Role of Ceramide in Mediating the Inhibition of Telomerase Activity in A549 Human Lung Adenocarcinoma Cells*

Besim OgretmenDagger , Deborah SchadyDagger , Julnar Usta§, Rachael WoodDagger , Jacqueline M. Kraveka, Chiara LubertoDagger , Helene Birbes||, Yusuf A. HannunDagger , and Lina M. Obeid||**

From the || Ralph H. Johnson Veterans Affairs Medical Center and Departments of Medicine, Dagger  Biochemistry and Molecular Biology, and  Pediatrics, Division of Hematology/Oncology, Medical University of South Carolina, Charleston, South Carolina 29425 and the § Department of Biochemistry, American University of Beirut, Beirut, Lebanon

Received for publication, January 12, 2001, and in revised form, March 26, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 C6-ceramide, but not by the biologically inactive analog dihydro-C6-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 µM C6-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 µM for 24 h) had no apoptotic effects. Trypan blue exclusion, [3H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 µM concentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G0/G1 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 µM 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 C6-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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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-alpha , interleukin 1-beta , nerve growth factor, and gamma -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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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 beta -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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INTRODUCTION
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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.

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.

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.

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."

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.

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.

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha , 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.

    ACKNOWLEDGEMENTS

We thank Dr. R. E. Pagano (Mayo Clinic, Rochester, MN) for providing the rabbit polyclonal antibody that recognizes GCS. We also thank Dr. Y. Hirabayashi (The Institute of Physical and Chemical Research, Saitama, Japan) for providing the full-length GCS cDNA. We thank Dr. Korey Johnson for helpful discussions and Kathy Wiita for administrative assistance. The flow cytometry analysis was performed in the Flow Cytometry Core Facility by C. Enockson in the Department of Microbiology and Immunology at the Medical University of South Carolina.

    FOOTNOTES

* This work was supported in part by research grants from the Department of Energy through the Environmental Biosciences Program at the Medical University of South Carolina, the American Cancer Society (IRG-97-151-01), and the Department of Defense, Coastal Carolina Program Project (Project #2) at Hollings Cancer Center (to B. O.) and by 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.

** To whom correspondence should be addressed: Medical University of South Carolina, Dept. of Medicine, P. O. Box 250779, Charleston, SC 29425. Tel.: 843-876-5169; Fax: 843-876-5172; E-mail: obeidl@musc.edu.

Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M100314200

    ABBREVIATIONS

The abbreviations used are: Rb, retinoblastoma; DNR, daunorubicin; PCR, polymerase chain reaction; TRAP, telomeric repeat amplification protocol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PS, phosphatidylserine; PARP, poly(ADP-ribose) polymerase; GCS, glucosylceramide synthase; b-SMase, bacterial sphingomyelinase; GFP, green fluorescent protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)).

    REFERENCES
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RESULTS
DISCUSSION
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