Biomedical subjects

Yusuf A Hannun

Publications and source records attributed to Yusuf A Hannun.

At least 19 recordsLinked to original sources

PubMed2006

Large-scale purification and characterization of recombinant Pseudomonas ceramidase: regulation by calcium.

Ceramidases (CDases) hydrolyze ceramide to sphingosine (SPH) and fatty acid. Pseudomonas CDase (pCDase) is a homolog of mammalian neutral ceramidases and may play roles in disease pathogenesis. In this study, pCDase was cloned and expressed in Escherichia coli (E. coli). The expressed recombinant pCDase was solubilized by optimizing several factors, including culture medium, the concentration of isopropyl-beta-thiogalactopyranoside (IPTG), temperature, and time of induction, which were identified to be critical for the optimal production of recombinant pCDase. The recombinant pCDase was purified using nickel-nitrilotriacetic acid affinity, phenyl-Sepharose, and Q-Sepharose column chromatography, which gave an overall yield of 0.45 mg/l purified protein of starting culture. The activity of the recombinant pCDase followed classical Michaelis-Menten kinetics, with optimum activity in the neutral pH range. Both the hydrolytic and the reverse activities of CDase were stimulated by calcium with an affinity constant (K(a)) of 1.5 microM. Kinetics studies showed that calcium caused a decrease of K(m) and an increase in V(max) of pCDase. Calcium and D-erythro-sphingosine caused significant changes in the near ultraviolet circular dichroism (CD) spectra and the changes were inhibited in the presence of EGTA. These results identify important interactions between calcium and pCDase, which may play an essential role in the interaction of pCDase and its substrate.

Amidohydrolases↗

PubMed2006

Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism.

Phospholipases are essential enzymes in cellular signalling processes such as cellular differentiation, proliferation and apoptosis. Based on its high degree of homology with sequences of prokaryote SMases, a type of Mg(2+)-dependent PLC (nSMase-1) was recently discovered which displayed strong redox dependence for activity in vitro [F. Rodrigues-Lima, A.C. Fensome, M. Josephs, J. Evans, R.J. Veldman, M. Katan (2000), J. Biol. Chem. 275 (36) 28316-28325]. The aim of this work was to test the hypothesis that glutathione could be a natural regulator of nSMase-1 activity ex vivo. We studied how altering glutathione levels and redox ratio modulate nSMase-1 activity in a HEK293 cell line that ectopically overexpressed the nSMase-1 gene. Diminishing total glutathione with BSO without altering significantly the GSH/GSSG ratio did not affect nSMase-1 activity. Treatment of cells with diamide produced a transient decrease of total glutathione and a sharp, but also transient, decrease of the GSH/GSSG ratio. Under these conditions, nSMase-1 activity was temporarily activated and then returned to normal levels. Simultaneous treatment with BSO and diamide that resulted in permanent decreases of total glutathione and GSH/GSSG redox ratio produced a sustained activation of nSMase-1 activity. Taken together, these data indicate that altering the GSH/GSSG ratio by increasing GSSG or decreasing GSH levels, but not the total concentration of glutathione, modulates nSMase-1 activity. Our findings are the first evidence supporting the ex vivo regulation of nSMase-1 through a redox glutathione-dependent mechanism.

Cell Line↗

PubMed2006

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2.

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.

Cell Line, Tumor↗

PubMed2006

Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease.

Previous work has identified sphingosine kinase-1 (SK1) as a substrate for the cysteine protease cathepsin B in vitro. In this study, the mechanism of SK1 cleavage by cathepsin B was investigated. We identified two initial cleavage sites for the protease, the first at histidine 122 and the second at arginine 199. Mutation analysis showed that replacement of histidine 122 with a tyrosine maintained the activity of SK1 while significantly reducing cleavage by cathepsin B at the initial cleavage site. The efficacy of cleavage of SK1 at arginine 199, however, was not affected. These studies demonstrate that SK1 is cleaved by cathepsin B in a sequential manner after basic amino acids, and that the initial cleavages at the two identified sites occur independently of each other.

Amino Acids, Basic↗

PubMed2006

Protein kinase C-induced activation of a ceramide/protein phosphatase 1 pathway leading to dephosphorylation of p38 MAPK.

Recently we showed that, in human breast cancer cells, activation of protein kinase C by 4beta-phorbol 12-myristate 13-acetate (PMA) produced ceramide formed from the salvage pathway (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). In this study, we investigated intracellular signaling events mediated by this novel activated pathway of ceramide generation. PMA treatment resulted in transient activation of mitogen-activated protein kinases (ERK1/2, JNK1/2, and p38) followed by dephosphorylation/inactivation. Interestingly, fumonisin B1 (FB1), an inhibitor of the salvage pathway, attenuated loss of phosphorylation of p38, suggesting a role for ceramide in p38 dephosphorylation. This was confirmed by knock-down of longevity-assurance homologue 5, which partially suppressed the formation of C(16)-ceramide induced by PMA and increased the phosphorylation of p38. These results demonstrate a role for the salvage pathway in feedback inhibition of p38. To determine which protein phosphatases act in this pathway, specific knock-down of serine/threonine protein phosphatases was performed, and it was observed that knock-down of protein phosphatase 1 (PP1) catalytic subunits significantly increased p38 phosphorylation, suggesting activation of PP1 results in an inhibitory effect on p38. Moreover, PMA recruited PP1 catalytic subunits to mitochondria, and this was significantly suppressed by FB1. In addition, phospho-p38 resided in PMA-stimulated mitochondria. Upon PMA treatment, a mitochondria-enriched/purified fraction exhibited significant increases in C(16)-ceramide, a major ceramide specie, which was suppressed by FB1. Taken together, these data suggest that accumulation of C(16)-ceramide in mitochondria formed from the protein kinase C-dependent salvage pathway results at least in part from the action of longevity-assurance homologue 5, and the generated ceramide modulates the p38 cascade via PP1.

Catalytic Domain↗

PubMed2006

The extended family of neutral sphingomyelinases.

The neutral sphingomyelinases (N-SMases) are considered major candidates for mediating the stress-induced production of ceramide, and N-SMase activity has been identified, characterized, and cloned from bacteria, yeast, and mammalian cells. Although the level of identity between these enzymes is low, a number of key residues thought to be involved in metal binding and catalysis are conserved. This has led to the suggestion of a common catalytic mechanism, and thus, these enzymes are considered to form an extended family of N-SMases. Despite considerable research into N-SMase activity in cell culture and various tissues, the lack, until recently, of molecular identification of specific N-SMase enzymes had precluded specific insights into the regulation, physiological, and pathological roles of these proteins. In this review, we summarize, for the first time, current knowledge of the N-SMase family, focusing on cloned members from bacteria, yeast, and mammalian cells. We also briefly consider the major future directions for N-SMase research which promises highly significant and specific insight into sphingolipid-mediated functions.

Amino Acid Sequence↗

PubMed2006

Necessary role for the Lag1p motif in (dihydro)ceramide synthase activity.

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

Amino Acid Sequence↗

PubMed2006

Involvement of sphingolipids in apoptin-induced cell killing.

The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.

Adenoviridae↗

PubMed2006

Tailoring structure-function and targeting properties of ceramides by site-specific cationization.

In the course of our studies on compartment-specific lipid-mediated cell regulation, we identified an intimate connection between ceramides (Cers) and the mitochondria-dependent death-signaling pathways. Here, we report on a new class of cationic Cer mimics, dubbed ceramidoids, designed to act as organelle-targeted sphingolipids (SPLs), based on conjugates of Cer and dihydroceramide (dhCer) with pyridinium salts (CCPS and dhCCPS, respectively). Ceramidoids having the pyridinium salt unit (PSU) placed internally (alpha and gamma- CCPS) or as a tether (omega-CCPS) in the N-acyl moiety were prepared by N-acylation of sphingoid bases with different omega-bromo acids or pyridine carboxylic acid chlorides following capping with respective pyridines or alkyl bromides. Consistent with their design, these analogs, showed a significantly improved solubility in water, well-resolved NMR spectra in D(2)O, broadly modified hydrophobicity, fast cellular uptake, and higher anticancer activities in cells in comparison to uncharged counterparts. Structure-activity relationship (SAR) studies in MCF-7 breast carcinoma cells revealed that the location of the PSU and its overall chain length affected markedly the cytotoxic effects of these ceramidoids. All omega-CCPSs were more potent (IC(50/48 h): 0.6-8.0 microM) than their alpha/gamma-CCPS (IC(50/48 h): 8-20 microM) or D-erythro-C6-Cer (IC(50/48 h): 15 microM) analogs. omega-DhCCPSs were also moderately potent (IC(50/48 h): 2.5-12.5 microM). Long-chain omega-dhCCPSs were rapidly and efficiently oxidized in cells to the corresponding omega-CCPSs, as established by LC-MS analysis. CCPS analogs also induced acute changes in the levels and composition of endogenous Cers (upregulation of C16-, C14-, and C18-Cers, and downregulation of C24:0- and C24:1-Cers). These novel ceramidoids illustrate the feasibility of compartment-targeted lipids, and they should be useful in cell-based studies as well as potential novel therapeutics.

Antineoplastic Agents↗

PubMed2006

New insights on the use of desipramine as an inhibitor for acid ceramidase.

Treatment of different cancer cell lines with desipramine induced a time- and dose-dependent downregulation of acid ceramidase. Desipramine's effect on acid ceramidase appeared specific for amphiphilic agents (desipramine, chlorpromazine, and chloroquine) but not other lysomotropic agents such as ammonium chloride and bafilomycin A1, and was not transcriptionally regulated. The cathepsin B/L inhibitor, CA074ME, but not the cathepsin D inhibitor, pepstatin A, blocked desipramine's effect on acid ceramidase. Desipramine led to a more pronounced downregulation of sphingosine compared to ceramide suggesting acid ceramidase inhibition is important to desipramine's mechanism of action. This study reveals a new mechanism of action for desipramine.

Antidepressive Agents, Tricyclic↗

PubMed2006

Neutral sphingomyelinases and nSMase2: bridging the gaps.

There is strong evidence indicating a role for ceramide as a second messenger in processes such as apoptosis, cell growth and differentiation, and cellular responses to stress. Ceramide formation from the hydrolysis of sphingomyelin is considered to be a major pathway of stress-induced ceramide production with magnesium-dependent neutral sphingomyelinase (N-SMase) identified as a prime candidate in this pathway. The recent cloning of a mammalian N-SMase-nSMase2- and generation of nSMase2 knockout/mutant mice have now provided vital tools with which to further study the regulation and roles of this enzyme in both a physiological and pathological context. In the present review, we summarize current knowledge on N-SMase relating this to what is known about nSMase2. We also discuss the future areas of nSMase2 research important for molecular understanding of this enzyme and its physiological roles.

Animals↗

PubMed2006

Acid ceramidase but not acid sphingomyelinase is required for tumor necrosis factor-{alpha}-induced PGE2 production.

Sphingolipids are well established effectors of signal transduction downstream of the tumor necrosis factor (TNF) receptor. In a previous study, we showed that the sphingosine kinase/sphingosine 1-phosphate (S1P) pathway couples TNF receptor to induction of the cyclooxygenase 2 gene and prostaglandin synthesis (Pettus, B. J., Bielawski, J., Porcelli, A. M., Reames, D. L., Johnson, K. R., Morrow, J., Chalfant, C. E., Obeid, L. M., and Hannun, Y. A. (2003) FASEB J. 17, 1411-1421). In this study, the requirement for acid sphingomyelinase and sphingomyelin metabolites in the TNFalpha/prostaglandin E(2) (PGE(2)) pathway was investigated. The amphiphilic compound desipramine, a frequently employed inhibitor of acid sphingomyelinase (ASMase), blocked PGE(2) production. However, the action of desipramine was independent of its action on ASMase, since neither genetic loss of ASMase (Niemann-Pick fibroblasts) nor knockdown of ASMase using RNA interference affected TNFalpha-induced PGE(2) synthesis. Further investigations revealed that desipramine down-regulated acid ceramidase (AC), but not sphingosine kinase, at the protein level. This resulted in a time-dependent drop in sphingosine and S1P levels. Moreover, exogenous administration of either sphingosine or S1P rescued PGE(2) biosynthesis after desipramine treatment. Interestingly, knockdown of endogenous AC by RNA interference attenuated cyclooxygenase 2 induction by TNFalpha and subsequent PGE(2) biosynthesis. Taken together, these results define a novel role for AC in the TNFalpha/PGE(2) pathway. In addition, the results of this study warrant careful reconsideration of desipramine as a specific inhibitor for ASMase.

Animals↗

PubMed2006

Bioactive sphingolipids in the modulation of the inflammatory response.

Inflammation is viewed as a protective response against insults to the organism. It involves the recruitment of many cell types and the production of various inflammatory mediators in attempts to contain and reverse the insult. However, inflammation can lead to irreversible tissue destruction by itself and, therefore, can represent a disease state that causes significant morbidity and mortality. Understanding the molecular mechanisms controlling the inflammatory response is essential to formulate therapeutic strategies for the treatment of inflammatory conditions. In fact, substantial research has unveiled important aspects of the inflammatory machinery, both at the cellular and molecular levels. Recently, sphingolipids (SLs) have emerged as signaling molecules that regulate many cell functions, and ample evidence emphasizes their role in the regulation of inflammatory responses. Here, we review the role of bioactive SL as regulators and mediators of inflammatory responses.

Animals↗

PubMed2006

Dynamic sequestration of the recycling compartment by classical protein kinase C.

It has been previously shown that upon sustained stimulation (30-60 min) with phorbol esters, protein kinase C (PKC) alpha and betaII become sequestered in a juxtanuclear region, the pericentrion. The activation of PKC also results in sequestration of transferrin, suggesting a role for PKC in regulating endocytosis and sequestration of recycling components. In this work we characterize the pericentrion as a PKC-dependent subset of the recycling compartment. We demonstrate that upon sustained stimulation of PKC, both protein (CD59, caveolin) and possibly also lipid (Bodipy-GM1) cargo become sequestered in a PKC-dependent manner. This sequestration displayed a strict temperature requirement and was inhibited below 32 degrees C. Treatment of cells with phorbol myristate acetate for 60 min led to the formation of a distinct membrane structure. PKC sequestration and pericentrion formation were blocked by hypertonic sucrose as well as by potassium depletion (inhibitors of clathrin-dependent endocytosis) but not by nystatin or filipin, which inhibit clathrin-independent pathways. Interestingly, it was also observed that some molecules that internalize through clathrin-independent pathways (CD59, Bodipy-GM1, caveolin) also sequestered to the pericentrion upon sustained PKC activation, suggesting that PKC acted distal to the site of internalization of endocytic cargo. Together these results suggest that PKC regulates sequestration of recycling molecules into this compartment, the pericentrion.

CD59 Antigens↗

PubMed2006

Potent antitumor activity of a novel cationic pyridinium-ceramide alone or in combination with gemcitabine against human head and neck squamous cell carcinomas in vitro and in vivo.

In this study, a cationic water-soluble ceramide analog L-threo-C6-pyridinium-ceramide-bromide (L-t-C6-Pyr-Cer), which exhibits high solubility and bioavailability, inhibited the growth of various human head and neck squamous cell carcinoma (HNSCC) cell lines at low IC50 concentrations, independent of their p53 status. Consistent with its design to target negatively charged intracellular compartments, L-t-C6-Pyr-Cer accumulated mainly in mitochondria-, and nuclei-enriched fractions upon treatment of human UM-SCC-22A cells [human squamous cell carcinoma (SCC) of the hypopharynx] at 1 to 6 h. In addition to its growth-inhibitory function as a single agent, the supra-additive interaction of L-t-C6-Pyr-Cer with gemcitabine (GMZ), a chemotherapeutic agent used in HNSCC, was determined using isobologram studies. Then, the effects of this ceramide, alone or in combination with GMZ, on the growth of UM-SCC-22A xenografts in SCID mice was assessed following the determination of preclinical parameters, such as maximum tolerated dose, clearance from the blood, and bioaccumulation. Results demonstrated that treatment with L-t-C6-Pyr-Cer in combination with GMZ significantly prevented the growth of HNSCC tumors in vivo. The therapeutic efficacy of L-t-C6-Pyr-Cer/GMZ combination against HNSCC tumors was approximately 2.5-fold better than that of the combination of 5-fluorouracil/cis-platin. In addition, liquid chromatography/mass spectroscopy analysis showed that the levels of L-t-C6-Pyr-Cer in HNSCC tumors were significantly higher than its levels in the liver and intestines; interestingly, the combination with GMZ increased the sustained accumulation of this ceramide by approximately 40%. Moreover, treatment with L-t-C6-Pyr-Cer/GMZ combination resulted in a significant inhibition of telomerase activity and decrease in telomere length in vivo, which are among downstream targets of ceramide.

Animals↗

PubMed2006

Distinct roles for de novo versus hydrolytic pathways of sphingolipid biosynthesis in Saccharomyces cerevisiae.

Saccharomyces cerevisiae produces the sphingolipid ceramide by de novo synthesis as well as by hydrolysis of complex sphingolipids by Isc1p (inositolphosphoceramide-phospholipase C), which is homologous with the mammalian neutral sphingomyelinases. Though the roles of sphingolipids in yeast stress responses are well characterized, it has been unclear whether Isc1p contributes to stress-induced sphingolipids. The present study was undertaken in order to distinguish the relative roles of de novo sphingolipid biosynthesis versus Isc1p-mediated sphingolipid production in the heat-stress response. Ceramide production was measured at normal and increased temperature in an ISC1 deletion and its parental strain (ISC1 being the gene that codes for Isc1p). The results showed that Isc1p contributes specifically to the formation of the C24-, C24:1- and C26-dihydroceramide species. The interaction between these two pathways of sphingolipid production was confirmed by the finding that ISC1 deletion is synthetically lethal with the lcb1-100 mutation. Interestingly, Isc1p did not contribute significantly to transient cell-cycle arrest or growth at elevated temperature, responses known to be regulated by the de novo pathway. In order to define specific contributions of ISC1, microarray hybridizations were performed, and analyses showed misregulation of genes involved in carbon source utilization and sexual reproduction, which was corroborated by defining a sporulation defect of the isc1Delta strain. These results indicate that the two pathways of ceramide production in yeast interact, but differ in their regulation of ceramides of distinct molecular species and serve distinct cellular functions.

Gene Deletion↗

PubMed2006

Identification of a novel amidase motif in neutral ceramidase.

Neutral CDases (ceramidases) are newly identified enzymes with important roles in cell regulation, but little is known about their catalytic mechanisms. In the present study the full-length human neutral CDase was cloned and expressed in the yeast double-knockout strain Dypc1Dydc1, which lacks the yeast CDases YPC1p and YDC1p. Biochemical characterization of the human neutral CDase showed that the enzyme exhibited classical Michaelis-Menten kinetics, with an optimum activity at pH 7.5. Activity was enhanced by Na+ and Ca2+. Mg2+ and Mn2+ were somewhat stimulatory, but Zn2+, Cu2+ and Fe2+ inhibited the enzyme. Dithiothreitol and 2-mercaptoethanol dose-dependently inhibited neutral CDase. In order to identify which amino acids were involved in the catalytic action of neutral CDase, the purified enzyme was subjected to chemical modifications. It was observed that the serine residue modifier di-isopropyl fluorophosphate dose-dependently inhibited activity, implicating a serine residue in the catalytic action. From an alignment of the sequences of the neutral CDases from different species, all conserved serine residues were selected for site-directed mutagenesis. Of the six aligned serine residues that were mutated to alanine, only the S354A mutant lost its activity totally. Ser354 falls within a very highly conserved hexapeptide sequence GDVSPN, which itself was in the middle of a larger conserved sequence, namely NXGDVSPNXXGP/XXC. Moreover, mutations of Asp352 and Cys362 in the consensus sequence to alanine resulted in loss of activity of neutral CDase. Hence the present study identified a novel amidase sequence containing a critical serine residue that may function as a nucleophile in the hydrolytic attack on the amide bond present in ceramide.

Amidohydrolases↗

PubMed2006

Regulation of the sphingosine-recycling pathway for ceramide generation by oxidative stress, and its role in controlling c-Myc/Max function.

In the present study, the regulation of the sphingosine-recycling pathway in A549 human lung adenocarcinoma cells by oxidative stress was investigated. The generation of endogenous long-chain ceramide in response to exogenous C6-cer (C6-ceramide), which is FB1 (fumonisin B1)-sensitive, was employed to probe the sphingosine-recycling pathway. The data showed that ceramide formation via this pathway was significantly blocked by GSH and NAC (N-acetylcysteine) whereas it was enhanced by H2O2, as detected by both palmitate labelling and HPLC/MS. Similar data were also obtained using a novel approach that measures the incorporation of 17Sph (sphingosine containing 17 carbons) of 17C6-cer (C6-cer containing a 17Sph backbone) into long-chain 17C16-cer in cells by HPLC/MS, which was significantly decreased and increased in response to GSH and H2O2 respectively. TNF (tumour necrosis factor)-a, which decreases the levels of endogenous GSH, increased the generation of C16-cer in response to C6-cer, and this was blocked by exogenous GSH or NAC, or by the overexpression of TPx I (thioredoxin peroxidase I), an enzyme that reduces the generation of intracellular ROS (reactive oxygen species). Additional data showed that ROS regulated both the deacylation and reacylation steps of C6-cer. At a functional level, C6-cer inhibited the DNA-binding function of the c-Myc/Max oncogene. Inhibition of the generation of longchain ceramide in response to C6-cer by FB1 or NAC significantly blocked the modulation of the c-Myc/Max function. These data demonstrate that the sphingosine-recycling pathway for the generation of endogenous long-chain ceramide in response to exogenous C6-cer is regulated by ROS, and plays an important biological role in controlling c-Myc function.

Acylation↗