
cells with DNR at 1 µM for 6 h, about 95% of the
cells were viable in trypan blue exclusion assays. Interestingly,
analysis of cell proliferation by a [3H]thymidine
incorporation assay showed that the growth of A549 cells was inhibited
about 90% after treatment with 1 µM DNR at 6 h
compared with vehicle matched controls (data not shown). As shown in
Fig. 5B, the absolute cellular ceramide levels were 1.10 and
1.70 pmol/nmol Pi in the absence or presence of 1 µM DNR at 6 h, respectively, showing around 40%
increased levels of endogenous ceramide. More importantly, telomerase
activity was found to be significantly inhibited (about 82%) in the
cells (Fig. 5C) following DNR treatment (1 µM
at 6 h). The IC50 value of DNR at 24 h was found
to be 8.4 µM in A549 cells as determined by the MTT assay as described above (data not shown). In addition, the possible effects
of DNR on apoptosis were analyzed by flow cytometry to detect the
translocation of PS using an annexin V-fluorescein isothiocyanate
antibody binding assay as described above (Fig. 5D). These
results confirmed that following DNR treatment (1 µM at
6 h) PS translocation was detected only in about 9% of the cell
population, whereas PS translocation was apparent in 2% of the
untreated controls (Fig. 5D, lower and
upper panels, respectively). These results were also
confirmed by the absence of PARP cleavage in Western blots after DNR
treatment of the cells (data not shown). Therefore, these data indicate
that DNR inhibits telomerase at subtoxic concentration, and this
inhibition, which precedes apoptotic cell death, is accompanied by the
elevation of endogenous ceramide and growth arrest in A549 cells.

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Fig. 5.
Analysis of the effects of DNR on cell
survival, endogenous ceramide levels, and telomerase activity in A549
cells. A, the survival of A549 cells grown in the
absence or presence of various concentrations of DNR was determined by
the trypan blue exclusion method as described. B, the
effects of 1 µM DNR on absolute endogenous ceramide
levels for 1-6 h in A549 cells were determined by the diacylglycerol
kinase method as described under "Experimental Procedures."
C, the effects of 1 µM DNR on telomerase
activity at 0, 1, 3, and 6 h time points were determined by the
TRAP assay (lanes 1, 2, 3, and
4, respectively). The samples were separated on 10%
acrylamide gels, and telomerase activity levels were normalized to the
internal standard (IS) levels of the assay as described
under "Experimental Procedures." D, the translocation of
PS from the inner to the outer plasma membrane in cells that remained
attached to the flasks after treatment in the absence or presence of
DNR (1 µM for 6 h) was determined by flow cytometry
using the human vascular phospholipid-binding protein annexin V
conjugated with fluorescein (upper and lower
panels, respectively). The results shown represent two independent
experiments. Standard deviations are represented by error bars.
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Effects of Overexpression of GCS on Telomerase Inhibition in
Response to C6-Ceramide and DNR--
The overexpression of
GCS in MCF-7 cells has been shown to attenuate endogenous ceramide by
converting ceramide to glucosylceramide and to induce a drug-resistant
phenotype (34). Therefore, to further determine whether ceramide plays
a role in the regulation of telomerase, we transfected A549 cells with
the pcDNA3 expression vector containing the full-length GCS
cDNA and then analyzed the effects of GCS overexpression on
endogenous ceramide generation and telomerase activity in response to
DNR and also C6-ceramide treatment. Our results show that
the stable overexpression of GCS resulted in about 3.5-fold increased
protein levels compared with control transfectants containing the
vector only, as determined by Western blot analysis using a rabbit
polyclonal antibody that recognizes human GCS protein (Fig.
6A, lanes 2 and
1, respectively). This GCS overexpression resulted in
increased GCS activity in the A549/GCS-16 transfectants (about
2.5-fold) compared with controls, as determined by an in
vitro enzyme assay (Fig. 6B, lanes 3 and 2, respectively), detecting the conversion of
NBD-C6-ceramide to NBD-C6-glucosylceramide in
the presence of UDP-glucose, using extracts from these
transfectants.

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Fig. 6.
The effects of overexpression of GCS on
telomerase activity in response to C6-ceramide and
DNR. The full-length GCS cDNA was cloned into the pcDNA3
expression vector, and A549 cells were stably transfected using the
Effectine transfection reagent as described under "Experimental
Procedures." A, the overexpression of GCS protein was
confirmed by Western blot analysis using the rabbit polyclonal anti-GCS
antibody in A549/GCS-16 and compared with that of A549/pcDNA3
control transfectants (lanes 2 and 1,
respectively). B, the activity of GCS was measured by an
in vitro enzyme assay measuring the conversion of
NBD-C6-ceramide to NBD-C6-glucosylceramide
(NBD-Gluc-C6) in the presence of UDP-glucose using the cell
extracts from A549/pcDNA3 and A549/GCS-16 transfectants
(lanes 2 and 3, respectively). Lane 1 contains NBD-C6-ceramide without any cell extract (used as
a control). SM, sphingomyelin. Telomerase activity
in A549/pcDNA3 and A549/GCS-16 transfectants in the absence or
presence of 20 µM C6-ceramide for 24 h
(C) and 1 µM DNR for 6 h (D)
(lanes 1 and 3, and 2 and
4, respectively) were measured by the TRAP assay as
described above. IS, internal standard. E,
endogenous ceramide levels in the absence or presence of DNR in
A549/pcDNA3 and A549/GCS-16 transfectants were measured by the
diacylglycerol kinase assay as described above. The total absolute
ceramide levels were normalized to the levels of phospholipid
phosphate. The results shown represent two independent experiments.
Error bars represent standard deviations.
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Next, the effects of C6-ceramide (20 µM for
24 h) and DNR (1 µM for 6 h) on telomerase
activity in A549/pcDNA3 and A549/GCS-16 cells were analyzed by the
TRAP assay. The overexpression of GCS prevented the inhibition of
telomerase activity in response to C6-ceramide and DNR in
A549/GCS-16 cells (Fig. 6, C and D, lanes 3 and 4, respectively). Specifically, in
A549/pcDNA3 cells telomerase activity was inhibited in response to
C6-ceramide by about 85%, whereas telomerase was inhibited
in A549/GCS-16 transfectants in the presence of C6-ceramide by about
20% (Fig. 6C, lanes 2 and 1, and
4 and 3, respectively). Similarly, overexpression
of GCS attenuated the inhibition of telomerase in response to DNR. Thus, telomerase activity was inhibited by about 82 and 38% in A549/pcDNA3 and A549/GCS-16 cells, respectively, compared
with untreated controls following DNR treatment (Fig. 6D,
lanes 2 and 1, and 4 and 3,
respectively). These results were correlated with the endogenous
ceramide levels in response to DNR in these cells, showing that
absolute cellular ceramide levels increased from 1.10 to 1.80 in
A549/pcDNA3, whereas cellular ceramide levels increased
from 0.85 to 1.25 pmol/nmol Pi in A549/GCS-16 cells (Fig.
6E). Thus, these results show that following DNR treatment (1 µM for 6 h), GCS overexpression resulted in a
significant reduction in the ceramide response (around 40%), which was
accompanied by a commensurate protection of telomerase inhibition
(around 44%) in A549/GCS-16 cells when compared with A549/pcDNA3
cells. Taken together, these results show that the attenuation
of ceramide levels by overexpression of GCS prevents the inhibition of
telomerase in response to C6-ceramide and DNR, showing for
the first time that ceramide is one of the key elements involved in the
regulation of telomerase activity.
 |
DISCUSSION |
In the present study, the possible role of ceramide in the
regulation of telomerase activity in A549 human lung adenocarcinoma cells was examined. Our results provide novel data showing for the
first time that both exogenous and endogenous ceramides are involved in
the inhibition of telomerase activity in A549 cells. Additional data
support the notion that endogenous ceramide elevation in
response to DNR also inhibits telomerase, and this inhibition can be
partially prevented by the overexpression of GCS.
Ceramide has been shown to exert growth-suppressive effects in various
human cancer cell lines. These effects have been associated with the
ability of ceramide to induce differentiation, cell cycle arrest due to
Rb dephosphorylation, apoptosis, and senescence, although the specific
responses vary among different cell types (24, 25). Thus, ceramide has
been suggested as a tumor suppressor lipid (25). On the other hand, it
has been shown that telomerase action on the stability of telomeres is
required for cells to escape their senescent phenotype, leading to
uncontrolled growth and thus to the development of continuing
proliferation of cancers (13). Taken together, these data suggest that
growth-suppressive effects of ceramide might be somehow related to the
action of telomerase in human cancer cells.
The data presented here show that the concentrations of exogenous
C6-ceramide and DNR that inhibited telomerase activity were not toxic to the cells and that the inhibition of telomerase precedes apoptotic cell death in response to ceramide or DNR, as determined by
the absence of PARP cleavage and PS translocation to the outer membrane. Therefore, the activation of a regulatory pathway by ceramide
or DNR leading to the inhibition of telomerase is either independent of
apoptosis or is selectively more sensitive to lower concentrations of
antiproliferative agents. These results are in agreement with a recent
study showing that telomerase activity is not related to apoptotic
signals induced by interferon-
but rather is closely related to the
cell cycle status in myeloma cells (37). Our results are in agreement
with this study, because telomerase inhibition by ceramide correlates
with the growth arrest in G0/G1 but is
independent of apoptotic cell death in A549 cells. The growth arrest in
response to C6-ceramide and increased endogenous ceramide
levels due to serum deprivation has been shown in Molt-4 cells
previously (38). Moreover, it has been well documented that Rb gene
product is a downstream target for a ceramide-dependent pathway of growth arrest (39).
There are data showing that the overexpression of Bcl-2 is associated
with increased telomerase activity in some human cancer cells (40, 41),
but its overexpression had no effect on telomerase activity in human
Jurkat T cells (42), suggesting that the effect of Bcl-2 on telomerase
might be cell line- or tissue-specific. Our results, however, showed
that overexpression of Bcl-2 did not have any significant effect on
telomerase activity in A549 cells (data not shown). Nevertheless, the
relationship between molecular signaling pathways involved in
ceramide-induced apoptotic cell death, cell cycle status, and
telomerase inhibition in the A549 cell line needs to be further evaluated.
One practical conclusion from these results concerns the apoptotic
effects of ceramide in various cells. There are clearly some cell
types, such as MCF-7 breast cancer and Molt-4 and HL-60 leukemia cells,
that are sensitive to the apoptotic effects of C6-ceramide
in the 3-10 µM range. However, there are other cell lines, including A549 lung carcinoma and lymphocytic Raji cells (43),
that are more resistant to its apoptotic effects, and in these
resistant cells, there are other effects of ceramide with no apoptosis,
such as cell cycle arrest (43) and the inhibition of telomerase, that
are detected in the 10-20 µM range at 24 h that may
be of physiologic and therapeutic value.
More importantly, our results provide strong evidence for the role of
endogenous ceramide in regulating telomerase activity. The
overexpression of b-SMase resulted in a significant decrease in
telomerase activity. However, treatment of cells extracellularly with
purified b-SMase did not affect telomerase activity. These results
suggest that ceramide accumulation in an intracellular compartment is
important for its role in regulating telomerase activity. This
compartment is most likely distinct from the plasma membrane, because
ceramide generated at the outer leaflet of the plasma membrane in
response to exogenous b-SMase is expected to flip-flop across the
plasma membrane, but it is not expected to gain access to intracellular
compartments. Similar results were published previously demonstrating
that exogenous b-SMase-induced ceramide generation at the plasma
membrane is not sufficient to mediate apoptosis, whereas ceramide
generated by overexpression of b-SMase led to apoptotic cell death in
Molt-4 leukemia cells, suggesting that the intracellular generation of
ceramide functions in a manner distinct from ceramide generated at the
outer leaflet of the plasma membrane (33, 44).
In addition, we show here that the elevation of endogenous ceramide in
response to non-toxic concentrations of DNR is also involved in
telomerase modulation. The inhibition of telomerase activity following
various chemotherapeutic agents in many different human cancer cell
lines has been reported previously (45, 46). Therefore, our results are
very interesting in demonstrating that endogenous ceramide generation
mediates a DNR-activated pathway involved in telomerase inhibition.
Indeed, our results with GCS, demonstrating that its overexpression
partially prevented DNR-induced telomerase inhibition, which correlated
with decreased ceramide levels, show that endogenous ceramide
is necessary for telomerase inhibition in response to DNR. Similarly,
GCS overexpression also prevented C6-ceramide-induced
telomerase inhibition, supporting the importance of ceramide in the
control of telomerase activity. These results are in agreement with
studies that showed that overexpressed GCS can convert
C6-ceramide to C6-glucosylceramide (34, 47). In
addition, it was shown previously that the overexpression of GCS
results in decreased ceramide levels in response to various chemotherapeutic agents, causing the development of a
multidrug-resistant phenotype in MCF-7 human breast cancer cells (34),
and down-regulation of GCS by antisense modification reverses
adriamycin resistance in MCF-7/Adr cells (48). However, it was also
suggested recently that GCS overexpression has a very limited role in
attenuating ceramide responses (47). Our results are much more in
agreement with those of Liu et al. (34, 48), because
GCS attenuates the responses not only to C6-ceramide but
also to DNR, one of the most widely used classes of chemotherapy agents.
These results also have additional implications. First, the
identification of ceramide as one of the key factors involved in
regulating telomerase activity provides a mechanistic tool to modulate
this enzyme in human lung cancer cells in vivo, which in
turn will help develop therapeutic strategies for the treatment of lung
cancers. In fact, many of the agents that are known to elevate cellular
ceramide levels, such as interferon-
, tumor necrosis factor, and
various differentiating agents (such as vitamin D3 and its analogs),
have also been shown to inhibit telomerase activity in various human
cancer cell lines (49, 50). However, the role of ceramide mediating the
effects of these agents on telomerase has not yet been investigated.
Second, because it has been shown that detection of telomerase
inhibition might be an important marker for measuring the tumor cell
killing of antineoplastic agents (45, 46), our results suggest that the
elevation of the endogenous ceramide level leading to telomerase
inhibition in response to various anticancer drugs might be an
important marker to predict the success or efficacy of the treatment of lung cancers. Third, our results are in parallel with the
antiproliferative role of ceramide observed in various human cancer
cells previously (24). Indeed, there are additional data demonstrating
that ceramides and ceramide metabolites play a preventive role when
used as dietary supplements inhibiting colon cancers in mice (51, 52).
Finally, the role of ceramide in cellular senescence has been well
established. Previous studies have demonstrated that the activity of
sphingomyelinase and ceramide levels are significantly elevated in
senescent human diploid fibroblasts, and the function of ceramide in
mediating cellular senescence is linked to its inhibitory effects on
DNA synthesis, activation of Rb, and the inhibition of AP1 function, all known parameters of cellular senescence (53, 54). The effect on AP1
was traced mechanistically to the ability of ceramide to inhibit
phospholipase D (54). It is also known that the down-regulation of
telomerase resulting in progressive shortening of telomeres is a
critical factor in mediating growth arrest and senescence (55). In
fact, to investigate whether telomerase inhibition by ceramide can be
detected also at the genomic DNA level in vivo, we performed
telomere restriction fragment length measurements by Southern blotting
as described (56), and our preliminary results showed that ceramide
treatment (20 µM at 24 h) resulted in a significant
shortening of the telomere restriction fragment length (data not
shown), indicating the in vivo effect of ceramide on the
inhibition of telomerase and the shortening of telomere length. The
molecular mechanisms of this accelerated reduction in telomere
restriction fragment length in response to C6-ceramide and
its implications in growth arrest in A549 cells need to be determined
in more detailed studies.
Therefore, our results, in light of these studies, suggest that the
inhibition of telomerase by ceramide might play an important role in
triggering signaling pathways that lead to growth arrest and cellular
senescence. The relationship between ceramide, telomerase, and
senescence, however, needs further investigation.
In conclusion, this study provides novel data indicating that ceramide
is a key factor involved in the control of telomerase activity and in
chemotherapy action on telomerase in A549 cells. However, the
mechanisms by which ceramide inhibits telomerase in A549 cells are
still unknown and need to be determined. We believe that further
investigation of ceramide-induced signaling pathways involved in
inhibiting telomerase activity will help develop mechanism-based
therapeutic strategies for the treatment of lung and other cancers in humans.