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At least 19 recordsLinked to original sources
PubMed2002

Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line. Role for endogenous ceramide in mediating the action of exogenous ceramide.

Treatment of A549 cells with C(6)-ceramide resulted in a significant increase in the endogenous long chain ceramide levels, which was inhibited by fumonisin B1 (FB1), and not by myriocin (MYR). The biochemical mechanisms of generation of endogenous ceramide were investigated using A549 cells treated with selectively labeled C(6)-ceramides, [sphingosine-3-(3)H]d-erythro-, and N-[N-hexanoyl-1-(14)C]d-erythro-C(6)-ceramide. The results demonstrated that (3)H label was incorporated into newly synthesized long chain ceramides, which was inhibited by FB1 and not by MYR. Interestingly, the (14)C label was not incorporated into long chain ceramides. Taken together, these results show that generation of endogenous ceramide in response to C(6)-ceramide is due to recycling of the sphingosine backbone of C(6)-ceramide via deacylation/reacylation and not due to the elongation of its fatty acid moiety. Moreover, the generation of endogenous long chain ceramide in response to C(6)-ceramide was completely blocked by brefeldin A, which causes Golgi disassembly, suggesting a role for the Golgi in the metabolism of ceramide. In addition, the generation of endogenous ceramide in response to short chain exogenous ceramide was induced by d-erythro- but not l-erythro-C(6)-ceramide, demonstrating the stereospecificity of this process. Interestingly, several key downstream biological activities of ceramide, such as growth inhibition, cell cycle arrest, and modulation of telomerase activity were induced by d-erythro-C(6)-ceramide, and not l-erythro-C(6)-ceramide (and inhibited by FB1) in A549 cells, suggesting a role for endogenous long chain ceramide in the regulation of these responses.

Adenocarcinoma↗
PubMed2001

Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.

Animals↗
PubMed1999

Role of ceramide in lipopolysaccharide (LPS)-induced signaling. LPS increases ceramide rather than acting as a structural homolog.

Ceramide and ceramide-activated enzymes have been implicated in responses to bacterial lipopolysaccharide (LPS) and the proinflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1). Although TNF and IL-1 cause elevation of cellular ceramide, which is thought to act as a second messenger, LPS has been proposed to signal by virtue of structural similarity to ceramide. We have investigated the relationship between ceramide and LPS by comparing the effects of a cell-permeable ceramide analog (C2-ceramide) and LPS on murine macrophage cell lines and by measuring ceramide levels in macrophages exposed to LPS. We found that while both C2-ceramide and LPS activated c-Jun N-terminal kinase (JNK), only LPS also activated extracellular signal-regulated kinases (ERKs). C2-ceramide was also unable to activate NF-kappaB, a transcription factor important for LPS-induced gene expression. Upon measurement of cellular ceramide in macrophage lines, we observed a small but rapid rise in ceramide, similar to that seen upon IL-1 or TNF treatment, suggesting LPS induces an increase in ceramide rather than interacting directly with ceramide-responsive enzymes. We found that C2-ceramide activated JNK and induced growth arrest in macrophages cell lines from both normal mice (Lpsn) and mice genetically unresponsive to LPS (Lpsd), whereas only Lpsn macrophages made these responses to LPS. Surprisingly, LPS treatment of Lpsd macrophages induced a rise in ceramide similar to that observed in LPS-responsive cells. These results indicate that the wild type Lps allele is not required for LPS-induced ceramide generation and suggest that ceramide elevation alone is insufficent stimulus for most responses to LPS.

Animals↗
PubMed1998

Ceramide can induce cell death in sensory neurons, whereas ceramide analogues and sphingosine promote survival.

Ceramide has been shown to induce apoptosis in leukaemic cells and some other cell types, but there are few data on its role in neuronal cells. We investigated the effect of ceramide and its analogues in cultured sensory neurons from neonatal mice. These cells undergo apoptosis in the absence of neurotrophins. Treatment with ceramide or its analogues increased survival, both in the presence and absence of NGF. Sphingosine treatment also increased survival. In the presence of the ceramidase inhibitor N-oleoyl ethanolamine, which blocks conversion of ceramide to sphingosine, the addition of natural ceramide-induced cell death, even in the presence of nerve growth factor (NGF). N-oleoyl ethanolamine did not cause cell death by itself. N-oleoyl ethanolamine did not alter the response to ceramide analogues, indicating that they were not ceramidase substrates. These results indicate that, in sensory neurons, exogenous ceramide is converted to sphingosine, which promotes cell survival. When conversion is blocked by ceramidase inhibition, exogenous ceramide causes cell death, presumably due to the high levels of ceramide itself. The ceramide analogues all mimicked the effect of sphingosine rather than ceramide, casting serious doubt on their validity as models of ceramide action. Ceramide analogues could prevent neuronal death even in the combined presence of N-oleoyl ethanolamine and natural ceramide. Surprisingly, dihydro C2-ceramide, which is frequently used as a control for C2-ceramide, had the same effect as ceramide analogues.

Animals↗
PubMed2005

Resistance to TRAIL is associated with defects in ceramide signaling that can be overcome by exogenous C6-ceramide without requiring down-regulation of cellular FLICE inhibitory protein.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor superfamily that selectively induces apoptosis in malignant cells. However, not all cancer cells are susceptible to TRAIL and mechanisms of resistance and new strategies to enhance sensitivity are an area of intense investigation. Glucose withdrawal or paclitaxel increase intracellular ceramide, down-regulate cellular FLICE inhibitory protein (cFLIP), and sensitize cells to TRAIL. Therefore, we investigated whether TRAIL resistance is due to ceramide levels and/or defects in ceramide generation following ligand binding. Colon cancer cells isolated from the primary tumor (SW480) and a subsequent metastasis (SW620) of the same patient have different sensitivities to TRAIL. Mass spectrometry was used to compare ceramide content in untreated and TRAIL-treated cells. Overall levels of ceramide were comparable in the cell lines but TRAIL-sensitive SW480 cells contained a higher percentage of C(16)-, and C(18)-ceramide and lower C(24)-ceramides than TRAIL-resistant SW620 cells. Upon TRAIL treatment, ceramide (primarily C(16)-ceramide) increased in SW480 but not SW620 cells. The increase in ceramide occurred with slow kinetics, paralleling caspase-3/7 activation. Combination of C(6)-ceramide with TRAIL resulted in apoptosis of SW620 cells. However, exogenous C(6)-ceramide did not affect levels of cFLIP nor did pretreatment sensitize cells to TRAIL. Exposure to TRAIL prior to ceramide was required to induce apoptosis, suggesting that ceramide plays a role in enhancing or amplifying TRAIL-mediated signaling. Our results suggest that ceramide plays a role in promoting TRAIL-mediated apoptosis and that TRAIL-resistant cancers may benefit from combination therapy with ceramide or agents that enhance ceramide accumulation.

Antineoplastic Agents, Phytogenic↗
PubMed1990

Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin.

Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from spingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate. In sum, these studies demonstrate the novel phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from sphingomyelin to ceramide 1-phosphate via the phosphorylation of ceramide.

Autoradiography↗
PubMed2003

A specific ceramide kinase assay to measure cellular levels of ceramide.

Human ceramide kinase was recently cloned and characterized. Recombinant ceramide kinase is highly active and ceramide is the only lipid that it phosphorylates, indicating that it should be useful for the measurement of ceramide levels in biological samples by conversion to ceramide-1-phosphate, in a manner analogous to that of the widely used Escherichia coli diacylglycerol kinase method. Using recombinant ceramide kinase, we have now developed a rapid and specific enzymatic method to quantify mass levels of long-chain ceramides in cellular lipid extracts. This new ceramide kinase assay is more specific than the commonly used diacylglycerol kinase method because the ubiquitous lipid diacylglycerol, the preferred substrate for diacyglycerol kinase which is usually present at higher concentrations than ceramide and can interfere with ceramide phosphorylation, is completely inactive with ceramide kinase. Moreover, this high specificity eliminates the need for analysis of the lipid product by thin-layer chromatography since ceramide-1-phosphate is the only radiolabeled lipid in organic solvent extracts of ceramide kinase reactions.

Biological Assay↗
PubMed2003

Inhibition of rat sympathetic neuron apoptosis by ceramide. Role of p75NTR in ceramide generation.

C6-ceramide protects sympathetic neurons from apoptosis caused by nerve growth factor (NGF) deprivation. Here, we report for the first time that ceramide generated "de novo" is also anti-apoptotic. Moreover, C6-ceramide is converted to long-chain ceramides in a process inhibited by fumonisin B1. The anti-apoptotic effect of C6-ceramide is due to the short analogue as to the long-chain ceramides. C6-ceramide shares mechanisms of action with NGF. C6-ceramide induces TrkA phosphorylation and selective activation of the phosphatidyl inositol 3-kinase (PI3-kinase)/Akt pathway but not the MAPK/ERK pathway. Importantly, the PI3-kinase inhibitor LY294002 abolishes the pro-survival effect of C6-ceramide. We identified a novel way to activate retrograde-mediated neuronal survival in the absence of NGF. Using compartmented cultures we show that addition of C6-ceramide exclusively to distal axons is sufficient to abort nuclear apoptosis. Our system offers a very unique alternative to understand the molecular bases of retrograde signaling in the absence of retrograde transport of neurotrophins. In search for a natural ligand that leads to ceramide generation we examined the activation of the sphingomyelin (SM) cycle downstream the p75 neurotrophin receptor (p75NTR). We found that in sympathetic neurons, selective activation of p75NTR by brain-derived neurotrophin factor or NGF plus K252a induces elevation of ceramide that correlates with SM hydrolysis. However, p75NTR activation does not generate sufficient ceramide to block apoptosis probably due to the rapid decrease in p75NTR expression that occurs upon NGF withdrawal.

Adrenergic Fibers↗
PubMed1995

Short-chain ceramide-1-phosphates are novel stimulators of DNA synthesis and cell division: antagonism by cell-permeable ceramides.

Ceramide and ceramide-1-phosphate are sphingolipid analogues of diacylglycerol and phosphatidate, respectively, and they are putative second messengers of agonist-stimulated sphingomyelin metabolism. The interactions of exogenous cell-permeable ceramides and ceramide-1-phosphates in modifying DNA synthesis and signal transduction were investigated in Rat-1 fibroblasts. C2- and C8-Ceramide-1-phosphates (N-acetylsphingosine-1-phosphate and N-octanoylsphingosine-1-phosphate, respectively) at 1-10 microM stimulated DNA synthesis and cell division. This effect was blocked by cell-permeable ceramides. C2-Ceramide stimulated the conversion of exogenous C8-ceramide-1-phosphate to C8-ceramide, with very little production of sphingosine or sphingosine-1-phosphate. This mechanism may be partly responsible for preventing the stimulation of DNA synthesis. Unlike phosphatidate or lyso-phosphatidate, concentrations of C8-ceramide-1-phosphate that stimulated DNA synthesis did not inhibit adenylate cyclase activity, nor did they increase the activities of phospholipase D or mitogen-activated protein kinases (42- and 44 kDa isoforms). Although ceramide-1-phosphate can be considered as an analogue of phosphatidate, the effects of this compound on signal transduction differ considerably from those of phosphatidate. This work demonstrates that short-chain ceramide-1-phosphates can be used as novel external agonists that can stimulate DNA synthesis. This effect can be counteracted by short-chain ceramides.

Animals↗
PubMed1994

Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction.

Extracellular agonists such as tumor necrosis factor-alpha (TNF-alpha) activate the sphingomyelin cycle leading to the generation of ceramide. Ceramide has been suggested as an important mediator of the effect of TNF-alpha on growth inhibition, c-myc down-regulation, apoptosis, and the activation of the nuclear factor kappa B. Although there is no clearly defined intracellular target for ceramide activity, previous studies have demonstrated the existence of a ceramide-activated protein phosphatase (CAPP) in vitro. Since c-myc is an early downstream cellular target for TNF-alpha, we examined the role of ceramide and CAPP in c-myc down-regulation. In intact HL-60 cells ceramide induced down-regulation of c-myc RNA levels. C2-ceramide was active at 1-10 microM and caused 40-80% inhibition of c-myc RNA levels at 30-120 min of treatment. In nuclear run-on studies, C2-ceramide induced a block to transcription elongation of the c-myc transcript without affecting transcription through the first exon. Therefore, ceramide appeared to inhibit c-myc expression via a mechanism identical with that of TNF-alpha. HL-60 cells contained CAPP which was inhibited by okadaic acid (0.1-10 nm). CAPP in HL-60 cells was activated by D-erythro-ceramide but not D-erythro-dihydroceramide. The specificity of activation of CAPP by ceramide in vitro was matched by a similar specificity of c-myc down-regulation in cells. Moreover, okadaic acid inhibited the effects of ceramide and TNF-alpha on c-myc down-regulation. On the other hand, okadaic acid did not inhibit the ability of phorbol 12-myristate 13-acetate to down-regulate c-myc, demonstrating the existence of at least two distinct pathways in the regulation of c-myc expression. These results demonstrate that CAPP is important for ceramide-induced down-regulation of c-myc in myeloid leukemia cells. The implications of these findings in further delineating a sphingomyelin signaling pathway with important anti-proliferative effects are discussed.

Ceramides↗
PubMed1998

Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis.

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. However, mechanisms responsible for ceramide-induced apoptosis remain unclear. We investigated the possibility that ceramide may decrease antiapoptotic signaling in cells by inhibiting Akt kinase activity. Our data show that C2-ceramide induces apoptosis in HMN1 motor neuron cells and decreases both basal and insulin- or serum-stimulated Akt kinase activity 65-70%. These results are consistent with decreased Akt kinase activity being involved in the apoptotic effects of ceramide. This possibility is further supported by studies showing that constitutively active Akt kinase decreases C2-ceramide-induced death of HMN1 cells as well as COS-7 cells. Decreased Akt activity is not due to ceramide activating the ceramide-activated protein phosphatase or to a direct inhibition of Akt kinase by ceramide, suggesting that ceramide acts upstream of Akt kinase to decrease its activity. Treating cells with C2-ceramide does not affect phosphorylation of insulin receptor substrate-1, interactions between insulin receptor substrate-1 and p85, or insulin-stimulated phosphatidylinositol 3-kinase activity, suggesting that the effects of C2-ceramide on Akt kinase are not mediated through modulating phosphatidylinositol 3-kinase. In sum, our results suggest that inhibition of the key antiapoptotic kinase, Akt, may play an important role in ceramide-induced apoptosis.

Animals↗
PubMed1997

C2-ceramide and C6-ceramide inhibited priming for enhanced release of superoxide in monocytes, but had no effect on the killing of leukaemic cells by monocytes.

Ceramide acts as an intracellular second messenger in cellular signal transduction. We examined the effects of two cell-permeable ceramides, C2-ceramide and C6-ceramide, on human monocyte functions. After monocytes were primed with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) for 18 hr in suspension culture, they produced a high amount of superoxide (O2-) when triggered by phorbol myristate acetate. C2- or C6-ceramide inhibited O2- release from monocytes primed with LPS (1 ng/ml) or IFN-gamma (100 U/ml), but did not affect unprimed monocytes. An analogue, C2-dihydroceramide, was inactive. C2-ceramide was most effective at 6 microM, and C6-ceramide at 60 microM. C2- or C6-ceramide at these concentrations was not toxic for monocytes, as assessed by trypan blue exclusion and by the 3-[4, 5-dimethylthiazol-2-y1]-2,5 diphenyl tetrazolium bromide (MTT) assay which measures the ability of live cells to produce formazan. C2-ceramide (20 microM) had no effect on the killing of leukaemic cells (HL-60 and K562 cells) by monocytes treated with IFN-gamma, LPS, or both for 18 hr, with killing assessed by an 111 Indium-releasing assay. C2-ceramide (20 microM) induced secretion of low amounts of tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) from the monocytes. But C2-ceramide did not alter the higher secretion of TNF-alpha or IL-1 beta from monocytes treated with IFN-gamma or LPS. Thus the cell-permeable ceramides acted like antagonists of LPS, rather than analogues of LPS, as has been proposed. The results here showed that the signal transduction pathway for O2- release by monocytes differed from that for the cytolysis of leukaemic cells, and confirmed that oxygen radicals are not involved in cytolysis.

Ceramides↗
PubMed1994

Quantification of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses.

Ceramides (sphingolipids) are the main polar lipids of the stratum corneum and play an important role in skin barrier function, cell adhesion and epidermal differentiation. In view of the function of ceramides in normal skin, this study aimed to assess their levels in patients with various types of hereditary ichthyosis, in which epidermal homeostasis is markedly abnormal. Stratum corneum samples were collected from 80 patients and 23 normal controls, and the intercellular and lipid envelope ceramides were analysed by high-performance thin-layer chromatography. The covalently bound ceramides (ceramides A and B) of the lipid envelope were present in all patients studied, and showed no significant differences from control samples. Total ceramides (ceramides 1-6) were decreased in bullous ichthyosiform erythroderma, which is presumably a secondary phenomenon similar to that seen in patients with atopic dermatitis. Patients with non-erythrodermic lamellar ichthyosis showed a marked decrease in levels of the important acylceramide, ceramide 1, whereas those with other types of autosomal recessive ichthyosis (limited lamellar ichthyosis and non-bullous ichthyosiform erythroderma) had mean levels similar to the controls. Ceramide 1 deficiency may therefore define a subgroup within the autosomal recessive ichthyoses. Sjögren-Larsson syndrome (SLS) shows a deficiency of both acyl-ceramides (ceramides 1 and 6), which would seem likely to disrupt the normal skin barrier function. Furthermore, glucosylceramides (cerebrosides) are known to be deficient in the neural tissue of patients with SLS. The relationship of these ceramide abnormalities to the underlying fatty alcohol oxidoreductase defect remains uncertain, but they may provide an interesting link between the nerve damage and cutaneous abnormalities seen in this rare neurodermatosis.

Adolescent↗
PubMed2006

Sphingosine, a product of ceramide hydrolysis, influences the formation of ceramide channels.

Ceramides are known to have a regulatory function in apoptosis, including the release of cytochrome c and other proapoptotic factors from the mitochondrial intermembrane space. Ceramides can form large, stable channels in the outer mitochondrial membrane, leading to the proposal that ceramide channels are the pathway through which these proteins are released. Here, we report that sphingosine, a product of ceramide hydrolysis by ceramidase, is capable of destabilizing ceramide channels, leading to their disassembly. Sphingosine is directly responsible for the disassembly of ceramide channels in planar membrane experiments and markedly reduces the ability of ceramide to induce the release of intermembrane space proteins from mitochondria in vitro. Low concentrations of both L and D sphingosine potentiate the release of intermembrane space proteins by long-chain ceramide and channel formation in liposomes. These results provide evidence for a mechanism by which the disassembly of ceramide channels, as initiated by ceramidase, could be accelerated by the direct interaction of the hydrolysis product with the ceramide channels themselves. This mechanism therefore could form a positive feedback loop for rapid shut-down of ceramide channels. However, potentiation of ceramide channel formation is also possible and thus both effects could influence the propensity for mitochondria-mediated apoptosis.

Ceramides↗
PubMed2002

Increase of ceramide in adriamycin-induced HL-60 cell apoptosis: detection by a novel anti-ceramide antibody.

We recently raised an IgM class of monoclonal antibody (Ab) for ceramide (NHCER-2), and examined its specificity and sensitivity. Enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) showed that NHCER-2 recognized ceramides but not other sphingolipids such as sphingosine, sphinganine, sphingomyelin, sphingosine-1-phosphate, ceramide-1-phosphate, glucosylceramide and cerebroside. In addition, N-hexanoyl, N-octanoyl and N-palmitoylsphingosine were detected by NHCER-2, but N-acetylsphingosine and dihydroceramide were not. Densities of ceramide detected by NHCER-2 were proportional to the amounts of ceramide standard up to 250 ng. When various concentrations of adriamycin (ADR) was added to induce apoptosis, the amounts of ceramide detected by NHCER-2 time- and dose-dependently increased in apoptosis-sensitive HL-60 cells as well as by DGK assay, but not in apoptosis-resistant HL-60/ADR cells. After cell fractionation, ceramide levels judged not only by diacylglycerol kinase (DGK) assay but also by NHCER-2 were shown to increase in the microsomal and the nuclear fraction in apoptosis-sensitive cells, but not in apoptosis-resistant cells. Moreover, absolute amounts of ceramide determined by NHCER-2 were well correlated with those by DGK assay. These results suggest that increase of ceramide in the nuclear fraction as well as in the microsomal fraction may play a role in ADR-induced apoptosis and that a novel anti-ceramide Ab NHCER-2 could be beneficial to investigate changes of ceramide content in the cells.

Antibodies, Monoclonal↗
PubMed1996

Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression.

Recent studies indicate that sphingolipids mediate several cellular processes. We assessed roles of sphingolipids in the regulation of E-selectin expression in human umbilical vein endothelial cells. All exogenously-added sphingolipids (sphingosine, C2-ceramide, sphingosine 1-phosphate, and N,N-dimethylsphingosine) failed to induce E-selectin expression by themselves. C2-ceramide at 5 micron enhanced interleukin-1 beta (IL-1 beta)-induced E-selectin expression 2.7-fold, whereas other sphingolipids tested had no effects on this process. Sphingomyelinase, but not phospholipases A2, C, or D, mimicked the enhancing effect of C2-ceramide. Northern blot analyses revealed that C2-ceramide and sphingomyelinase increased interleukin-1 beta-induced E-selectin gene transcription levels. C2-ceramide and sphingomyelinase induced NF-kappaB activation by themselves and enhanced activation by IL-1 beta, which is essential for E-selectin expression. Immunological analyses with anti-NF-kappaB antibodies showed that subunit composition of NF-kappaB activated by IL-1 beta differs from that activated by C2-ceramide, suggesting that signaling pathways utilized by these stimuli may be different. Treatment with C2-ceramide or sphingomyelinase did not alter NF-ELAM1 specific binding activity. IL-1 beta induced sphingomyelin hydrolysis to ceramide; intracellular ceramide level increased to 182% of control value at 30 min. Taken together, these findings suggest that (i) sphingomyelin hydrolysis to ceramide does not trigger, but rather enhances cytokine-induced E-selectin expression, in part through NF-kappaB; (ii) sphingomyelin hydrolysis to ceramide does not mediate all the effects of IL-1 beta, although it may play important roles in IL-1 beta signal transduction in human umbilical vein endothelial cells.

Base Sequence↗
PubMed2001

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase.

The retinoid N-(4-hydroxyphenyl)retinamide (4-HPR; fenretinide) is cytotoxic to a variety of cancer cell lines, and we previously showed an association between ceramide generation and 4-HPR cytotoxicity for neuroblastoma cell lines (B. J. Maurer et al., J. Natl. Cancer Inst. (Bethesda), 91: 1138-1146, 1999). Here we determine whether the increased ceramide mediated by 4-HPR in the CHLA-90 human neuroblastoma cell line results from de novo ceramide synthesis. Treatment of CHLA-90 with 4-HPR for 2 h, in the presence of [(3)H]palmitic acid, caused sequential formation of [(3)H]sphinganine (220% over control) and [(3)H]ceramide (160% over control), with sphinganine returning to baseline at 4 h, and ceramide continuing to increase (215% over control). 4-HPR treatment did not accelerate cellular decay of sphingomyelin. Preincubation of cells with either L-cycloserine, an inhibitor of serine palmitoyltransferase (SPT), or fumonisin B(1), an inhibitor of ceramide synthase, retarded ceramide formation in response to 4-HPR treatment, although sphinganine was still generated when 4-HPR and FB(1) were present. Data from in vitro enzyme assays using microsomes showed that preexposure of intact cells to 4-HPR resulted in a time (175% over control; 6 h)- and dose-dependent increase (173% over control; 10 microM) in SPT activity as well as a time (265% over control)- and dose-dependent increase (215% above control; 10 microM) in ceramide synthase activity. Our results show that 4-HPR-mediated ceramide generation is derived from the de novo synthetic pathway by coordinate activation of SPT and ceramide synthase. Knowledge of these biochemical events is of utility when downstream modulators of ceramide metabolism are used to heighten the cytotoxic response to chemotherapy.

Acyltransferases↗
PubMed2003

On-line solid-phase extraction of ceramides from yeast with ceramide III imprinted monolith.

A molecularly imprinted polymeric monolith (MIPM) was prepared by in situ polymerization using styrene, glycidyl methacrylate and methacrylic acid as monomers, divinylbenzene and triallyl isocyanurate as cross-linking agents, and ceramide III as print molecule. The texture, pore size distribution, mobile phase flow characteristic, and chromatographic performance of the MIPM and a control monolith synthesized without the print molecule were examined, respectively. The results showed that using ceramide III as print molecule significantly affected the pore structure and pore distribution of the monolith, and greatly improved the retention of ceramide III and its analogues used in cosmetics as well. The retention of ceramide III on the MIPM could be reduced by increasing the ratio of chloroform to hexane in eluting buffer. The workability of the MIPM was firstly demonstrated through the separation of a model lipid mixture containing ceramide III and ergosterol, the main sterol impurity in yeast lipid extracts. The application of the ceramide III imprinted monolith to the isolation of ceramides from yeast lipid extracts was attempted and resulted in a considerable enrichment of ceramides, as shown by FIIR analysis. This indicates the potential of ceramide III imprinted monolith synthesized in the present study in the on-line solid-phase extraction of ceramides from yeast.

Ceramides↗