248 INVOLVEMENT OF THE SPHINGOLIPID, CERAMIDE, IN HEAT SHOCK-INDUCED APOPTOSIS IN BOVINE OOCYTE

2009 ◽  
Vol 21 (1) ◽  
pp. 222 ◽  
Author(s):  
D. Kalo ◽  
Z. Roth
Keyword(s):  
Heat Shock ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Developmental Competence ◽  
Immunofluorescent Staining ◽  
Blastocyst Formation ◽  
Short Term ◽  
Oocyte Developmental Competence ◽  
Induced Apoptosis

Programmed cell death through the sphingomyelin pathway has been suggested to underlie the mechanism by which heat shock disturbs oocyte developmental competence. Serial experiments were performed to characterize the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation could be regulated. In all experiments, bovine cumulus–oocyte complexes (COC) were aspirated from ovaries collected at the abattoir. Cumulus–oocyte complexes were matured (22 h, 38.5°C, 5% CO2), in vitro fertilized (18 h, 38.5°C, 5% CO2), and cultured for 8 days (KSOM; 38.5°C, 5% CO2, 5% O2). In the first experiment, COC were matured at 38.5°C (n = 673) or 41°C (heat shock; n = 718). Exposure of COC to heat shock during maturation reduced cleavage (92.3 v. 84.2%; P < 0.05) and blastocyst formation rates (24.1 v. 14.6%; P < 0.05) relative to the control groups. In the second experiment, COC (n = 563) were exposed to either long-term (22 h) or short-term (0.5 to 3.5 h) heat shock during maturation. An Annexin V-FITC assay (Bender) was used to identify the turnover of phosphatidylserine, one of the features of early-stage apoptosis. In addition, a subgroup of matured oocytes (n = 384) were denuded and fixed in 2% paraformaldehyde, followed by immunofluorescent staining using anti-ceramide (Alexis) to detect ceramide levels and counterstaining with Hoechst (Sigma). Immunofluorescent staining showed no difference in endogenous ceramide levels between groups. Similarly, annexin expression did not differ between groups at the end of the maturation but was higher (P < 0.05) in oocytes exposed to short-term heat shock than in those subjected to long-term heat shock, suggesting that early features of apoptosis should be examined at a specific time of heat exposure. Another experiment was performed to examine the effect of ceramide on oocyte developmental competence. Cumulus–oocyte complexes (n = 1185) were matured with or without 10, 30, and 50 μm C2-ceramide (Sigma). An Annexin V-FITC assay was used with a subgroup of oocytes (n = 137) that were matured with or without 50 μm C2-ceramide for 2 h. To examine the major pathway of ceramide generation, heat-shocked COC were matured with or without 5 μm fumonisin B1 (FB1; Sigma), a specific inhibitor of dihydroceramide synthase (i.e. de novo formation) and ceramide synthase (i.e. salvage pathway), or with 5 μm desipramine hydrochloride (DH; Sigma), a specific inhibitor of the acid sphingomyelinase (i.e. hydrolysis). Results revealed that maturation in 50 μm C2-ceramide increased (P < 0.05) annexin expression in association with reducing cleavage rate and blastocyst formation (P < 0.05). Although not significant, maturation with FB1 moderated the heat-shock effect on oocyte developmental competence. Similarly, 5 μm DH increased the blastocyst formation rate for heat-shocked oocytes from a level of 17% to the control level (22.5%). In summary, ceramide appears to play an important role in heat-shock-induced apoptosis because blocking ceramide formation alleviated its deleterious effect. Research was supported in part by USDA Grant 2007-35203 and BARD Grant 3986-07

Involvement of the sphingolipid ceramide in heat-shock-induced apoptosis of bovine oocytes

2011 ◽  
Vol 23 (7) ◽  
pp. 876 ◽  
Author(s):  
Dorit Kalo ◽  
Zvi Roth
Keyword(s):  
Heat Shock ◽  
De Novo ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Oocyte Developmental Competence ◽  
Induced Apoptosis

Programmed cell death via the sphingomyelin pathway has been suggested to underlie heat-shock disturbance of oocyte developmental competence. A series of experiments were performed to characterise the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation can be regulated. Bovine cumulus–oocyte complexes (COCs) were aspirated from ovaries collected in the cold season (November–April), in vitro-matured, fertilised and cultured for 8 days. Exposure of COCs to heat shock (41°C) during maturation reduced cleavage rate and blastocyst formation relative to the control group (38.5°C). Annexin-V binding (V-FITC assay), which is associated with the early apoptotic event of membrane phosphatidylserine turnover, was higher in oocytes exposed to short-term versus long-term heat shock, suggesting that heat-shock-induced apoptosis involves membrane alterations. Similar to heat exposure, oocyte maturation with C2-ceramide had a dose-dependent deleterious effect on the first cleavages and subsequent embryonic development in association with increased annexin-V binding. Blocking endogenous ceramide generation with fumonisin B1, a specific inhibitor of dihydroceramide synthase (i.e. de novo formation), moderated, to some extent, the effects of heat shock on oocyte developmental competence, suggesting that ceramide plays an important role in heat-shock-induced apoptosis.

scholarly journals Curcumin Enhanced Busulfan-Induced Apoptosis through Downregulating the Expression of Survivin in Leukemia Stem-Like KG1a Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Guangyang Weng ◽  
Yingjian Zeng ◽  
Jingya Huang ◽  
Jiaxin Fan ◽  
Kunyuan Guo
Keyword(s):  
Specific Inhibitor ◽  
Annexin V ◽  
Chemotherapeutic Agents ◽  
Combination Group ◽  
Hematopoietic Stem ◽  
Leukemia Relapse ◽  
Conditioning Regimens ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Related Proteins

Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CUR- and CUR+BUS-treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin.

scholarly journals Protein kinases influence bovine oocyte competence during short-term treatment with recombinant human follicle stimulating hormone

Reproduction ◽  
2005 ◽  
Vol 130 (3) ◽  
pp. 303-310 ◽  
Author(s):  
Atef Ali ◽  
Marc-André Sirard
Keyword(s):  
Protein Kinase ◽  
Follicle Stimulating Hormone ◽  
Term Treatment ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Short Term ◽  
Short Term Treatment ◽  
Developmental Capacity ◽  
Bovine Oocytes

The aim of this study was to investigate the effect of short-term treatment (first 2 or 6 h) with recombinant human follicle-stimulating hormone (r-hFSH) during in vitro maturation (IVM) on the developmental competence of bovine oocytes. The roles of protein kinase A (PKA) and protein kinase C (PKC) (possibly involved in FSH response), were investigated using activators (Sp-cAMPS, PMA) or inhibitors (Rp-cAMPS, sphingosine) of these two protein kinases, respectively. The developmental competence of bovine oocytes was measured by the rate of blastocyst formation after in vitro fertilization (IVF). Our results showed that when cumulus–oocyte complexes (COCs) were cultured with r-hFSH for the first 6 h, a highly significant (P < 0.0001) improvement is seen in blastocyst development rate as a proportion of oocytes in culture compared with those matured with r-hFSH for the first 2 or 24 h. A transient exposure (6 h) to the highest dose (100 μM) of forskolin (an activator of adenylate cyclase) increased (P < 0.05) the rate of blastocyst formation. But the PKA inhibitors (Rp-cAMPS) did not affect the stimulatory effects of r-hFSH on the blastocyst yield. However, stimulation of PKC by low doses of PMA (0.1–0.5 μM) during short-term treatment, enhanced (P < 0.0001) the developmental capacity of oocytes, while sphingosine (a specific inhibitor of PKC) inhibited (P < 0.05) the stimulatory effects of r-hFSH on the rate of blastocyst formation. Our results indicate that although the developmental capacity of bovine oocytes in vitro can be modulated by both the PKA, and the PKC pathways, the activation of PKC during short-term treatment can mimic the effect of r-hFSH on the cytoplasmic maturation in bovine oocytes in vitro.

Apoptosis induced by lipid-associated membrane proteins fromMycoplasma penetransis mediated by nuclear factor κB activation in mouse macrophage

2008 ◽  
Vol 54 (2) ◽  
pp. 150-158 ◽  
Author(s):  
Yanhua Zeng ◽  
Yimou Wu ◽  
Zhongliang Deng ◽  
Xiaoxing You ◽  
Cuiming Zhu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Molecular Mechanisms ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Fluorescein Isothiocyanate ◽  
Nuclear Factor Κb ◽  
Pyrrolidine Dithiocarbamate ◽  
Pathogenic Potential ◽  
Mouse Macrophages ◽  
Induced Apoptosis

Mycoplasma penetrans was shown to be involved in alteration of several eukaryotical cells functions and a causative agent in urogenital infectious diseases. Lipid-associated membrane proteins (LAMPs) may be responsible for the pathogenicity of some mycoplamas. In this study, we investigated whether M. penetrans LAMPs have pathogenic potential by inducing apoptosis in mouse macrophages. As analyzed by annexin-V – fluorescein isothiocyanate staining, significant early- and late-stage apoptosis was induced in M. penetrans LAMPs-challenged mouse macrophages. And agarose gel electrophoresis of the DNA of M. penetrans LAMPs-challenged cells revealed a ladder-like pattern of migration of DNA indicative of apoptosis. The possible molecular mechanisms responsible for the induction of apoptosis were also investigated by characterizing the activation of nuclear transcription factor κB (NFκB). NFκB was activated and translocated into the nucleus in mouse macrophages stimulated by M. penetrans LAMPs. The activation of NFκB and M. penetrans LAMPs-induced apoptosis in mouse macrophages was partially inhibited by the NFκB-specific inhibitor pyrrolidine dithiocarbamate. Thus, this study demonstrates that M. penetrans LAMPs may be an important etiological factor owing to their ability to induce apoptosis in mouse macrophages, which is probably mediated through the activation of NFκB.

scholarly journals A Novel Role of IGF1 in Apo2L/TRAIL-Mediated Apoptosis of Ewing Tumor Cells

Sarcoma ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Frans van Valen ◽  
Henning Harrer ◽  
Marc Hotfilder ◽  
Uta Dirksen ◽  
Thomas Pap ◽  
...  
Keyword(s):  
De Novo ◽  
Fumonisin B1 ◽  
Chemotherapeutic Agents ◽  
Ceramide Synthase ◽  
Short Term ◽  
Igf1 Receptor ◽  
Induced Apoptosis ◽  
Survival Effect

Insulin-like growth factor 1 (IGF1) reputedly opposes chemotoxicity in Ewing sarcoma family of tumor (ESFT) cells. However, the effect of IGF1 on apoptosis induced by apoptosis ligand 2 (Apo2L)/tumor necrosis factor (TNF-) related apoptosis-inducing ligand (TRAIL) remains to be established. We find that opposite to the partial survival effect of short-term IGF1 treatment, long-term IGF1 treatment amplified Apo2L/TRAIL-induced apoptosis in Apo2L/TRAIL-sensitive but not resistant ESFT cell lines. Remarkably, the specific IGF1 receptor (IGF1R) antibodyα-IR3 was functionally equivalent to IGF1. Short-term IGF1 incubation of cells stimulated survival kinase AKT and increased X-linked inhibitor of apoptosis (XIAP) protein which was associated with Apo2L/TRAIL resistance. In contrast, long-term IGF1 incubation resulted in repression of XIAP protein through ceramide (Cer) formation derived from de novo synthesis which was associated with Apo2L/TRAIL sensitization. Addition of ceramide synthase (CerS) inhibitor fumonisin B1 during long-term IGF1 treatment reduced XIAP repression and Apo2L/TRAIL-induced apoptosis. Noteworthy, the resistance to conventional chemotherapeutic agents was maintained in cells following chronic IGF1 treatment. Overall, the results suggest that chronic IGF1 treatment renders ESFT cells susceptible to Apo2L/TRAIL-induced apoptosis and may have important implications for the biology as well as the clinical management of refractory ESFT.

Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3406-3406
Author(s):  
Louie Naumovski ◽  
Jason Ramos ◽  
Jun Chen ◽  
Mint Sirisawad ◽  
David Lucas ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Motexafin Gadolinium ◽  
Akt Phosphorylation ◽  
Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

scholarly journals 121 HEAT SHOCK TO PIG OOCYTES DOES NOT INDUCE APOPTOSIS BUT REDUCES EMBRYO DEVELOPMENT

2005 ◽  
Vol 17 (2) ◽  
pp. 211
Author(s):  
J.-K. Tseng ◽  
P.-C. Tang ◽  
J.-C. Ju
Keyword(s):  
Heat Shock ◽  
Apoptotic Cell ◽  
Electric Pulse ◽  
Annexin V ◽  
Developmental Competence ◽  
Control Group ◽  
Hsp 70 ◽  
Cell Numbers ◽  
Significant Difference

Oocytes are susceptible to heat shock (HS) during the maturation process. It has been demonstrated that HS induces apoptosis and/or the expression of HS protein 70 (hsp 70) in in vitro-produced oocytes and embryos. The objectives of this study were to analyze the effects of HS on the development and apoptosis of pig oocytes and embryos. Porcine ovaries were collected from a local slaughterhouse and the cumulus-oocyte complexes (COCs) were aspirated from follicles 3–6 mm in diameter and subjected to standard in vitro maturation procedures at 39°C for 42 h. The in vitro matured oocytes were then randomly allocated to different HS treatments at 41.5°C for 0 (control, C0h), 1 (HS1h), 2 (HS2h), or 4 h (HS4h). An additional control group of oocytes was cultured for 4 h without HS (C4h). Data were analyzed by chi-square test. In Experiment 1, anti-hsp 70 (SPA-810AP, Stressgen, San Diego, CA, USA) and Western blotting were used to examine the expression of hsp 70. Results indicated that no significant difference of hsp 70 expression in metaphase II porcine oocytes occurred between controls and HS groups (P > 0.05, 7 replicates). In Experiment 2, apoptosis of metaphase II oocytes after HS was identified by annexin V-FITC (Sigma, St. Louis, MO, USA) staining and TUNEL (Roche, Indianapolis, IN, USA). No significant apoptotic signal was detected in the HS groups compared to the controls. The intensity of annexin V staining was not affected by HS, but it increased with the time of culture (P < 0.05, n = 24–37). In Experiment 3, the apoptotic rate and developmental competence of the HS-oocytes were evaluated by TUNEL assay (n = 123–137, 4 replicates). Parthenogenetic activation (n = 123–137) was performed by an electric pulse (2.2 kV cm−1) combined with 6-dimethyaminopurine treatment (6-DMAP, 2.5 μM, 4 h, Sigma). The cleavage rates in HS2h (43 ± 29%) and HS4h (35 ± 28%) decreased (P < 0.05) compared to those in C0h (62 ± 12%) and C4h (66 ± 8%). In addition, the blastocyst formation rates and total cell numbers reduced (P < 0.05) after 2 h (11 ± 10%, 20 ± 16) and 4 h (11 ± 8%, 19 ± 8) of HS treatments compared to those in C0h (23 ± 14%, 32 ± 22) and C4h (21 ± 11%, 27 ± 17), all respectively. The numbers of blastocysts with TUNEL-positive signals were not significantly different between the HS and control groups, but the signals increased (P < 0.05) before the 8-cell stage in HS groups (22–24%) compared to the C0h and C4h controls (16 and 11%), respectively. These results indicate that reduction in developmental competence of in vitro-matured pig oocytes after heat shock is not closely correlated to the expression of hsp 70 in the oocytes and to the apoptotic cell numbers in the blastocyst. Whether detection of apoptosis by TUNEL or annexin V-FITC in oocytes is a good indicator requires further investigation.

186 THE EFFECT OF SHORT VERSUS LONG TERM ELEVATED NONESTERIFIED FATTY ACID CONCENTRATIONS DURING MURINE IN VITRO FOLLICLE GROWTH ON OOCYTE DEVELOPMENTAL COMPETENCE

2015 ◽  
Vol 27 (1) ◽  
pp. 184
Author(s):  
S. D. M. Valckx ◽  
L. Jordaens ◽  
R. Cortvrindt ◽  
P. E. J. Bols ◽  
J. L. M. R. Leroy
Keyword(s):  
Oocyte Maturation ◽  
Developmental Competence ◽  
Nonesterified Fatty Acid ◽  
Follicular Growth ◽  
Final Phase ◽  
Follicle Growth ◽  
Final Oocyte Maturation ◽  
Oocyte Developmental Competence

Metabolic disorders, like obesity and type 2 diabetes are characterised by lipolysis-linked elevated nonesterified fatty acid (NEFA) concentrations. Exposure to high NEFA concentrations during the final phase of bovine in vitro oocyte maturation (24 h) impairs oocyte developmental competence and subsequent embryo quality. However, because elevated NEFA concentrations in vivo are often present for a longer period of time, our recent research focused on a more in vivo-like long-term NEFA exposure (12 d) of whole murine follicles in vitro. The model covers follicular growth from the early secondary to the antral stage in vitro, including final oocyte maturation after an ovulatory stimulus (OS). Results showed an altered follicular growth and physiology (steroid synthesis, gene expression) and a subsequent reduced oocyte developmental competence. However, it remains unclear what specific time frame in follicular development is the most sensitive to such metabolic insult. Therefore, we hypothesised that chronic elevated NEFA concentrations throughout follicle growth affect oocyte developmental competence more severely than short-term NEFA exposure limited to the final phase of oocyte maturation. The aim was to study the effect of elevated NEFA concentrations 1) during the final phase of follicular oocyte maturation (after the OS) and 2) during the whole period of in vitro murine follicle growth until the antral stage, including final oocyte maturation. Early secondary follicles, isolated from the ovaries of 13-day-old B6CBAF1 mice, were cultured in vitro until the antral stage (3 replicates). Follicles were exposed to: BASAL NEFA mix for 12 days (BASAL-BASAL, control); HIGH stearic acid (SA) for 12 days (SA-SA); HIGH NEFA mix for 12 days (NEFA-NEFA); HIGH SA after the OS (BASAL-SA); and HIGH NEFA mix after the OS (BASAL-NEFA). Oocytes were isolated out of antral follicles 20 hours after the OS, routinely fertilized and presumptive zygotes cultured. Cleavage (n cleaved zygotes/n oocytes) and blastocyst (n blastocysts/n oocytes) rates were documented and analysed by means of binary logistic regression. Cleavage rate was reduced for BASAL-BASAL (37%) compared to BASAL-NEFA embryos (52%; P = 0.045). The BASAL-NEFA treatment (43%) presented with a higher blastocyst percentage than BASAL-BASAL (23%; P = 0.004), NEFA-NEFA (26%; P = 0.037) and SA-SA (15%; P = 0.001) treatments. The BASAL-SA (30%) treatment performed better than the SA-SA treatment (P = 0.049). Even though BASAL-BASAL (control) embryo development was surprisingly low, the results indicate that long-term NEFA exposure during follicle growth in vitro affects oocyte developmental competence more severely than an exposure limited to the final phase of maturation after the OS. The results thus emphasise that the maternal micro-environment throughout follicular growth and not only during final oocyte maturation is essential for optimal oocyte quality.

Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates

Blood ◽  
2000 ◽  
Vol 96 (13) ◽  
pp. 4118-4123 ◽  
Author(s):  
Veronique Maguer-Satta ◽  
Robert Oostendorp ◽  
Dianne Reid ◽  
Connie J. Eaves
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Specific Inhibitor ◽  
Hematopoietic Cell ◽  
Leukemic Cells ◽  
Short Term ◽  
Cell Fate Decisions ◽  
Short Term Exposure ◽  
Necrosis Factor

Abstract In this study, it is shown that short-term exposure of normal human marrow CD34+CD38− cells to low concentrations of tumor necrosis factor (TNF) in the presence of 100 ng/mL Flt3 ligand and Steel factor and 20 ng/mL interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor, in either bulk or single-cell serum-free cultures, markedly reduces their ability subsequently to generate colony-forming cells (CFCs) in 6-week stromal cell–containing long-term cultures without affecting their viability, mitogenic response, or short-term ability to produce CFCs. A similar differential effect on the functional attributes of CD34+CD38− cells was seen when C2- or C6-ceramide, but not dihydro-C2-ceramide (an inactive analog of ceramide), was substituted for TNF. The addition of D-erythro-MAPP (a specific inhibitor of intracellular ceramide degradation) enhanced the ability of TNF to selectively eliminate long-term culture–initiating cell (LTC-IC) activity. These findings indicate that TNF can directly modulate the ability of CD34+CD38− cells to maintain their LTC-IC function at doses below those required to initiate apoptosis, cell cycle arrest, or both, and they suggest that this may be mediated by the TNF-induced generation of intracellular ceramide. Identification of a signaling intermediate that can influence primitive hematopoietic cell fate decisions offers a new approach to the investigation of signaling mechanisms in normal stem cell populations and to how these may be altered in leukemic cells.

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