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Blood, 1 November 2005, Vol. 106, No. 9, pp. 3200-3205. Prepublished online as a Blood First Edition Paper on July 14, 2005; DOI 10.1182/blood-2005-04-1386. This Article Abstract Full Text (PDF) All Versions of this Article: 2005-04-1386v1 106/9/3200    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Beyne-Rauzy, O. Articles by Mansat-De Mas, V. Search for Related Content PubMed PubMed Citation Articles by Beyne-Rauzy, O. Articles by Mansat-De Mas, V. Related Collections Neoplasia Signal Transduction Gene Expression Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  NEOPLASIA Tumor necrosis factor- inhibits hTERT gene expression in human myeloid normal and leukemic cells Odile Beyne-Rauzy, Naïs Prade-Houdellier, Cécile Demur, Christian Recher, Jacques Ayel, Guy Laurent, and Véronique Mansat-De Mas From the Institut National de la Santé et de la Recherche Médicale (INSERM) U563, Centre Hospitalier et Universitaire (CHU) Purpan, Toulouse, France; the Service de Médecine Interne, CHU Purpan, Toulouse, France; the Laboratoire d'Hématologie Biologique, CHU Purpan, Toulouse, France; the Service d'Hématologie, CHU Purpan, Toulouse, France; and the Service d'Orthopédie, CHU Purpan, Toulouse, France.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Telomerase catalytic subunit (hTERT) has been shown to play a critical role not only in telomere homeostasis but also in cellular survival, DNA repair, and genetic stability. In a previous study, we described that tumor necrosis factor-x (TNFx) induced in the leukemic KG1 cells a senescence state characterized by decreased hTERT activity followed by prolonged growth arrest, increasedx -galactosidase activity, telomere shortening, and major chromosomal instability. Interestingly, granulocyte-macrophage colony-stimulating factor (GM-CSF) abrogated all these events. In the present study, we show for the first time that TNFx acts by inhibiting the hTERT gene in both normal CD34x+ cells and fresh leukemic cells. Using KG1 cells as a representative cellular model, we show that TNFx induced sphingomyelin hydrolysis, ceramide production, and c-Jun N-terminal kinase (JNK) activation, all of which are critical components of TNFx signaling, resulting in hTERT gene inhibition. Moreover, we provide evidence that the protective effect of GM-CSF is related to its capacity to interfere with both ceramide generation and ceramide signaling. Negative regulation of the hTERT gene may represent one mechanism by which TNFx interferes with normal hemopoiesis.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   Telomerase is a large ribonucleoprotein complex containing 2 major subunits contributing to enzymatic activity: a RNA component (hTR) that serves as a template for the polymerase activity of the enzyme and a catalytic subunit with reverse transcriptase activity (hTERT). Previous studies have documented that hTERT represents the rate-limiting step in telomerase function.1 For this reason, and because this enzyme has been found to play an essential role in the regulation of telomere elongation and cellular protection, hTERT regulation has been the subject of intense investigation in recent years. From these studies, it appears that hTERT is tightly regulated both at transcriptional and posttranscriptional levels. For example, the cloning and characterization of the hTERT 5' gene regulatory elements have identified more than 20 transcription factor binding sites acting as activators or repressors (for a review, see Poole et al2). Despite increasing knowledge about hTERT transcription regulation, much less is known about which type of internal or external stimuli could interfere with hTERT gene regulation. Recent studies have described that, in epithelial cells, oncogenes, such as Her2/Neu,3 growth factors, such as epidermal growth factor (EGF),4 or steroids5 could activate hTERT at the transcriptional level. In contrast, transforming growth factor- (TGF) acts as a repressor of hTERT gene transcription in a variety of cellular models.6-8 In a previous study, we described that, in leukemic myeloid KG1 cells, tumor necrosis factor- (TNF) induced premature senescence, characterized by cellular growth arrest, increased -galactosidase activity, reduced hTERT activity, telomeric disturbances (shortening, losses, fusions), and intense chromosomal instability.9 Moreover, increased -galactosidase activity was also found in normal CD34+ as well as in fresh acute myeloid leukemia (AML) cells treated with TNF.9 To the best of our knowledge, the role of TNF in hTERT regulation in hematopoietic cells had not been previously documented. This question may be of great significance. Indeed, hTERT plays a critical role not only in telomere homeostasis but also in cellular survival, DNA repair, and genetic stability, whereas TNF accumulates in a number of pathologic situations, including not only chronic inflammation and neoplastic disorders but also leukemia, myelodysplasia, and aplastic anemia.10-13 The present study was aimed at evaluating whether TNF could interfere with hTERT expression in normal progenitor cells and in leukemic cells derived from patients with AML. In this study, we also investigated the signaling pathways by which TNF influences hTERT gene regulation or which could interfere with TNF.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Normal progenitor purification and culture Normal bone marrow CD34+ hematopoietic progenitor cells (HPCs) were obtained from discarded fragments of hematologically healthy patients undergoing hip surgery after informed consent was provided per the Declaration of Helsinki. Mononuclear cells from bone marrow were obtained by Ficoll-Hypaque density gradient centrifugation after which isolation of HPCs was performed by positive selection of CD34-expressing cells. Briefly, CD34+/HPCs were magnetically labeled using magnetic-activated cell sorting (MACS) CD34 microbeads then isolated by positive selection through medium-size (MS) separation column (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the CD34+ cells was evaluated by flow cytometry using anti-CD34 and anti-CD45 monoclonal antibodies and reached 85% to 98%. Human progenitors (5 x 105 cells/mL) were cultured in Iscoves modified Dulbecco medium (IMDM) containing 10% of a commercial mixture of bovine serum albumin (BSA), insulin, and transferrin (BIT9500; StemCell Technologies, Grenoble, France) supplemented with 100 ng/mL stem-cell factor, 1 U/mL interleukin 3, 100 ng/mL fms-like tyrosine kinase type 3 ligand (FLT3-L), and 5 pg/mL thrombopoietin (R&D Systems, Oxon, United Kingdom). Human recombinant TNF (PeproTech-TEBU, Rocky Hill, NJ) was added to fresh medium containing cytokines every 2 days. Patient sample culture Approval for these studies was obtained from the Institut National de la Santé et de la Recherche Médicale (INSERM) U563 institutional review board. Fresh AML cells were obtained from patients diagnosed at the Hematology Department of Toulouse University Medical Center (France), after informed consent was provided, per the Declaration of Helsinki. AML cells were isolated from bone marrow by Ficoll-Hypaque density gradient centrifugation and were cryopreserved in IMDM medium with dimethyl sulfoxide (10% final concentration) and fetal calf serum (FCS; 50% final concentration) or immediately processed for assays. Leukemic cells (1 x 106 cells/mL) were maintained in IMDM containing 10% 5637-conditioned medium (CM-5637) (5637 is a bladder carcinoma cell line). Human recombinant TNF was added to fresh medium containing CM-5637 every 2 days. Cell line The human leukemic cell line KG1 was purchased from American Type Culture Collection (ATCC, Rockville, MD), cultured in IMDM medium supplemented with 20% FCS (Invitrogen, Gibco, Cergy Pontoise, France), and incubated in a humidified incubator containing 5% CO2 at 37°C. The cells were split with fresh medium every 2 or 3 days and maintained at 4 x 105 cells/mL. Cell viability was assessed by trypan blue exclusion. Cells were treated with TNF (20 ng/mL), human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D Systems), or cell-permeant exogenous ceramide, C6-ceramide (10 µM; Sigma Aldrich, Saint Quentin Fallavier, France). Immunophenotypic analysis Anti-CD34 and anti-CD45 antibodies (Beckman Coulter, Villepinte, France) were used to examine surface antigen expression according to the manufacturer's recommendations. Analysis was performed with an EPICS XL-MCL (Beckman Coulter). Quantitative RT-PCR Total RNA were isolated by using Trizol Reagent (Invitrogen, Cergy Pontoise, France). Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) was performed using the LightCycler TeloTAGGGhTERT quantification kit (Roche Diagnostics, Mannheim, Germany) as indicated by the manufacturer. Briefly, hTERT mRNA was reverse transcribed and amplified with specific primers in a one-step RT-PCR reaction. The amplicon was detected by fluorescence, using a specific pair of probes that hybridize to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. The emitted fluorescence was measured by the LightCycler instrument. In a separate one-step RT-PCR, mRNA encoding for porphobilinogen deaminase (PBGD) was used as a housekeeping gene. Results were expressed as the ratio between hTERT and PBGD transcripts normalized to untreated cells as previously described.9 Telomerase activity Quantitative determination of telomerase activity was performed using the Telomeric Repeat Amplification Protocol (TRAP) with teloTAGGG telomerase PCR enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics) following the manufacturer's instructions as previously described.9 The relative telomerase activity (RTA) was determined as absorbance of the sample compared with absorbance of the control template. Western blot Cells were harvested and lysed with Laemmli buffer, sonicated, and boiled for 5 minutes at 95°C. Samples were separated on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred onto nitrocellulose membrane, and immunostained. The following primary antibodies were used: anti-ERK (extracellular signal-regulated kinase), anti-JNK (c-jun N-terminal kinase) (Santa Cruz Biotechnology, Le Perray en Yvelines, France), anti–phospho-MAPK (mitogen-activated protein kinase), anti–phospho-JNK, anti–phospho-p38, anti-p38, anti–phospho-MAPKAPK-2 (MAPK-activated protein kinase 2), anti–MAPKAPK-2 (Cell Signaling, Ozyme, Saint Quentin en Yvelines, France), and horseradish peroxidase–labeled antimouse and antirabbit antibodies. Proteins were visualized using the electrochemiluminescence (ECL) detection system (Pierce, Rockford, IL). Metabolic cell labeling and ceramide quantitation Total cellular ceramide quantitation was performed by labeling cells to isotopic equilibrium with 1 µCi/mL [0.037 MBq] [9, 10-3H] palmitic acid (Amersham Biosciences, Orsay, France) for 48 hours in complete medium as previously described.14 Cells were then washed and resuspended in complete medium for time-course experiments. Lipids were extracted and resolved by thin-layer chromatography. Ceramide was scraped and quantified by liquid scintillation spectrometry. Statistics Results are expressed as mean values ± standard deviations. Statistical analysis of the data was performed by the Student t test. Differences were considered as significant for P values less than .05.    Results Top Abstract Introduction Materials and methods Results Discussion References   Effect of TNF on hTERT gene expression in CD34+ normal cells In a first set of experiments, we tested whether TNF altered hTERT expression in normal bone marrow progenitors. Normal marrow CD34+ cells were cultured in medium containing various cytokines (stem-cell factor [SCF], interleukin-3 [IL3], FLT3-L, and thrombopoietin [TPO]) in the presence or not of TNF for 14 days. According to a previous study, we used the dose of 20 ng/mL.15 In control cells, this mixture of cytokines induced 5.4-fold and 32-fold expansion at 7 and 14 days, respectively. As expected from previous studies using a similar mixture of cytokines,15 we found that TNF reduced cellular expansion compared with controls. However, this difference became significant only after 14 days of culture (Figure 1A). We did not observe apoptosis in TNF-treated cells (data not shown). Moreover, TNF was found to facilitate differentiation as reflected by reduced CD34 expression compared with control cells after 7 days of culture (Figure 1B), whereas CD34 became undetectable after 14 days of culture in both TNF-treated cells and control cells (data not shown). In parallel, as shown in Figure 1C, treatment with TNF resulted in a dramatic decrease in hTERT gene expression compared with untreated cells as revealed by real-time quantitative PCR (93.8% ± 3.4%) followed by a time-dependent reduction in hTERT activity which became almost undetectable after 10 days of culture (Figure 1D). These results show that TNF inhibits hTERT gene expression in normal myeloid lineage. View larger version (9K): [in this window] [in a new window]   Figure 1.. Influence of TNF on normal bone marrow progenitors. Fresh CD34+ were cultured in medium containing various cytokines as described in "Materials and methods" in the presence or absence of TNF (20 ng/mL) for 7 to 14 days. (A) Viable cells were counted by trypan blue dye exclusion. Results represent the mean ± SD of 5 independent experiments. ***P < .001. (B) Differentiation was analyzed by flow cytometry using anti-CD34 monoclonal antibody at 7 days of culture. Results are representative of 3 independent experiments. (C) Transcripts of hTERT were quantified using the LightCycler TeloTAGGGhTERT quantification kit as described in "Materials and methods." hTERT/PBGD represents the ratio between hTERT and PBGD transcripts normalized to untreated control cells at 7 days of culture. They are expressed as the mean hTERT/PBGD values from 3 independent experiments ± SDs; ****P < .001. (D) Telomerase activity was measured by using the TeloTAGGG telomerase PCR ELISA kit as described in "Materials and methods." Results are expressed as mean RTA values ± SDs of 3 independent experiments. *P = .014; **P = .002 and .004.   Effect of TNF on hTERT gene expression in leukemic cells We next investigated whether TNF could also regulate hTERT gene expression in myeloid leukemic cells. These experiments were performed with both KG1 cells9 and in fresh AML cells cultured in CM-5637 for 7 days. They revealed that, as for normal myeloid cells, 7 days of exposure to TNF resulted in a dramatic reduction in hTERT gene expression in KG1 cells.9 However, in these cells, hTERT inhibition was detectable as soon as 2 hours, with a maximum at 6 hours and remained stable over 7 days.9 Dose-effect analysis revealed a maximum reduction of hTERT gene at 20 ng/mL (data not shown). In initial experiments, we obtained similar results with fresh AML cells but experimental conditions based on 7-day exposure were often questionable because of the loss of viability in control cells. For this reason, further experiments were conducted with freshly thawed AML cells or with cells immediately harvested from patients with AML and incubated in short-term liquid culture (2-6 hours) in the presence of CM-5637 with or without TNF. As depicted in Table 1, treatment with TNF resulted in rapid hTERT gene reduction in virtually all cases, hTERT transcripts becoming undetectable in 2 of 10 cases. These results show that, as in myeloid normal cells, TNF negatively influences hTERT expression in myeloid leukemic cells. View this table: [in this window] [in a new window]   Table 1.. Influence of TNF on hTERT gene expression in AML cells   Role of MAPK in TNF-induced inhibition of hTERT in KG1 cells To investigate the signaling pathway regulating hTERT, we used KG1 cell line as a representative model of myeloid cells. Previous studies have described that TNF activates a complex network of signaling pathways among which MAPK may represent an important functional component through regulation of a wide variety of genes. Therefore, we hypothesized that ERK, JNK, or p38MAPK might play an important role in mediating the inhibitory effect of TNF on hTERT gene regulation. As a matter of fact, we found that, in KG1 cells, treatment with TNF induced a rapid increase (as soon as 15 minutes) in JNK and p38MAPK, but not ERK phosphorylation as revealed by immunoblotting with antibodies directed against phosphorylated forms of p44/p42ERK, p56/p44JNK, and p38MAPK (Figure 2A,B,C, respectively). Moreover, we found that JNK activation was abrogated by pretreatment (one hour) with the JNK inhibitor SP600125 (20 µM), whereas the p38MAPK inhibitor SB203580 (1 µM) abrogated the activation of MAPKAPK-2, a substrate of p38MAPK (Figure 2B,D). Then, we investigated whether the JNK or p38MAPK pathway inhibition may interfere with TNF-induced hTERT gene inhibition. As shown in Table 2, we found that pretreatment with the JNK inhibitor SP600125 (20 µM) abrogated the inhibitory effect of TNF on hTERT, whereas pretreatment with the p38MAPK inhibitor SB203580 (1 µM) had no effect. These results suggest that JNK is specifically involved in TNF-mediated hTERT gene regulation. View this table: [in this window] [in a new window]   Table 2.. Influence of MAPK inhibitors on TNF-induced inhibition of hTERT in KG1 cells   Role of ceramide in TNF-induced inhibition of hTERT in KG1 cells Previous studies have documented that, in myeloid leukemic cells, TNF stimulates the JNK pathway by activating the sphingomyelin cycle.16 This pathway consists in the stimulation of a neutral Mg2+-dependent sphingomyelinase responsible for sphingomyelin hydrolysis and subsequent generation of ceramide that activates the JNK cascade through a redox-dependent mechanism.17 Moreover, independent studies have shown that, at least in nonhematopoietic cellular models, ceramide interferes with hTERT gene expression.18,19 For all these reasons, we speculated that ceramide could mediate the effect of TNF on hTERT expression in KG1 cells. As shown in Figure 3A, labeling studies revealed that, in KG1 cells, intracellular ceramide concentration increased on TNF stimulation with a maximum of 145% at 3 to 5 minutes, and then returned to basal line. These results suggest that TNF may indeed activate the sphingomyelin cycle in KG1 cells. To evaluate the functional consequence of endogenous ceramide release, we treated KG1 cells with cell-permeant exogenous ceramide (C6-ceramide) (10 µM) for 2 hours and hTERT transcripts were measured using real-time quantitative PCR (RQ-PCR). As shown in Figure 3B, treatment with C6-ceramide resulted in a 40% decrease in hTERT PCR products. Ceramide-mediated hTERT inhibition was therefore in a same range as that observed with TNF. The fact that C6-ceramide mimicked the effect of TNF strongly suggests that TNF-induced ceramide production is responsible for hTERT regulation. Moreover, the effect of C6-ceramide was inhibited by the JNK inhibitor SP600125, suggesting that, like for TNF, JNK mediated the effect of C6-ceramide on hTERT (Figure 3B). These results suggest that ceramide plays an important role in mediating the effect of TNF on hTERT through a JNK-dependent mechanism. View larger version (23K): [in this window] [in a new window]   Figure 2.. Role of MAPK in TNF-induced inhibition of hTERT in KG1 cells. KG1 cells were treated or not with TNF for 15 to 120 minutes after overnight serum deprivation. MAPK expression was evaluated by Western blot analysis as described in "Materials and methods." (A) Phospho-p42/44 ERK and p42/44 ERK expression. FCS was used as positive control. (B) Phospho-p46/54 JNK and p46/54 JNK expression. (C) Phospho-p38 and p38 expression. (D) Phospho-MAPKAPK-2 and MAPKAPK-2 expression.   View larger version (12K): [in this window] [in a new window]   Figure 3.. Role of ceramide in TNF-induced activation of JNK. (A) KG1 cells were prelabeled with [3H]-palmitic acid for 48 hours, washed, and treated with TNF (20 ng/mL) at the indicated time. Intracellular levels of ceramide were analyzed as described in "Materials and methods." Results are expressed as the mean values from 3 independent experiments ± SDs. *P < .05. (B) KG1 cells were preincubated or not for 1 hour with JNK inhibitor SP600125 (20 µM) then treated or not with C6-ceramide (10 µM) for 2 hours. Transcripts of hTERT were quantified using the LightCycler TeloTAGGGhTERT quantification kit as previously described. Results are expressed as mean ± SD from 4 independent experiments. ****P < .001; *P = .03.   Influence of GM-CSF on TNF effects In further experiments, we considered the possibility that GM-CSF could interfere with the inhibitory effect of TNF on the hTERT gene. This hypothesis was supported by the fact that GM-CSF has been found to negatively regulate stress-induced ceramide production.20 For this reason, KG1 cells were cotreated with TNF (20 ng/mL) and GM-CSF (13 ng/mL), and ceramide production was investigated. These experiments revealed that GM-CSF inhibited TNF-induced ceramide generation as illustrated in Figure 4A. Moreover, GM-CSF inhibited the effect of both TNF and ceramide on the hTERT gene (Figure 4B), suggesting that GM-CSF acted both upstream and downstream of ceramide production.    Discussion Top Abstract Introduction Materials and methods Results Discussion References   This study shows that TNF regulates telomerase in both normal and leukemia cells. This regulation involves ceramide and JNK and is inhibited by GM-CSF. Concerning CD34+ normal progenitors, previous studies have demonstrated that telomerase activity is detectable at low levels in hematopoietic progenitor cells and is up-regulated in response to cytokine stimulation.21,22 Our study suggests that, in hematopoietic cells, regulation of hTERT enzymatic activity is under the control of the hTERT gene as described in other cellular models, although we cannot rule out complementary posttranscriptional mechanisms. Our study shows for the first time that, in vitro, TNF inhibited hTERT gene and enzymatic activity in normal myeloid progenitors. Although the functional consequence of this regulation remains uncertain, it is possible that hTERT inhibition mediates the negative effect of TNF in cellular expansion,23 through 2 hypothetic mechanisms. First, based on the protective function of hTERT in many tissues,24 it is conceivable that TNF-induced hTERT inhibition results in an increase in cell loss along myeloid differentiation even though we found no increase in apoptosis. Second, it is possible that hTERT plays by itself an important function in differentiation commitment as recently suggested.25 View larger version (14K): [in this window] [in a new window]   Figure 4.. Influence of GM-CSF on ceramide pathway. (A) Intracellular ceramide variations measured at the peak of stimulation in KG1 cells treated with TNF (20 ng/mL) and GM-CSF (13 ng/mL). Results are expressed as mean ± SD from 5 independent experiments. **P = .008. (B) KG1 cells were incubated with or without TNF, C6-ceramide (10 µM), or GM-CSF for 2 hours. Transcripts of hTERT were quantified using the LightCycler TeloTAGGGhTERT quantification kit as previously described. Results are expressed as mean ± SD from 4 independent experiments. ***P < .001; **P = .004.   To investigate the signaling pathway regulating hTERT, we used the KG1 cell line as a representative model of myeloid cells. We show that TNF induces early and transient ceramide generation in KG1 cells. Others and we have documented that, in other AML cell lines such as U937 cells, TNF induced ceramide generation due to stimulation of a neutral sphingomyelinase and subsequent sphingomyelin (SM) hydrolysis (SM cycle).26,27 The kinetics and the magnitude of ceramide production observed in KG1 cells are very similar to that of TNF-treated U937 cells. These results suggest that TNF activated the SM cycle in KG1 cells. Previous studies showed that ceramide inhibited hTERT in epithelial tumor cells.18,19 Therefore, we hypothesized that, in KG1 cells, ceramide could mediate the effect of TNF. Actually, cell-permeant ceramide inhibited hTERT gene expression in KG1 cells. This result suggests that the SM cycle is an important component of TNF signaling leading to hTERT inhibition. If this is the case, it can be assumed that any negative regulators of this pathway, including increased protein kinase C activity or enhanced antioxidative defenses,28 may confer a significant protection of AML cells toward TNF. In additional studies, we investigated the effect of TNF on MAPK. Indeed, previous studies have documented that TNF may activate the 3 MAPK modules, that is, ERK/MAPK, JNK/SAPK (stress-activated protein kinase), and p38MAPK in various AML cellular models. Moreover, p38MAPK has been implicated in cellular senescence.29 For this reason, we used specific pharmacologic inhibitors to investigate the respective contribution of these pathways in TNF-induced hTERT regulation. We found that JNK, but not ERK or p38MAPK, is involved in TNF signaling leading to hTERT gene inhibition. This result contrasts with other studies, which showed that, at least in epithelial cells, JNK is an activator of the hTERT gene,30,31 suggesting that the role of JNK in hTERT regulation is cell specific. Further studies are needed to identify the regulatory mechanisms, which operate downstream of JNK and interfere with hTERT regulation in myeloid cells. GM-CSF was found to protect cells from hTERT inhibition by TNF. GM-CSF is not a unique growth factor capable to positively regulate hTERT. Indeed, it has been reported that insulin-like growth factor 1 (IGF-1) or IL6 abrogated dexamethasone-induced down-regulation of hTERT activity in multiple myeloma cells, and that phosphatidylinositol 3-kinase (PI3K) and nuclear factor B (NF-B) pathways mediated protective effects of IGF-1 and IL6.32 In KG1 cells, we found that GM-CSF inhibited ceramide generation. Moreover, this growth factor inhibits C6-ceramide inhibition of hTERT. These results suggest that GM-CSF exerts a dual mechanism that involves both abrogation of ceramide generation induced by TNF and interruption of ceramide signaling. In summary, our study shows that TNF down-regulates the hTERT gene in normal and leukemic cells through the ceramide-JNK pathway. Hematopoietic growth factors efficiently counterregulate this signaling. On the basis of the role of hTERT in cellular protection, chromosome stability, and perhaps differentiation, the negative effect of TNF may have important functional consequences, including abnormal regulation of hematopoietic cell differentiation, genetic instability, and marrow insufficiency.    Acknowledgements   We thank Monique Laroche and Nicole Lhermie for technical assistance and Jean-Pierre Jaffrézou for improving the English manuscript.    Footnotes   Submitted April 5, 2005; accepted June 30, 2005. Prepublished online as Blood First Edition Paper, July 14, 2005; DOI 10.1182/blood-2005-04-1386. Supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Association Laurette Fugain, the Association Cent pour Sang La Vie and by a fellowship from the Ligue Nationale contre le Cancer (N.P.-H.). N.P.-H., O.B.-R., and V.M.-D.M. performed the research and analyzed the data; J.A. provided the discarded fragment from hip surgery; C.R. and C.D. provided the fresh AML cells; G.L., O.B.-R., and V.M.-D.M. designed the research and wrote the paper. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Véronique Mansat-De Mas, Laboratoire d'Hématologie, Pavillon Caubet, CHU Purpan, Place du Dr Baylac, 31059 Toulouse Cedex, France; e-mail: demas.v{at}chu-toulouse.fr.    References Top Abstract Introduction Materials and methods Results Discussion References   Kirkpatrick KL, Clark G, Ghilchick M, Newbold RF, Mokbel K. hTERT mRNA expression correlates with telomerase activity in human breast cancer. 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