[Mucopolysaccharidosis: A review]. / Mucopolysaccharidoses : quand y penser ?

Mucopolysaccharidosis are lysosomal storage diseases, secondary to the accumulation of mucopolysaccharides. Type 1 mucopolysaccharidosis is the most common form and affects between 0.69 and 1.66 newborns per 100,000. The severity of mucopolysaccharidosis is variable with lethal forms in utero and attenuated forms diagnosed in adults. The most common symptoms are short stature, facial dysmorphism, chronic articular pains that can mimic chronic inflammatory rheumatism, axial and peripheral bone involvement, hepatosplenomegaly and an early carpal tunnel. Depending on the type of mucopolysaccharidosis, corneal, cerebral or cardiac involvements are possible. Screening is based on the analysis of urinary glycosaminoglycans. The deficient enzyme assay and the gene analysis confirm the diagnosis. Mucopolysaccharidosis recognition is important for patient management and family screening. In addition, specific enzyme replacement therapy exists for certain types of mucopolysaccharidosis. Role of clinician is important to evoke and diagnose mucopolysaccharidosis.

[Papillon-Lefèvre syndrome: A new case]. / Le syndrome de Papillon-Lefèvre, à propos d'une nouvelle observation.

Papillon-Lefèvre syndrome (PLS) is a rare primary immunodeficiency, which combines severe periodontal disease with edentulism and palmoplantar keratosis (PPK). PLS is inherited as an autosomal recessive trait and is due to mutations in the cathepsin C gene. The biological properties of the neutrophils (PN) are altered, leading to a gingival dysbiosis and bacterial overgrowth, with intense inflammation of the periodontium. We report the observation of a 4-year-old girl who presented to the clinic with gingivitis, partial edentulism, and PPK, whose diagnosis, raised after a long delay, was suggested by null cathepsin C activity and confirmed by the presence of heterozygous mutations in exon 4: c.628C>T, pArg210* and in exon 7: c.1286G>A, p.Trp429*. A multidisciplinary approach transformed the functional and esthetic prognosis and psychological behavior of this child. This classical observation describes this poorly known phenotype.

A sequential bioorthogonal dual strategy: ManNAl and SiaNAl as distinct tools to unravel sialic acid metabolic pathways.

Recent methodological developments in metabolic oligosaccharide engineering (MOE) pave the way for tremendous advances in glycobiology. Herein, we propose a Sequential Bioorthogonal Dual Strategy (SBDS) combining the use of two unprotected alkyne-tagged monosaccharide reporters (ManNAl and SiaNAl) with the bioligation of fluorescent probes by copper-catalysed azide-alkyne cycloaddition (CuAAC). With SBDS, we are able to shed light on trafficking and cellular uptake mechanisms of sialic acid. Using their corresponding analogues, we visualized that SiaNAl enters via endocytosis, whereas its biosynthetic intermediate ManNAl uptake is mediated by a yet unknown but specific plasma membrane transporter. Sialin, a lysosomal protein, is shown to be crucial for the export of exogenous sialic acid from lysosomes to the cytosol. Metabolic labeling with alkyne-tagged derivatives of N-acetylneuraminic acid (Neu5Ac) or N-acetylmannosamine (ManNAc) could thus be used to follow endocytosis in physiological vs. pathological conditions.

Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

[Cerebrotendinous xanthomatosis: a multicentric retrospective study of 15 adults, clinical and paraclinical typical and atypical aspects]. / Étude rétrospective multicentrique de 15 cas adultes de xanthomatose cérébrotendineuse : aspects cliniques et paracliniques typiques et atypiques.

INTRODUCTION: Cerebrotendinous xanthomatosis, a metabolic leukodystrophy with an autosomal recessive inheritance, is secondary to deficiency of sterol 27-hydroxylase, an enzyme involved in cholesterol catabolism. Classical symptoms include clinical or infraclinical xanthomas affecting the skin and tendons, early cataracts, neurological signs and diarrhea. Brain imaging reveals involvement of the dentate nuclei and periventricular white matter hyperintensities. The diagnosis is based on an increased cholestanol level in serum, confirmed by the presence of a mutation in the CYP27A1 gene. Treatment is based on chenodeoxycholic acid. METHOD: We report a retrospective multicentric study of 15 cases of cerebrotendinous xanthomatosis diagnosed in French adults. Clinical, molecular and MRI findings were recorded in all patients. RESULTS: The average age at diagnosis was 39years (range 27-65). Disease onset occurred in childhood in 73% of patients and in adulthood in 27%. All patients with a pediatric onset were diagnosed during adulthood (age range 28-65years). Clinical symptoms variably associated cerebellar syndrome, pyramidal syndrome, cognitive decline, epilepsy, neuropathy (sought in 10 of our patients, present in forms in 8), psychiatric disorders, cataract and xanthomas. One patient had an atypical presentation: monoparesis associated with xanthomas. Brain MRI was abnormal in all: findings consisted in T2-weighted hyperintensity of the dentate nuclei (47%), periventricular leuoencephalopathy (73%) which preferentially involved the posterior cerebral part (60%), leucoencephalopathy with a vascular pattern (7%), hyperintensity of the cortico-spinal tracts (53%), globi pallidi, corpus callosum and cerebral atrophy (33%). Serum cholestanol was elevated in 93% of patients. The most frequent mutation was 1183C>T (n=5/15). Under treatment with chenodeoxycholic acid, eight patients improved initially, followed by stabilization in five of them, and worsening in the others. Four patients died. CONCLUSION: Patients with the xanthoma-neurological disorder association should be tested for cerebrotendinous xanthomatosis. The disease often begins in childhood with a diagnostic delay but also in adulthood. Involvement of the dentate nuclei is specific but not sensitive and the supratentorial leucoencephalopathy is not specific but with an antero-posterior gradient. A vascular distribution and involvement of the corpus callosum are possible. Serum cholestanol assay is very reliable: an elevated level provides the diagnosis, which must nevertheless be confirmed by molecular biology.

Dual role of sphingosine kinase-1 in promoting the differentiation of dermal fibroblasts and the dissemination of melanoma cells.

Despite progress in the understanding of the biology and genetics of melanoma, no effective treatment against this cancer is available. The adjacent microenvironment has an important role in melanoma progression. Defining the molecular signals that control the bidirectional dialog between malignant cells and the surrounding stroma is crucial for efficient targeted therapy. Our study aimed at defining the role of sphingosine-1-phosphate (S1P) in melanoma-stroma interactions. Transcriptomic analysis of human melanoma cell lines showed increased expression of sphingosine kinase-1 (SPHK1), the enzyme that produces S1P, as compared with normal melanocytes. Such an increase was also observed by immunohistochemistry in melanoma specimens as compared with nevi, and occurred downstream of ERK activation because of BRAF or NRAS mutations. Importantly, migration of melanoma cells was not affected by changes in SPHK1 activity in tumor cells, but was stimulated by comparable modifications of S1P-metabolizing enzymes in cocultured dermal fibroblasts. Reciprocally, incubation of fibroblasts with the conditioned medium from SPHK1-expressing melanoma cells resulted in their differentiation to myofibroblasts, increased production of matrix metalloproteinases and enhanced SPHK1 expression and activity. In vivo tumorigenesis experiments showed that the lack of S1P in the microenvironment prevented the development of orthotopically injected melanoma cells. Finally, local tumor growth and dissemination were enhanced more efficiently by coinjection of wild-type skin fibroblasts than by fibroblasts from Sphk1(-/-) mice. This report is the first to document that SPHK1/S1P modulates the communication between melanoma cells and dermal fibroblasts. Altogether, our findings highlight SPHK1 as a potential therapeutic target in melanoma progression.

Evidence for clinical, genetic and biochemical variability in spinal muscular atrophy with progressive myoclonic epilepsy.

Spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) is a recently delineated, autosomal recessive condition caused by rare mutations in the N-acylsphingosine amidohydrolase 1 (acid ceramidase) ASAH1 gene. It is characterized by motor neuron disease followed by progressive myoclonic seizures and eventual death due to respiratory insufficiency. Here we report an adolescent female who presented with atonic and absence seizures and myoclonic jerks and was later diagnosed as having myoclonic-absence seizures. An extensive genetic and metabolic work-up was unable to arrive at a molecular diagnosis. Whole exome sequencing (WES) identified two rare, deleterious mutations in the ASAH1 gene: c.850G>T;p.Gly284X and c.456A>C;p.Lys152Asn. These mutations were confirmed by Sanger sequencing in the patient and her parents. Functional studies in cultured fibroblasts showed that acid ceramidase was reduced in both overall amount and enzymatic activity. Ceramide level was doubled in the patient's fibroblasts as compared to control cells. The results of the WES and the functional studies prompted an electromyography (EMG) study that showed evidence of motor neuron disease despite only mild proximal muscle weakness. These findings expand the phenotypic spectrum of SMA-PME caused by novel mutations in ASAH1 and highlight the clinical utility of WES for rare, intractable forms of epilepsy.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.

Farber lipogranulomatosis type 1--late presentation and early death in a Croatian boy with a novel homozygous ASAH1 mutation.

BACKGROUND: We report a boy with an unusually late presentation of Farber lipogranulomatosis type l. CASE STUDY: The first symptoms appeared at the end of the first year of life in the form of joint swelling; other symptoms such as cherry-red spot, hoarseness, subcutaneous nodules appeared much later. The history of the disease, from the first symptoms till his early death, lasted 26.5 months. The neuronal dysfunction accompanied by the rapid neurological deterioration with seizures and myoclonias, rather than the general dystrophy, seemed to limit the duration of disease in our patient and provoked his early death. Diagnosis was confirmed by analysis of ceramide metabolism in cultured fibroblasts and of the ASAH1 gene, which indicated homozygosity for a novel point mutation. CONCLUSION: The deficient activity of acid ceramidase correlated well with poor prognosis of the disease in our boy, in contrast to late appearance of dermal nodules and the subsequent severe clinical course with fatal outcome. Farber lipogranulomatosis should be suspected in children with joint swelling as the first and only symptom of disease. In order to advance our knowledge towards establishing genotype-phenotype correlations in Farber's disease, detailed analysis of the ASAH1 gene is needed.

Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death.

Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose- and time-dependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.

[Adult leukoencephalopathy caused by alpha-mannosidosis deficiency]. / Leucoencéphalopathie de l'adulte associée à un déficit en alpha-mannosidase.

Adult leukoencephalopathy caused by alpha-mannosidosis deficiency (MIM248500) is a recessive inherited lysosomal storage disease associated with decreased activity of alpha-mannosidase. This enzyme degrades oligosaccharides and glycoproteins in neural and visceral tissues. There are two different disease phenotypes, type-I or severe infantile phenotype and type 2, which progresses more slowly and is compatible with survival into adulthood. We report the case of a 51-year-old man with gait disorders beginning at the age of 40 years associated with leukoencephalopathy due to alpha-mannosidosis deficiency.

Caspase-dependent and -independent cell death of Jurkat human leukemia cells induced by novel synthetic ceramide analogs.

Ceramide metabolism has emerged as a potential target for anticancer therapy. Here, the potential usefulness of two novel synthetic ceramide analogs as anti-leukemic drugs was investigated. Compounds AD2646 and AD2687 were able to dose-and time-dependently decrease the viability of Jurkat leukemic cells. This was accompanied by an accumulation of endogenous ceramide owing to perturbed ceramide metabolism. Cytotoxicity involved caspase activation but also necrotic-like features, as evidenced by phosphatidylserine externalization, membrane permeability, hypodiploidy, caspase processing and only partial protection from cell death by a pan-caspase inhibitor. Ceramide analogs also induced cell death in Jurkat mutants that are deficient in cell death signaling proteins, including FADD, caspase-8 and 10, and RIP. While overexpression of Bcl-xL did not suppress ceramide accumulation, it conferred robust protection from caspase activation and cell death. Altogether, these novel ceramide analogs are able to kill leukemic cells through distinct pathways implicating caspase activation and mitochondrial events, and represent a new group of bioactive molecules with potential applications in anticancer therapy.

Prevention of posterior capsule opacification by the induction of therapeutic apoptosis of residual lens cells.

Posterior capsule opacification (PCO) is a common complication of cataract surgery. Using adenovirus(Ad)-mediated gene transfer, we overexpressed the proapoptotic molecules p53, procaspase 3, Bax, and TRAIL to induce therapeutic programmed cell death of residual lens cells to prevent PCO. Overexpressed TRAIL did not induce apoptosis in cultured rabbit lens cells or in human lens cells. Overexpressed p53 induced apoptosis of lens cells in vitro and ex vivo, but was unable to prevent PCO in vivo. Overexpressed procaspase 3 was associated with engagement of many components of the apoptotic pathway, including cleavage of intracellular caspase targets such as PARP and inter-nucleosome DNA fragmentation. Even when only slightly overexpressed, Bax caused apoptosis of transduced rabbit and human lens cells by engaging the mitochondrial pathway, including catalytic activation of the caspases. A single in vivo injection of Ad vectors expressing either Bax or procaspase 3 into the capsular bag at the end of phacoemulsification prevented PCO in rabbits. These experiments show that Ad-mediated Bax or procaspase 3 overexpression is capable of inducing therapeutic programmed cell death in vitro and in vivo in residual lens cells and preventing PCO in a rabbit model of PCO. Manipulation of proapoptotic molecule expression could be a novel gene therapy approach for prevention of PCO.

Overcoming MDR-associated chemoresistance in HL-60 acute myeloid leukemia cells by targeting sphingosine kinase-1.

We examined the involvement of sphingosine kinase-1, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed sphingosine kinase-1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment. Enforced expression of sphingosine kinase-1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a sphingosine kinase-1 inhibition and apoptosis both prevented by sphingosine kinase-1 over-expression. Furthermore, F-12509a, a new sphingosine kinase inhibitor, led to ceramide accumulation, decrease in sphingosine 1-phosphate content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding sphingosine 1-phosphate or overexpressing sphingosine kinase-1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide.

In endothelium, reoxygenation after hypoxia (H/R) has been shown to induce production of reactive oxygen species (ROS) by complex III of the mitochondrial respiratory chain. The purpose of the present study was to test the involvement of ceramide in this phenomenon. Human umbilical vein endothelial cells underwent 2 h of hypoxia (PO2, approximately 20 mmHg) without glucose and 1 h of reoxygenation (PO2, approximately 120 mmHg) with glucose. ROS production was measured by the fluorescent marker 2',7'-dichlorodihydrofluorescein diacetate, and cell death by propidium iodide. We showed that 1) after 1 h of reoxygenation, fluorescence had risen and that ROS production was inhibited by desipramine, an inhibitor of sphingomyelinase, an enzyme responsible for ceramide production (126 +/- 7% vs. 48 +/- 12%, P < 0.05); 2) administration of ceramide (N-acetylsphingosine) per se (i.e., in the absence of H/R) induced ROS production (65 +/- 3%), which was inhibited by complex III inhibitor: antimycin A (24 +/- 3%, P < 0.0001), or stigmatellin (31 +/- 2%, P < 0.0001); 3) hypoxia/reoxygenation-induced ROS production was not affected by either ceramide-activated protein kinase inhibitor dimethyl aminopurine or mitochondrial permeability transition inhibitor cyclosporin A but was significantly inhibited by the antiapoptotic protein Bcl-2 (82 +/- 8%, P < 0.05); 4) ceramide-induced ROS production was also inhibited by Bcl-2 (41 +/- 4%, P < 0.0001). These results demonstrate that in endothelial cells submitted to hypoxia and glucose depletion followed by reoxygenation with glucose, the pathway implicated in mitochondrial complex III ROS production is ceramide dependent and is decreased by the antiapoptotic protein Bcl-2.

Stress-induced apoptosis is impaired in cells with a lysosomal targeting defect but is not affected in cells synthesizing a catalytically inactive cathepsin D.

The role of cathepsin D in stress-induced cell death has been investigated by using ovine fibroblasts exhibiting a missense mutation in the active site of cathepsin D. The cathepsin D (lysosomal aspartic protease) deficiency did not protect cells against toxicity induced by doxorubicin and other cytotoxic agents, neither did it protect cells from caspase activation. Moreover, the cathepsin D inhibitor, pepstatin A, did not prevent stress-induced cell death in human fibroblasts or lymphoblasts. The possible role of lysosomal ceramide or sphingosine-mediated activation of cathepsin D in apoptosis was also excluded by using human cells either overexpressing or deficient in acid ceramidase. However, a normal lysosomal function seems to be required for efficient cell death, as indicated by the finding that fibroblasts from patients with mucolipidosis II were partially resistant to staurosporine, sphingosine and TNF-induced apoptosis, suggesting a key role of lysosomes in cell death.

Sphingolipid mediators in cardiovascular cell biology and pathology.

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.

Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in cytoplasmic droplets.

Multidrug-resistant (MDR) cancer cells have been shown to have an accumulation of glucosylceramide (GlcCer). In this study, we aim at localizing, at subcellular level, where these lipids accumulate. Neutral lipids and phospholipid containing organelles have been identified using confocal fluorescence microscopy and microspectrofluorometry by monitoring the emission of the fluorescent probe Nile-red. Data from confocal fluorescence microscopy analysis shows accumulation of neutral lipids in cytoplasmic droplets of MDR human carcinoma MCF7R cells. Microspectrofluorometric measurements show an increase of the gold-yellow emission intensity in MCF7R cells, corresponding to neutral lipids. Similar observations were made in human MDR vincristine-HL60 and doxorubicin-KB selected cells. Total cellular glucosylceramide (GlcCer) measurements using [(3)H]-palmitic acid and thin layer chromatography show a significant increase of GlcCer in MCF7R cells. Moreover, MCF7R cells treated with fluorescent GlcCer-bodipy exhibit an accumulation of this lipid in cytoplasmic droplets. Treatment of MCF7R cells with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanolol (PPMP), a potent inhibitor of GlcCer synthase, attenuates the Nile-red fluorescence emission emanating from these structures and reverses MDR. Moreover, Golgi compartments stained with fluorescent PPMP-bodipy, show an increase in the Golgi compartments density. Treatment of MCF7R cells with cyclosporine A (CSA), tamoxifen (TMX) and 3'-azido-3'deoxythymidine (AZT) leads to the same effect observed in the presence of PPMP. Treatment of MCF7 and MCF7R with the beta-glucosidase inhibitor conduritol beta-epoxide (CBE) significantly increases resistance to daunorubicin only in MCF7R cells. These data demonstrate also that: (i) CSA, an inhibitor of MDR, has an additional target in addition to P-glycoprotein; and (ii) TMX (used in breast cancer treatment and prevention) and AZT (used in the treatment of HIV) could have side effects by disturbing lipid metabolism and inhibiting many cellular functions required in normal cells.

Sphingosine 1-phosphate antagonizes apoptosis of human leukemia cells by inhibiting release of cytochrome c and Smac/DIABLO from mitochondria.

Sphingosine 1-phosphate (S-1P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-1P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-alpha (TNF-alpha), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which stimulates sphingosine kinase, the enzyme responsible for S-1P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethylsphingosine (DMS), a specific inhibitor of sphingosine kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-alpha, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-1P cotreatment. Hence, S-1P, likely generated through a protein kinase C- mediated activation of sphingosine kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells.